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and and regime. AG

Release of Information

Monsanto is submitting the information in this petition for review by the USDA as part of the regulatory process. By submitting this information, Monsanto does not authorize its release to any third party. In the event the USDA receives a Freedom of Information Act request, pursuant to 5 U.S.C., § 552 and 7 CFR Part 1, covering all or some of this information, Monsanto expects that, in advance of the release of the document(s), USDA will provide Monsanto with a copy of the material proposed to be released and the opportunity to object to the release of any information based on appropriate legal grounds, e.g., responsiveness, confidentiality, and/or competitive concerns. Monsanto expects that no information that has been identified as CBI (confidential business regime.and information), will be provided to any third party. Monsantoand understands that a CBI- AG deleted copy of this information may be made available to the public in a reading room and by individual request, as part of a public comment period. Except in accordance with the foregoing, Monsanto does not authorizeBayer the release,property public ationprotection orpublishing other distribution of this information (including websiteof pos ting) without Monsanto's priorcontents notice and its consent. parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject the violate be copy of and may documentpublication, It exploitation this any permission prohibited

This document is protected under copyright law. This document is for use only by the regulatory authority to which this has been submitted by Monsanto Company, and only in support of actions requested by Monsanto Company. Any other use of this material, without prior written consent of Monsanto, is strictly prohibited. By submitting this document, Monsanto does not grant any party or entity any right or license to the information or intellectual property described in this document.

Monsanto Company 06-CR-166U Page 2 of 262 and and regime. AG

Petition for the Determination of Non-regulated Status for MON 89034

Summary

The Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture (USDA) has responsibility under the Plant Protection Act (Title IV Pub. L. 106-224, 114 Stat. 438, 7 U.S.C. § 7701-7772) and the Plant Quarantine Act (7 U.S.C. § 151-167) to prevent the introduction and dissemination of plant pests into the United States. The APHIS regulation 7 CFR Part 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated article does not regime.and present a plant pest risk and should no longer be regulated. If andAPHIS determines that the AG regulated article does not present a plant pest risk, the petition is granted, thereby allowing unrestricted introduction of the article. Bayer property protectionpublishing Monsanto Company is submitting thisof request to APHIS for a determinationcontents of non- its regulated status for the new biotechnology-derivedparties. corndata product, MONtherefore 89034, any progeny derived from crosses between MON 89034 and conventionaland/oror corn, and any affiliates. progeny derived from crossesproperty of MON intellectual89034third with other biotechnology-derivedmay corn its which has been granted non-regulated statusas under 7 CFR Part 340. the of and is regulatory owner. a document Corn (Zea mays L.) is the largestany such crop grown in the U.S. in terms ofits acreage planted and reproductiondocument net value. Insect pests in the corn fields,owner if not properlythis controllofed, significantly reduce corn yields and grain quality. In 1997,under Monsanto of commercialized a biotechnology- rightsthe this derived corn documentproduct,and/or YieldGard ® Corn Borer corn (i.e., MONrights 810) which contains the to of fall use of Bacillus thuringiensis (Bt) cry1Ab gene. The expressionthe of Cry1Ab protein in corn plants providesThis effective protectionmay distribution,againstand damage caused by lepidopteran insect pests, rights owner such as Europeansubject corn borer. Since the launch of MON 810, several other Bt corn products have also been commercializedthe in theviolate U.S., including YieldGard® Rootworm be copy corn which expresses Cry3Bb1 proteinof that confers protection against coleopteran pests. and In 2005,may approximately document publication,35% of the total U.S. corn acreage was planted with corn seed It exploitation possessing insectthis resistance trait. any permission Monsanto has recently developedprohibited MON 89034 as a second generation insect protection corn product to provide enhanced benefits for the control of lepidopteran insect pests. the be MON 89034 producescommercial the Cry1A.105 and Cry2Ab2 proteins derived from Bacillus thuringiensisFurthermore,any, which are active against lepidopteran insect pests. Compared to MON 810, MONConsequently, 89034 willwithout better serve corn growers’ need for controlling a wider spectrum of lepidopteran pests and help assure the durability of Bt corn. MON 89034 provides outstanding control of Ostrinia species such as European corn borer (ECB) and Asian corn borer, and Diatraea species such as southwestern corn borer (SWCB) and sugarcane borer. Control of these insects provided by MON 89034 is comparable to MON 810. MON 89034 also provides a high level control of fall armyworm (FAW) throughout the season, whereas MON 810 principally controls fall armyworm larvae during vegetative growth stage. Furthermore, MON 89034 provides significantly improved protection from

Monsanto Company 06-CR-166U Page 4 of 262 CBI Deleted Version damage caused by corn earworm (CEW) than MON 810. In addition to the wider spectrum of insect control, the combination of the Cry1A.105 and Cry2Ab2 insecticidal proteins in a single plant, MON 89034, provides a much more effective insect resistance management (IRM) tool. The results of mathematical modeling indicate that biotechnology-derived plants expressing two Cry proteins will have significantly greater durability than plants producing either of the single proteins if the cross-resistance between the Cry proteins is low and the mortality of susceptible insects caused by each of the individual proteins is at least 90%. Comparative biophysical studies indicate that the Cry1A.105 and Cry2Ab2 proteins have important differences in their mode of action, specifically in the way in which they bind to the lepidopteran midgut. Therefore, the probability of cross-resistance between these two proteins is low. Furthermore, in vitro and in planta studies with Cry1A.105 and Cry2Ab2 demonstrate that both proteins are regime.and highly active against the primary lepidopteran pests of corn and(ECB, SWCB, CEW, and AG FAW), particularly ECB, achieving close to or greater than the critical 95% level of control in all cases. With these properties, MON 89034 should be durable with a significantly smaller structured refuge thanBayer is necessary property for Bt cornprotection productspublishing producing a single insecticidal protein. of contents its parties.data therefore The data and information presented in this application demonstrateand/oror that MON 89034 is affiliates. not likely to pose an increasedproperty plant pest intellectual potentialthird or to have an adversemay environmental its impact compared to conventional corn.as This conclusion is based on multiple lines of the of and evidence. The first is isthe detailed molecularregulatory characterization of theowner. inserted DNA. a document Results confirm the insertionany ofsuch a single functional copy of cry1A.105its and cry2Ab2 reproductiondocument expression cassettes at a single locusowner within the cornthis genome. Theof second is a detailed biochemical characterization of the Cry1Aunder.105 andof Cry2Ab2 proteins produced in MON rightsthe this 89034. In addition,documentand/or the data demonstrate that the Cry1A.105rights and Cry2Ab2 proteins to of fall use of produced in MON 89034 are equivalent to the respectivethe Cry1A.105 and Cry2Ab2 proteins Thisproduced by recombinantmay strainsdistribution,and of Escherichia coli, which were used in the rights owner various safety assessmentsubject studies. The third line of evidence is an assessment of the toxicity and allergenicity potential of thethe Cr y1A.105violate and Cry2Ab2 proteins based on be copy extensive information collected and studiesof performed on the two proteins. The results and demonstratemay with reasonabledocumentpublication, certainty that the Cry1A.105 and Cry2Ab2 proteins are It exploitation unlikely to be allergensthis or toxins. The fourth line of evidence is the compositional and nutritional assessmentany which confirms that MON 89034 grain and forage are permission compositionally equivalent to andprohibited as safe as those of conventional corn. The fifth line of evidence is the extensive evaluation of the MON 89034 phenotypic and agronomic the be characteristics andcommercial ecological interactions, which demonstrates that MON 89034 is not likelyFurthermore, to haveany an increased plant pest potential compared to the conventional corn. Finally,Consequently, an assessmentwithout on the potential impact on non-target organisms (NTO) and endangered species concludes that MON 89034 is unlikely to have adverse effects on these organisms under the conditions of use.

MON 89034 was produced by Agrobacterium-mediated transformation of corn with PV- ZMIR245, which is a binary vector containing 2T-DNAs. The first T-DNA, designated as T-DNA I, contains the cry1A.105 and the cry2Ab2 expression cassettes. The second T-DNA, designated as T-DNA II, contains the nptII (neomycin phosphotransferase II)

Monsanto Company 06-CR-166U Page 5 of 262 CBI Deleted Version expression cassette. During transformation, both T-DNAs were inserted into the genome. The nptII gene was used as the selectable marker which was needed for selection of the transformed cells. Once the transgenic cells were identified, the selectable marker gene was no longer needed. Therefore, traditional breeding was used to isolate plants that only contain the cry1A.105 and cry2Ab2 expression cassettes (T-DNA I) and do not contain the nptII expression cassette (T-DNA II), thereby, producing marker-free corn MON 89034. Molecular characterization of MON 89034 by Southern blot analyses demonstrated that the DNA inserted into the corn genome is present at a single locus and contains one functional copy of the cry1A.105 and the cry2Ab2 expression cassettes. All genetic elements are present in the inserted DNA as expected with the exception that the e35S promoter, which regulates expression of the cry1A.105 gene, has been modified and that the Right Border sequence present in PV-ZMIR245 was replaced by a Left Border regime.and sequence in MON 89034. No backbone plasmid DNA or nptIIand sequences were detected. AG PCR and DNA sequence analyses provided the complete DNA sequence of the insert and confirmed the organization of the elements within the insert. Bayer property protectionpublishing The stability of the integrated DNA wasof demonstrated by the fact that thcontentse Southern blot its fingerprint of MON 89034 was maintained for sevenparties. generationsdata tested intherefore the breeding history. Additionally, T-DNA II analysis of multiple generaand/ortionsor of MON 89034 affiliates. indicated that there were no propertyT-DNA II intellectualelementhirdts present other thanmay those which are its common to T-DNA I, including 35S aspromoter, nos 3´ end sequence, and Left Border the of and sequence. Furthermore, isthese generations haveregulatory been shown not to containowner. any backbone a document sequence from plasmid PV-ZMIR245.any such The stability was further confirmedits by the fact reproductiondocument that the inheritance of the lepidopteranowner protection traitthis in MON 89034of follows Mendelian segregation principle. under of rightsthe this documentand/or rights to of fall use of The expression levels of Cry1A.105 and Cry2Ab2 proteinsthe were determined in MON 89034 tissuesThis produced frommay multiple distribution, andfield sites in the major U.S. corn production rights owner regions. The resultssubject demonstrated that both Cry1A.105 and Cry2Ab2 proteins were expressed in all tissues collected, includingthe leaf,violate root, forage, silk, pollen, grain and be copy stover. The mean Cry1A.105 protein oflevels (µg/g dwt) across all test sites were 5.9 in and grain, 42may in forage,document 12 inpublication, pollen, 520 in leaves of plants at V2-V4 stage, 120 in leaves of It exploitation plants at pre-VTthis stage, 12 in forage root, and 50 in stover. In tissues harvested throughout the growingany season, mean Cry1A.105 protein levels (µg/g dwt) across all test permission sites varied from 72-520 in leaf,prohibited 11-79 in root, and 42-380 in whole plant. In general, levels of the Cry1A.105 protein declined over the growing season. The mean Cry2Ab2 the be protein levels (µg/gcommercial dwt) acr oss all test sites were 1.3 in grain, 38 in forage, 0.64 in pollen,Furthermore, 180 anyin leaves of plants at V2-V4 stage, 160 in leaves of plants at pre-VT stage, 21 in forageConsequently, root, withoutand 62 in stover. In tissues harvested throughout the growing season, mean Cry2Ab2 protein levels (µg/g dwt) across all test sites varied from 130-180 in leaf, 21-58 in root, and 38-130 in whole plant. In general, levels of the Cry2Ab2 protein declined over the growing season.

Safety assessment studies require large amount of the Cry1A.105 and Cry2Ab2 proteins. The expression levels of the two proteins in MON 89034 were too low to allow for purification of sufficient quantities of the two proteins directly from MON 89034 for use

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in the safety assessment studies. Therefore, it was necessary to produce the Cry1A.105 and Cry2Ab2 proteins in a high-expressing recombinant host organism, E. coli. The proteins produced by E. coli were engineered to match the amino acid sequences of their counterparts expressed in MON 89034. Thus, the physicochemical and functional equivalence of MON 89034-produced and E. coli-produced proteins was examined to ensure that the proteins from the two host sources were equivalent so that the E. coli- produced proteins could be used as surrogates in the studies. Small quantities of the Cry1A.105 and Cry2Ab2 proteins were purified from the grain of MON 89034. Large quantities of Cry1A.105 and Cry2Ab2 proteins were produced and purified from E. coli. The proteins from the two sources were characterized and the equivalence was evaluated based a panel of analytical tests and assays, including Western blot analysis; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); matrix-assisted laser regime.and desorption/ionization time-of-flight mass spectrometry (MALDI-TOFand MS); N-terminal AG sequence analysis; glycosylation analysis; and insect activity bioassay. The results provide a detailed characterization of the Cry1A.105 and Cry2Ab2 proteins isolated from MON 89034 and confirmed their equivalenceBayer to theproperty E. coli -producedprotectionpublishing Cry1A.105 and Cry2Ab2 proteins. of contents its parties.data therefore The history of safe use and data from multiple studies supportand/or the safetyor of MON 89034 affiliates. and the Cry1A.105 and Cry2Ab2property proteins. intellectual Thethird tw o proteins belong mayto a family of Cry its proteins from Bacillus thuringiensis, anas organism which has been used commercially in the of and the U.S. for over four isdecades to produce miregulatorycrobial pesticides. Btowner. corn expressing a document several Cry1 proteins and anyBt cotton such expressing both Cry1 and Cry2Ab2its proteins have reproductiondocument been cultivated in large areas in theowner U.S. and otherthis countries forof up to a decade. The extremely low mammalian toxicity of underBt-based microbialof insecticides and Cry proteins rightsthe this has been demonstrateddocumentand/or in numerous safety studies, and thererights are no confirmed cases of to of fall use of allergic reactions to Cry proteins. Cry1A.105 and Cry2Ab2the proteins do not share any amino acidThis sequence similaritiesmay withdistribution, knownand alle rgens, gliadins, glutenins, or protein rights owner toxins which havesubject adverse effects to mammals. This has been shown by extensive assessments with bioinformatic tools, suchthe as FASTAviolate sequence alignment search and an be copy eight-amino acid sliding window search.of Cry1A.105 and Cry2Ab2 proteins are rapidly and digestiblemay in simulateddocument gastricpublication, fluids (SGF). Greater than 95% to 99% of these proteins It exploitation were digested thisin SGF in less than 30 seconds. Proteins that are rapidly digestible in mammalian gastrointestinalany systems are unlikely to be allergens when consumed. Mice permission acute oral toxicity studies demonstrateprohibited that the Cry1A.105 and Cry2Ab2 proteins are not acutely toxic and do not cause any adverse effects even at the highest does levels tested, the be which are 2072commercial and 2198 mg/kg body weight for Cry1A.105 and Cry2Ab2 proteins, respectively.Furthermore,any The dietary safety assessment based on the acute toxicity data and corn productConsequently, dietarywithout pattern establishes that the margins of exposure (MOE) for the overall U.S. population are ≥199,000 and 981,000 for the Cry1A.105 and Cry2Ab2 proteins, respectively. And the MOEs are ≥79,400 and 390,000 for the Cry1A.105 and Cry2Ab2 proteins, respectively, for children aged 3-5 years old, an age group with the highest corn consumption per unit body weight. For poultry and livestock, the MOEs range between 1,930–13,500 and 2,160–47,600 for the Cry1A.105 and Cry2Ab2 proteins, respectively. Taken together, these data indicate that food and feed drived from MON 89034 which contains the Cry1A.105 and Cry2Ab2 proteins are safe for consumption.

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Compositional assessment of the grain and forage from multiple field sites in the major U.S. corn production regions demonstrate that MON 89034 is nutritionally and compositionally equivalent to, and as safe and nutritious as its conventional counterpart. The compositional analyses were conducted on a total of 77 components, nine in forage and 68 in grain, including protein, fat, carbohydrate, fiber, ash, moisture, amino acids, fatty acids, vitamins, anti-nutrients, secondary metabolites and minerals. The results were within the range of data expected for these types of analyses. Of the 77 components analyzed, the results of 16 components were below the limit of quantitation for over half of the analyses conducted. As such, these 16 components were not included in the statistical analyses. Among the remaining 61 components, analyses of the data across all test sites (combined sites) indicate that there were no statistical differences for 58 regime.and components. The three analytes with statistical differences wereand phosphorus in forage, AG and stearic acid (C18:0) and arachidic acid (C20:0) in grain. The differences observed are generally small (3.4 – 19.2%) considering the range of natural variability, and the mean levels and ranges of these three analBayerytes of MONproperty 89034 areprotection wellpublishing within the ranges of values observed for the 15 reference ofcommer cial corn hybrids grown alongcontents the side of its MON 89034, and within the ranges in the Internaparties.tionaldata Life Sciences thereforeInstitute crop composition database, as well as within the published literatureand/or ranges.or Therefore, it is affiliates. concluded that the slight differenceproperty observed intellectual forthird these 3 components mayis not biologically its meaningful, and MON 89034 and the controlas corn are compositionally equivalent. the of and is regulatory owner. a document The phenotypic, agronomic,any and such ecological interaction assessmentits indicates that MON reproductiondocument 89034 is comparable to conventionaownerl corn and is unlikelythis to haveof any increased plant pest risk. An important element in assessingunder plan t pestof potential and environmental impact of rightsthe this MON 89034 documentis to compareand/or MON 89034 to conventional corn.rights The assessment is based to of fall use of initially on the concept of familiarity, which USDA recognizesthe plays an important role in assessments.This Familiarity is basedmay distribution,on theand fact that the biotechnology-derived plant is rights owner developed fromsubject a conventional plant variety whose biological properties and plant pest potential are known to experts. Familiartheity violate considers the biology of the crop, the be copy introduced trait, the receiving environmentof and the interaction among these factors, and and providesmay a basis fordocument comparativepublication, risk assessment between a biotechnology-derived plant It exploitation and its conventionalthis counterpart. Results from the phenotypic and agronomic characteristics assessmentsany indicate that MON 89034 does not possess characteristics that permission would confer an increased plantprohibited pest risk compared to conventional corn. The assessments are based on a combination of laboratory experiments and field studies the be conducted by scientistscommercial who are familiar with the production and evaluation of corn. In eachFurthermore, of theseany studies, MON 89034 was compared to an appropriate conventional corn hybridConsequently, which haswithout a genetic background similar to MON 89034 but does not possess the lepidopteran-protection trait. In addition, multiple commercial corn hybrids were also employed to provide a range of values that are common to the commercial corn hybrids for each measured characteristic. These assessments included five seed germination parameters, two pollen characteristics, 14 plant growth and development characteristics, and more than 70 observations for each of the plant-insect, plant-disease and plant responses to abiotic stressor interactions.

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Seed dormancy and germination characterization observed no viable hard seed in any of the temperature regimes tested. No statistically significant differences were detected between MON 89034 and the control corn for pollen diameter or viability. The phenotypic and agronomic characteristics data collected from 18 field test sites in 2004 and 2005 demonstrate that no significant differences were detected between MON 89034 and the control corn for seedling vigor, early stand count, final stand count, days to 50% pollen shed, days to 50% silking, stay green, ear height, dropped ears, root lodged plants, grain moisture, test weight and yield. Differences in plant height and number of stalk lodged plants were detected in 2004 trials but not in 2005 trials. In 2004 trials, plant height was slightly lower for MON 89034 compared to control (84.1 vs. 85.4 inches), and stalk lodged plants were less for MON 89034 than for the control (0.8 vs. 2.4 per plot). The magnitude of these differences are generally small, similar differences are not regime.and detected in 2005 trials, and the mean values of MON 89034 falland well within the ranges of AG values observed for the 23 reference commercial corn hybrids grown along the side of MON 89034. Therefore, these two differences observed in 2004 field trials are not considered biologically meaningful and areBayer unlikely toproperty contribute protection to increasedpublishing plant pest potential. In addition to the phenotypic ofand agronomic characteristics, observationalcontents data its on the presence of and differential response to bioticparties. (insects,data diseases)therefore and abiotic (drought, wind, nutrient deficiency) stressors were collected and/orin theor two years of field affiliates. trials to examine the ecologicalproperty interactions intellectual of thirdMON 89034 comparedmay with those of the its conventional control corn. Based on as255 comparative observations recorded over two the of and years, no repeatable differencesis were observed regulatoryacross sites between MONowner. 89034 and the a document control in their susceptibilityany or tolerancesuch to the ecological stressorsits assessed. The result reproductiondocument supports the conclusion that comparedowner to conventionalthis corn, theof eco logical interactions between MON 89034 and insects, diseases,under and abofiotic stressors were not altered except rightsthe this for the introduceddocument lepidopteran-protectionand/or trait. rights to of fall use of the The environmentalThis assessmentmay of MONdistribution, and89034 and Cry1A.105 and Cry2Ab2 proteins rights owner indicates that thesesubject two proteins pose no adverse effect on non-target organisms (NTOs) and endangered species under the conditionsthe violate of use. The assessment took into be copy consideration several components, includingof the familiarity with the mode of action of and Cry proteins,may the activitydocumentpublication, spectra of the Cry1A.105 and Cry2Ab2 proteins, the expression It exploitation levels of the twothis proteins in MON 89034, the environmental fate of the proteins, the lack of interaction betweenany the two proteins, and feeding tests of the two proteins or MON permission 89034 corn materials to represenprohibitedtative NTOs. The tested NTOs include one mammalian species (mice), two avian species (broiler chicken and bobwhite quail), one aquatic the be species (Daphnia),commercial two species of soil decomposers (Collembola and earthworm), and fourFurthermore, beneficialany insect species (honeybee, minute pirate bugs, ladybird beetle, and parasitic wasp).Consequently, The estimateswithout of MOEs for the non-target insects exposed to Cry1A.105 and Cry2Ab2 proteins are ≥14. Risk assessments of potential effects on the federally listed threatened or endangered species indicated that only Karner blue butterfly (Lycaeides melissa samuelis) has the potential to occur in proximity to corn fields. There are only two Wisconsin counties in the Midwestern corn belt where potential temporal overlap between Karner blue butterfly larvae and corn pollen shed is likely. Based on a conservative risk analysis model, it is shown that the margin of safety for Karner blue is ≥12 fold using the highest possible pollen concentration from MON 89034. Taken

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together, these data support the conclusion that MON 89034 is unlikely to have adverse effects on NTOs and endangered species under the conditions of use.

The potential for MON 89034 outcrossing to sexually compatible species is unlikely in the U.S. Corn and annual teosinte (Zea mays subsp. mexicana) are genetically compatible, wind-pollinated and, in areas of Mexico and Guatemala, freely hybridize when in close proximity to each other. However, teosinte is not present in the U.S. other than as an occasional botanical garden specimen. Differences in factors such as flowering time, geographical separation and development factors make natural crosses in the U.S. highly unlikely. In contrast with corn and teosinte, special techniques are required to hybridize corn and Tripsacum. With the exception of Tripsacum floridanum, it is difficult to cross Tripsacum with corn, and the open literature indicates that the regime.and offspring of the cross show varying levels of sterility. Tripsacumand -corn hybrids have not AG been observed in the field. Therefore, the environmental consequence of pollen transfer from MON 89034 to other wild plant species is considered negligible. Bayer property protectionpublishing Finally, an assessment of current cornof agronomic practices determinedcontents that the its introduction of MON 89034 will not impact cultivationparties. practicesdata and the managementtherefore of weeds, diseases and insects except for the control of lepidopteranand/or insor ect pests. As with affiliates. other lepidopteran-protected Btproperty corn, MON intellectual 89034third provides a simple andmay highly effective its means for controlling lepidopteran pests.as The approach is environmentally benign and the of and helps to preserve beneficialis insects and decreaseregulatory cultiva tion input, andowner. requires fewer a document chemical insecticide applications.any such Th e combination of the Cry1A.105its and Cry2Ab2 reproductiondocument insecticidal proteins in a single plantowner provides not onlythis a wider ofspectrum of pest control but also a better insect resistance managementunder tool.of rightsthe this documentand/or rights to of fall use of Based on the data and information presented in this submission,the it is concluded that MON 89034 isThis not likely to pose mayan increased distribution,and plant pest potential or to have an adverse rights owner environmental impactsubject compared to conventional corn. The successful adoption of MON 89034 is expected to increase economic,the enviro violatenmental and health benefits due to the be copy protection of corn yields, decrease of chemiof cal insecticide usage, reduction of mycotoxin and levels inmay corn grain,document andpublication, increase of Bt corn product durability. Therefore, Monsanto It exploitation Company requeststhis a determination from APHIS that MON 89034 and any progeny derived from crossesany between MON 89034 and other commercial corn varieties be permission granted non-regulated status underprohibited 7 CFR Part 340. the commercial be Furthermore,any Consequently,without

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Abbreviations, Acronyms and Definitions

Note: Standard abbreviations, e.g., units of measure, are used according to the format described in ‘Instructions to Authors’ in the Journal of Biological Chemistry.

2T-DNA Plasmid vector containing two separate T-DNA regions each surrounded by left and right borders of the Ti plasmid 35S The promoter and leader from the cauliflower mosaic virus (CaMV) 35S RNA AACC American Association of Cereal Chemists and aadA Bacterial promoter and coding sequence andfor an aminoglycoside-regime. modifying enzyme, 3'(9)-O-nucleAG otidyltransferase from the transposon Tn7 ACB Asian corn borer, OstriniaBayer funicalisproperty protectionpublishing ADF Acid detergent fiberof contents its parties.data therefore ANOVA Analysis of Variance and/oror AOCS American Oil Chemistsaffiliates. Society property intellectualthird may AOSA Association itsof Official Seed Analysts as the of and BCW Blackis cutworm, Agrotis ipsilonregulatory owner. a document BSA Bovineany serum such albumin its reproductiondocument of Bt Bacillus thuringiensisowner this under of this Cab The rights5' untranslatedthe leader of the gene encoding wheat chlorophyll documentand/or of rights a/b-bindingto of proteinfall use the CaMV This cauliflowermay mosaic distribution,and virus rights owner CAPS subjectN-Cyclohexyl-3-aminopropanesulfonic acid CBI be Confidential businessthe information violate copy of CEW Corn earworm, Helicoverpaand zea may documentpublication, CFR It Code ofexploitation Federal Regulations this Cry anyCrystal proteins from Bacillus thuringiensis permission Cry1A.105 A chimericprohibited protein comprised of domains from the naturally occurringthe be Cry1Ab, Cry1F, and Cry1Ac proteins of Bacillus commercialthuringiensis cry1A.105Furthermore, any Coding sequence for Cry1A.105 protein Cry2Ab2Consequently, without A Cry2 class crystal protein from Bacillus thuringiensis subsp. kurstaki. cry2Ab2 Coding sequence for Cry2Ab protein CTAB Hexadecyltrimethylammonium bromide CTP Chloroplast transit peptide CV Column volume DAP Days after planting

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dCTP Deoxycytidine triphosphate DNA Deoxyribonucleic acid dNTP Deoxynucleotide triphosphate

DT50 Time to 50% dissipation of a protein in soil

DT90 Time to 90% dissipation of a protein in soil DTT Dithiothreitol DW Dry weight dwt Dry weight of tissue E. coli Escherichia coli e35S The promoter and leader from cauliflower mosaic virus (CaMV) regime.and 35S RNA containing the duplicated enhancerand region AG EC50 Effective protein concentration to inhibit the growth of the target insect by 50% Bayer property protectionpublishing ECB European corn borer,of Ostrinia nubilalis contents ECL Enhanced chemiluminescence its parties.data therefore EDTA Ethylenediaminetetraacetic acid and/oror affiliates. ELISA Enzyme-linkedproperty immunosorbent intellectualthird assay may its as EPA Unitedthe States of Environmentaland Protection Agency is regulatory owner. EPPS 4-(2-Hydroxyethyl)-1-pipa erazinepropanesulfonicdocument acid any such its reproductiondocument EPSPS 5-Enolpyruvylshikimate-3-phosphateowner this synthaseof FA Fatty acid under of rightsthe this documentand/or rights FASTA Algorithmto of usedfall to find localuse highof scoring alignments between a pair of protein or nucleotide sequencesthe This may distribution,and FAW Fallrights armyworm, Spodopteraowner frugiperda subject FDA United States Food and Drug Adminstration be the violate FIFRA copy Federal Insecticide,of Fungicide and Rodenticide Act and FMV may documentFigwortpublication, mosaic virus 35S promoter It exploitation FW Fresh weight this any fwt Fresh weight of tissue permission GDU Growing prohibiteddegree units the HEPES commercialN-[2-(Hydroxyethyl)piperazine be -N'-(2-ethanesulfonic acid)] HRPFurthermore, any Horseradish peroxidase Hsp17Consequently, without The 3' nontranslated region of the gene for wheat heat shock protein 17.3, which ends transcription and directs polyadenylation Hsp70 Maize heat shock protein 70 gene IgG Immunoglobulin G ILSI-CCD International Life Sciences Institute crop composition database IRM Insect resistance management

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LB buffer Laemmli buffer [62.5mM Tris-HCl, 5% (v/v) 2-mercaptoethanol, 2% (w/v) sodium dodecyl sulfate, 0.005% (w/v) bromophenol blue, 10% (v/v) glycerol, pH 6.8]

LC50 LC stands for lethal concentration. LC50 is the concentration of a substance which causes the death of 50% (one half) of a group of test organisms Left Border DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA LOD Limit of detection LOQ Limit of quantitation LSD Least significant difference and and regime. MALDI-TOF MS Matix-assisted laser desorption/ionizationAG time-of-flight mass spectrometry MEEC Maximum expected environmental concentration Bayer property protectionpublishing MH+ Protonated mass ionof contents its MMT Million metric tones parties.data therefore and/oror MOE Margin of exposure affiliates. property intellectualthird may MON 810 A Monsantoits corn product, producing the insecticidal Bt Cry1Ab as proteinthe of and is regulatory owner. MON 863 A Monsanto corn product,a producingdocument the insectic idal Bt Cry3Bb1 any such its protein reproductiondocument owner this of MON 89034 A Monsanto cornunder product, andof the subject of this application, rightsthe this documentand/orwhich produces Bt Cry1A.105 and Cry2Ab2rights proteins to of fall use of MS Mass spectrometry the This may distribution,and MW Molecularrights weight owner MWCO subjectMolecular weight cut-off be the violate MWM copy Molecular weightof marker N/A documentNot applicable and may publication,exploitation NCGAIt National Corn Growers Association this any NDF Neutral detergent fiber permission NFDM Non-fat driedprohibited milk the NK603 commercialA Monsanto be corn product producing the glyphosate-tolerant CP4 EPSPS protein Furthermore,any NMWCConsequently, without Nominal molecular weight cut-off NOEC No Observable Effect Concentration NOEL No Observable Effect Level nos The nopaline synthase gene from Agrobacterium tumefaciens nptII Coding sequence of neomycin phosphotransferase II gene that confers resistance to neomycin and kanamycin NPTII Neomycin phosphotransferase II

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NTO Non-target organism OECD Organization for Economic Co-operation and Development ori-PBR322 Origin of replication from pBR322 for maintenance of plasmid in E. coli ori-V Origin of replication for Agrobacterium derived from the broad host range plasmid RK2 OSL Overseason leaf OSR Overseason root OSWP Overseason whole plant PAGE Polyacrylamide gel electrophoresis and PBS Phosphate buffered saline and regime. AG PBST Phosphate buffered saline containing 0.05% (v/v) Tween-20 PCR Polymerase chain reaction Bayer property protectionpublishing PIP Plant-incorporatedof protectant contents PMSF Phenylmethanesulfonyl fluoride its parties.data therefore PTH Phenylthiohydantoin and/oror affiliates. PVDF Polyvinylideneproperty difluoride intellectualthird may its as PVPP Polyvinylpolypyrrolidonethe of and is regulatory owner. PV-ZMIR245 Plasmid vector used to developa MONdocument 89034 any such its reproductiondocument Ract1 the rice actin geneowner this of Right Border DNA region fromunder Agrobacterium of tumefaciens containing the right rightsthe this documentand/orborder sequence used for transfer of rightsthe T-DNA to of fall use of rop Coding sequence for repressor ofthe primer protein for maintenance This of plasmidmay copy distribution, numberand in E. coli rights owner SAP subjectScientific Advisory Panel organized by US EPA be the violate SAS copy Statistical Analysisof System SCB documentSugarcane borer, Diatraeaand saccharalis may publication,exploitation SD It Standard deviation this any SDS Sodium dodecyl sulfate permission SDS-PAGE Sodium dodecylprohibited sulfate polyacrylamide gel electrophoresis the SE commercialStandard be error SGF simulated gastric fluid Furthermore,any SSU-CTPConsequently, without The DNA encoding the transit peptide region of maize ribulose 1,5-bisphosphate carboxylase small subunit including the first intron SWCB Southwestern corn borer, Diatraea grandiosella TDF Total dietary fiber T-DNA I Transfer DNA containing the cry1A.105 and cry2Ab2 expression cassettes in plasmid vector PV-ZMIR245

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T-DNA II Transfer DNA containing the nptII gene cassette in plasmid vector PV-ZMIR245 T-DNA Transfer DNA TFA Trifluoroacetic acid TMB 3,3’,5,5’-tetramethylbenzidene Tris Tris(hydroxymethyl)aminomethane Tween-20 Polyoxyethylenesorbitan monolaurate USDA-APHIS United States Department of Agriculture – Animal and Plant Health Inspection Service USDA-ERS United States Department of Agriculture – Economic Research and Service and regime. USDA-NASS United States Department ofAG Agriculture – National Agricultural Statistics Service USDA-NRCS United States DepartmentBayer of Agricultureproperty –protection Nationalpublishing Resources Conservation Serviceof contents its USFWS United States Fish and Wildlifeparties. Servicedata therefore and/oror WBCW Western bean cutworm,affiliates. Striacosta albicosta property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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I. Rationale for the Development of MON 89034

I.A. Basis for the Request for a Determination of Nonregulated Status under 7 CFR Part 340.6

The Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture (USDA) has responsibility, under the Plant Protection Act (7 U.S.C. § 7701- 7772) and the Plant Quarantine Act (7 U.S.C. § 151-167), to prevent the introduction and dissemination of plant pests into the United States. The APHIS regulation 7 CFR Part 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and should regime.and no longer be regulated. If APHIS determines that the regulatedand article does not present a AG plant pest risk, the petition is granted, thereby allowing unrestricted introduction of the article. Bayer property protectionpublishing I.B. MON 89034 of contents its parties.data therefore Monsanto has developed a biotechnology-derived corn, MON 89034,and/oror which produces the affiliates. Cry1A.105 and Cry2Ab2 insecticidalproperty proteinsintellectualthird and is protected frommay feeding damage its caused by European corn borer (Ostriniaas nubilalis) and other lepidopteran insect pests. the of and Cry1A.105 is a modifiedis Bacillus thuringiensisregulatory (Bt) Cry1A protein.owner. Cry2Ab2 is a a document protein derived from Bt (subsp.any such kurstaki ). The combination ofits the Cry1A.105 and reproductiondocument Cry2Ab2 insecticidal proteins in a singleowner plant providesthis effectiveof insect control against a broad range of lepidopteran insect pestsunder a nd offersof an effective and durable insect- rightsthe this resistance managementdocumentand/or tool. rights to of fall use of the I.C. RationaleThis for the Develomaypment distribution, of MONand 89034 and Benefits rights owner subject Corn is the largest crop grown in the U.S.the in termsviolate of acreage planted and net value. In be copy 2005, corn was planted on 81.8 millionof acres and grain harvested from 75.1 million acres and (NCGA,may 2006). Thedocument cornpublication, grai n harvested had an average yield of 148 bushels per acre, It exploitation with a total productionthis of 11 billion bushels valued at $ (NCGA, 2006). In 2005, approximatelyany 42.5 million acres of corn planting area in the U.S. (or 52% of the permission total U.S. corn acreage) was plantedprohibited with biotechnology-derived corn seed, and 28.6 million acres (or 35% of the total corn acreage) were planted with corn seed possessing the be insect resistance commercial(Bt) trait (USDA-NASS, 2006). Furthermore,any In theConsequently, U.S., bothwithout corn yields and grain quality are significantly negatively impacted by insect pests (James, 2003). In 1997, Monsanto commercialized the biotechnology- derived corn product, YieldGard® Corn Borer corn (i.e., MON 810) which contains Bt cry1Ab gene that encodes the Cry1Ab protein, providing effective protection against damage caused by lepidopteran insect pests, especially the European corn borer (ECB, Ostrinia nubilalis) and the corn earworm (CEW, Helicoverpa zea). At that time in the U.S., the combination of crop losses and management costs resulting from lepidopteran

® YieldGard is a registered trademark of Monsanto Technology LLC.

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pests were estimated to be over $ per year (Mason et al., 1996). The introduction of MON 810 and other Bt corn products provided corn growers with a more effective solution for the control of lepidopteran pests such as ECB and CEW. In addition to the benefits as an effective insect pest management tool, MON 810 also resulted in lower levels of harmful mycotoxins in corn grain, which has resulted in improved food and feed safety of the corn products. The mycotoxin reduction effect was due to the decrease of insect damage to the Bt corn ears and thereby reducing the entering routes by mycotoxin- producing fungi. The mycotoxin reduction has been consistently demonstrated in countries around the world where corn borers are the predominant insect pests (Clements et al., 2003; Dowd, 2000 and 2001; Hammond et al., 2002 and 2004; de la Campa et al., 2005; Bakan et al., 2002; Magg et al. 2002; Munkvold et al., 1999; Munkvold, 2003; Papst et al., 2005; Pietri and Piva 2000; Wu, 2006). Furthermore, the use of MON 810 regime.and and other Bt corn products has reduced the use of chemical insecticidesand (Carpenter et al., AG 2004; Brookes and Barfoot, 2005). Within ten years since the first Bt corn product introduction, the safe and effective use of Bt corn has been adopted globally on over 44 million acres in 2005 to control several Bayerprimary insectproperty pests protection in 12publishing countries (James, 2005). of contents its parties.data therefore Recently, Monsanto developed MON 89034 as a second generationand/or insector protection corn affiliates. to provide enhanced benefits forproperty the control intellectual ofthird lepidopteran pests. Comparedmay to MON its 810, MON 89034 will even better serveas corn growers’ need for controlling a wider the of and spectrum of lepidopteranis pests, reducing the leveregulatoryls of mycotoxins in grain,owner. enabling more a document efficient plant breeding of anythis multi-genicsuch trait into superior hybrids,its and reducing the reproductiondocument refuge acreage needed for insect resistanceowner management.this By producingof efficacious levels of two insecticidal proteins – Cry1A.105under and Cry2Ab2,of MON 89034 increases the rightsthe this durability of thedocument productand/or against the primary lepidopteran rightspests of corn. In addition, the to of fall use of individual proteins extend the spectrum of control againsthet lepidopteran insects commonly present inThis corn fields. Specifically,may distribution, the andCry1A.105 protein provides increased activity rights owner against fall armywormsubject (FAW, Spodoptera frugiperda) and black cutworm (BCW, Agrotis ipsilon) compared to Cry1Ab. Thethe Cry2Ab2 violate protein provides improved control be copy over Cry1Ab products from damage causedof by corn earworm. This wider spectrum of and activity mayalso will potentiallydocumentpublication, contribute to the further reduction of mycotoxins in grain It exploitation that result fromthis fungal invasion after insect feeding damage. any permission Taken together, adoption of MONprohibited 89034 will enhance the economic benefits to growers and improve the quality of grain and the safety of food and feed products derived from the be the grain. In addition,commercial MON 89034 was developed to allow the efficient introgression of twoFurthermore, insect protectionany genes into improved corn germplasm, which will reduce the time and Consequently,costs for thewithout introduction of new improved varieties into the marketplace. MON 89034 was developed using a single transformation vector containing both the cry1A.105 and cry2Ab2 genes. This approach, known as vector stacking, increases the efficiency of breeding multiple traits into new corn hybrids, thereby providing growers an earlier access to improved germplasm containing these traits rather than through conventional inbred stacking.

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The benefits of MON 89034 are multi-faceted and interact in several ways to provide four key advantages, which are more specifically described below:

1) Extended Spectrum: A major benefit of MON 89034 is the protection of corn plants from feeding damage caused by lepidopteran insect pest larvae. MON 89034 provides outstanding control of Ostrinia species such as European corn borer and Asian corn borer (ACB), and Diatraea species such as southwestern corn borer (SWCB) and sugarcane borer (SCB). Control of these insects provided by MON 89034 is comparable to MON 810. MON 89034 also provides a high level of control of fall armyworm (FAW) throughout the season, whereas MON 810 principally controls damage caused by FAW larvae during vegetative growth. In addition, MON 89034 provides significant protection from damage caused by regime.and corn earworm. Although earworm control is not complete,and it is superior to that AG provided by MON 810. Introduction of MON 89034 will offer farmers a safe and effective alternative to the use of chemical insecticides for the control of the lepidopteran pests. Bayer property protectionpublishing of contents its 2) Improved Insect Resistant Management (IRM)parties.: data MON 89034 producestherefore two different Bt proteins, Cry1A.105 and Cry2Ab2, bothand/or areor highly efficacious affiliates. against a variety of lepidopteranproperty pests.intellectual thirdThe mechanism of insecticidalmay activity or its mode of action of Cry proteinsas consists of a number of steps e.g., solubilization, the of and activation, receptoris binding, oligomerization,regulatory and pore formationowner. (English and a document Slatin, 1992; Gill etany al., such1992; Schnepf et al., 1998; Zhuangits and Gill, 2003). reproductiondocument There are important differencesowner in each stepthis of the ofmode of action, which influences the interactions of these proteinsof with susceptible insects without rights under this qualitatively and/orinfluencing thetheir host range. Several lines of evidence establish that document to of fall of rights Cry1A.105 and Cry2Ab2 have importantuse differences in their mode of actions, the particularlyThis in the waymay in whichdistribution, andthey bind to the lepidopteran midgut. These rights owner proteins subjecthave different primary structures, share only 14% of amino acid sequence identity,be and bind to distinct proteinsthe inviolate the midgut of target species at different rates withcopy different affinities. of Therefore, the probability of cross-resistance between thesedocument proteins is very low.and Furthermore, in vitro and in planta studies of may publication,exploitation It the Cry1A.105 and Cry2Ab proteins demonstrate that both proteins are highly this active againstany the primary lepidopteran pests of corn: ECB, SWCB, CEW, and FAW. In view of thepermission dual effective dose and the distinct mode of action of the prohibited two proteins in MON 89034, the likelihood of resistance evolution is significantly the reduced comparedcommercial tobe singl e protein products. Therefore, MON 89034 is expected to be sustainable using a reduced structured refuge. Based on conservative Furthermore,any Consequently,mathematicalwithout models, a 5% structured refuge in the U.S. corn belt and a 20% structured refuge in cotton growing regions will ensure the durability of MON 89034.

3) Mycotoxin Reduction: Lepidopteran pests such as ECB, CEW and FAW cause substantial damage to stalks, ears, and leaves of developing corn plants resulting in reduced yields. MON 89034 has been shown to provide protection against a wider variety of lepidopteran pests than MON 810, as such it will provide greater yield protection and result in continuous reduction for potential mycotoxin

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contamination of the corn products. Corn ears that are protected from feeding damage caused by ECB, CEW, and FAW would have fewer ports of entry for invasion by mycotoxin-producing fungi, which will make the grain even safer to consume. As regulations are implemented globally that limit mycotoxin levels in food and feed, the economic impacts of mycotoxin contamination in grain will become increasingly important. In the U.S., the FDA has set guidelines for acceptable levels of the mycotoxin, fumonisin, in corn used for food and feed. In the recent study by Wu (2006), the economic benefit of mycotoxin reduction in Bt corn grain to meet this FDA standard resulted in an annual benefit in the U.S. of $ . With the expanded insect control spectrum of MON 89034 compared to MON 810, it is expected that there will be a greater benefit to food and feed consumed globally. and and regime. 4) Efficient Trait Integration: The rapidAG development of elite corn hybrids containing the cry1A.105 and cry2Ab2 genes is made possible by vector stack technology, i.e., the incorporation Bayerof multip leproperty genes into protection a singlepublishing transformation event. This approach increases theof efficiency of introduction of bothcontents proteins into its new corn germplasm by conventional breeding,parties. therebydata providing thereforegrowers early access to a variety of elite corn germplasm containing and/orboth orinsecticidal proteins. affiliates. Corn hybrids containingproperty multiple intellectual Bt proteithirdns conferring insectmay protection traits its have been developed previouslyas using traditional breeding techniques, i.e., two the of and inbreds containingis individual traits wereregulatory crossed to produce owner.the combined trait a document product. However,any such such breeding programs are generallyits costly because of reproductiondocument duplicated work to introgressowner two transformationthis eventsof into new germplasms. MON 89034 can reduce the undertime and costof factors in breeding programs to rightsthe this introgressdocument onlyand/or a single transformation event. rights to of fall use of the This may distribution,and rights owner In summary, thesubject introduction of MON 89034 will provide superior corn hybrids with higher yields, better quality grain, reduced potential for the development of insect be the violate resistance, and willcopy enhance breeding efficiencies.of and may documentpublication, I.D. AdoptionIt of MON 89034exploitation this any Corn is the largest crop grownpermission in the U.S. in terms of acreage planted and net value. In 2006, approximately 40% of theprohibited total corn acreage in the U.S. was planted with corn seed the possessing insectcommercial resistance be(B t) trait (USDA-NASS, 2006). Growers are expected to adopt MON 89034 in those regions of the U.S where lepidopteran pest pressure is high. TheFurthermore, major areaany of adoption is likely to be the U.S. corn belt including Illinois, Indiana, Consequently,without Iowa, Kansas, Kentucky, Michigan, Minnesota, Missouri, Nebraska, North Dakota, Ohio, South Dakota and Wisconsin.

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I.E. Submissions to Other Regulatory Agencies

I.E.1. Submissions to the EPA

Substances that are pesticides, as defined under the Federal Insecticide, Fungicide and Rodenticide Act [7 U.S.C. §136(u)], are subject to regulation by the Environmental Protection Agency (EPA). Pesticides produced in planta, referred to as plant- incorporated protectants (PIP’s), are also subject to regulation by the EPA.

Pursuant to §408(d) of the Federal Food Drug and Cosmetic Act [21 U.S.C. 346 a(d)] and Monsanto petitioned EPA for temporary exemptions from the andrequirement regime. of a tolerance for Cry1A.105 and Cry2Ab2 proteins in 2005.AG In July 2006, EPA established a temporary exemption from the requirement of a tolerance for the plant-incorporated protectant Bt Cry1A.105 protein and the genetic material necessary for its production in Bayer property protectionpublishing all corn commodities including field corn,of sweet corn, and popcorn (40 CFRcontents §174.453). Likewise, temporary exemption from the requirement of a toleranceits for Bt Cry2Ab2 parties.data therefore protein and the genetic material necessary for its production inand/or cornor was also granted by EPA in July 2006 (40 CFR §174.454).affiliates. property intellectualthird may its as Monsanto has filed an application,the of datedand September 28, 2006, to the EPA for the is regulatory owner. registration of the plant-incorporated protectanta Bacillusdocument thuringiensisits Cry1A.105 and Cry2Ab2 proteins, and theany genetic such material (vectorreproduction PV-ZMIR245) necessary for their this documentof production including field corn, sweetowner corn, and popcorn. under of rightsthe this documentand/or rights I.E.2. Submission to theto FDA of fall use of the This may distribution,and rights owner MON 89034 fallssubject within the scope of the Food and Drug Administration’s (FDA) policy statement concerningbe regulation of productsthe deviolaterived from new plant varieties, including those produced throughcopy genetic engineering.of Monsanto has voluntarily initiated and will complete a consultationdocument process with FDAand prior to commercial distribution of this may publication,exploitation product.It A safety and nutritional assessment of food and feed derived from MON 89034 this was submitted to theany FDA dated October 13, 2006. permission prohibited I.E.3. Submissions to Foreign Government Agencies the commercial be Regulatory submissions for import and production approvals will be made to countries thatFurthermore, import anyU.S. corn grain and have regulatory approval processes in place. These will includeConsequently, submissionswithout to a number of foreign government regulatory agencies, including Japan’s Ministry of Agriculture, Forestry and Fisheries (MAFF) and the Ministry of Health, Labor and Welfare (MHLW); the Canadian Food Inspection Agency (CFIA) and Health Canada; Argentina’s National Advisory Committee on Agricultural Biotechnology (CONABIA) and the National Service of Agricultural and Food Health and Quality (SENASA); the European Food Safety Authority (EFSA), and the regulatory authorities in many other countries. As appropriate, notifications of import will be made to importing countries that do not have a formal approval process.

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I.E.4. References

Bakan, B., D. Melcion, D. Richard-Molard, and B. Cahagnier. 2002. Fungal growth and Fusarium mycotoxin content in isogenic traditional maize and genetically modified maize grown in France and Spain. J. Agric. Food Chem. 50:728-731. Brookes, G. and P. Barfoot. 2005. GM crops: the global economic and environmental impact – the first years 1996-2004. AgBioForum 8(2&3):187-196. Carpenter, J.E., S. Sankula, C.S. Silvers, and L.P. Gianessi. 2004. Insecticidal Bacillus thurgingiensis plants versus chemical insecticides. ACS Symposium Series 866. and Agricultural Biotechnology. Chapter 3: 37-51. and regime. Clements, M.J., K.W. Campbell, C.M. Maragos, AGC. Pilcher, J.M. Headrick, J.K. Patacky, D.G. White. 2003. Influence of Cry1Ab protein and hybrid genotype on fumonisin contamination and fusariumBayer ear rot propertyof corn. Cropprotection Sci.publishing 43:1283-1293. of contents de la Campa, R., D.C. Hooker, J.D. Miller, A.W. Schaafsma, and B.G. Hammond. 2005. its parties.data therefore Modeling effects of environment, insect damage, and Btand/or genotypesor on fumonisin accumulation in maize in Argentina and the Philippines. Mycopathologia affiliates. may 159:539-552. property intellectualthird its as Dowd, P.F. 2000. Indirect thereduction of of earand molds and associated mycotoxins in Bacillus is regulatory owner. thuringiensis corn under controlleda and opendocument field conditions: utility and any such its limitations. J. Econ. Entomol. 93:1669-1679.reproduction document owner this of Dowd, P.F. 2001. Biotic and abiotic underfactors limitingof efficacy of Bt corn in indirectly rightsthe this reducingdocument mycotoxinand/or levels in commercial fields. J. Econ.rights Entomol. 94:1067-1074. to of fall use of English, L. and S.L. Slatin. 1992. The mode of actionthe of delta-endotoxins from Bacillus This may distribution,and thuringiensis - rightsa comparison with otherowner bacteria l toxins. Insect Biochem. Mol. Biol. 22:1-7.subject be the violate Gill, S.S., E.A. Cowles,copy and P.V. Pietrantonio.of 1992. The mode of action of Bacillus thuringiensis endotoxins. Annu. Rev.and Entomol. 37:615-636. may documentpublication, It exploitation Hammond, B.,this K. Campbell, T. Degooyer, A. Robinson, J. Richard, J. Segueira, C. Rubinstein, anyJ. Cea, M. Plancke, L. Pinson, C. Radu, H. Esin, F. Tatli, and R. permission Grogna. 2002. Reductionprohibited of fumonisin levels in grain from YieldGard corn borer. Proceedings of the 2nd Fungal Genomics, 3rd Fumonisin Elimination and 15th the be Aflatoxincommercial Elimination Workshops, Oct 23-25, 2002, San Antonio, Texas. Hammond,Furthermore, anyB.G., K.W. Campbell, C.D. Pilcher, T.A. Degooyer, A.E. Robinson, B.L. Consequently,McMillen,without S.M. Spangler, S.G. Riordan, L.G. Rice, and J.L. Richard. 2004. Lower fumonisin mycotoxin levels in the grain of Bt corn grown in the United States in 2000-2002. J. Agric. Food Chem. 52:1390-1397. James, C. 2003. Global review of commercialized transgenic crops: 2002; Feature: Bt Maize. ISAAA Briefs. No. 24. ISAAA. Ithaca, NY. http://www.isaaa.org/bin/Briefs/29/briefs.htm. Accessed September 14, 2006.

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Magg, T., A.E. Melchinger, D. Klein, and M. Bohn. 2002. Relationship between European corn borer resistance and concentration of mycotoxins produced by Fusarium spp. in grains of transgenic Bt maize hybrids, their isogenic counterparts and commercial varieties. Plant Breed. 121:146-154. Mason, C.E., M.E. Rice, D.D. Calvin, J.W. Van Duyn, W.B. Showers, W.D. Hutchinson, J.F. Witkowski, R.A. Higgins, D.W. Onstad, G.P. Dively. 1996. European Corn Boreer: Ecology and Management. North Central Regional Extension Publication 327. Ames, IA. Iowa State University. Munkvold, G.P. 2003. Cultural and genetic approaches to managing mycotoxins in maize. Annu. Rev. Phytopathol. 41:99-116. Munkvold, G.P., R.L. Hellmich, and L.G Rice. 1999. Comparison of fumonisinand and regime. concentration in kernels of transgenic AGBt maize hybrids and nontransgenic hybrids. Plant Disease. 83:130-138. NCGA. 2006. The World of Corn. National Corn Growers Association. Bayer property protectionpublishing http://www.ncga.com/03world/main/index.htmlof . Accessed Augustcontents 1, 2006. its Papst, C., H.F. Utz, A.E. Melchinger, J. Elder, T. Magg,parties. dataD. Klein, and M.therefore Bohn. 2005. Mycotoxins produced by Fusarium spp. in isogenic Btand/or vs. non-Btor maize hybrids affiliates. under European corn borerproperty pressure. intellectual Agronomythird J. 97:219-224. may its Pietri, A. and G. Piva. 2000. Occurrenceas and control of mycotoxins in maize grown in the of and Italy. In: Proceedingsis of the 6th Internationalregulatory Feed Productionowner. Conference, Food a document safety, current situationany andsuch perspectives in the European community,its edited by reproductiondocument G. Piva and F. Masoero, 27- owner28 November 2000,this Piacenza,of Italy, pp. 226-236. under of Schnepf, E.; N. Crickmore,rights J. Vanthe Rie, D. Lereclus, J. thisBaum, J. Feitelson, D.R. Zeigler, documentand/or of rights and D.H. Dean. to1998. of Bacillusfall thuringiensisuse and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:775-806. the This may distribution,and rights owner USDA-NASS. 2006.subject National Agricultural Statistics Service, United States Department of Agriculture.be Acreage Report, Crthe Pr2-5 violate (6-06) a. June 30, 2006. http://usda.mannlib.cornell.edu/usdacopy of /nass/Acre/2000s/2006/Acre-06-30-2006.pdf and Wu. F. 2006.may Mycotoxindocumentpublication, reduction in Bt corn: potential economic, health, and regulatory It exploitation impacts. Transgenic Res. 15: 227-289. this any Zhuang, M. and S.S. Gill, permissionS. S. 2003. Mode of action of Bacillus thuringiensis toxins. Pages 213-236 in Chemistryprohibited of Crop Protection, Progress and Prospects in the Science andcommercial Regulation. be Voss, G. and Ramos, G. (ed), Wiley-VCH, Weinheim. Furthermore,any Consequently,without

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II. The Biology of Corn

This section summarizes the biology of corn based on: 1) the consensus document on the biology of corn (maize) developed as part of the Organization for Economic Co-operation and Development (OECD, 2003); 2) a summary prepared by the Biotechnology Regulatory Services of the United States Department of Agriculture-Animal and Plant Health Inspection Service (USDA-APHIS, 2006); 3) information provided in the USDA petition for MON 88017 (Monsanto Company petition No. 04-CR-108U); and 4) other published literature.

II.A. Corn as a Crop regime.and and AG Corn (Zea mays L.), or maize, is one of the few major crop species indigenous to the Western Hemisphere. It is a member of the Maydeae tribe of the grass family, Poaceae. It is a robust monoecious annual plant, whicBayerh requiresproperty the help protection of manpublishing to disperse its seeds for propagation and survival. Cornof is an efficient plant for capturingcontents the energy of its the sun and converting it into food, and adapts readilyparties. to didatafferent conditionstherefore of humidity, sunlight, altitude, and temperature. and/oror affiliates. property intellectualthird may its Corn is grown in nearly all areas of theas world and ranks third behind rice (Oryza sativa the of and L.) and wheat (Triticumis sp.) in total global regulatoryproduction. In 2004, owner.corn was planted a document globally on 146.7 million haany with such a total production of 723.9 millionits metric tones (MMT) reproductiondocument (FAOSTAT, 2006). The top threeowner productio n countriesthis in 2004of were: USA (299.9 MMT), China (130.4 MMT) and Brazilunder (41.8 MMT).of In the U.S., corn is grown in rightsthe this almost all thedocument states,and/or and in 2004, it produced 11.8 billionrights bushels of corn grain with a to of fall use of market value of $ (USDA-NASS, 2006). the This may distribution,and rights owner In industrializedsubject countries corn has two major uses: 1) as animal feed in the form of grain, forage or silage; and 2) as a raw materithe alviolate for wet- or dry-milled processed products be copy such as high fructose corn syrup, oil,of star ch, glucose, and dextrose (Tsaftaris, 1995). and These processedmay productsdocumentpublication, are used as ingredients in many industrial applications and in It exploitation human food products.this In developing countries, corn is used in a variety of ways. In Latin American countriesany such as Mexico, one of the main uses of corn is for food. In permission Africa, corn is consumed as a foodprohibited in the sub-Saharan region, and in Asia it is generally used to feed animals (Morris, 1998). the be commercial CornFurthermore, has beenany studied extensively, and it seems that the domestication of corn occurred in southernConsequently, Mexicowithout 7,000 to 10,000 years ago. The putative parents of corn have not been recovered, but it is likely that teosinte played an important role in the genetic background of corn. The transformation from a wild, weedy species to one dependent on humans for its survival probably evolved over a long period of time by the indigenous inhabitants of the Western Hemisphere. Corn, as we know it today, cannot survive in the wild because the female inflorescence (the ear) restricts seed dispersal. Although grown extensively throughout the world, corn is not considered a persistent weed or one difficult to control. A summary of the history, taxonomy, genetics, and life cycle of corn is

Monsanto Company 06-CR-166U Page 31 of 262 CBI Deleted Version presented below, followed by a discussion of gene flow between cultivated corn and its wild relatives.

II.B. History of Corn

Corn originated in the highlands of Mexico 7,000 to 10,000 years ago. By the time Columbus discovered the Western Hemisphere, corn was being grown by the indigenous people from Chile to southern Canada. Columbus noted the presence of corn on the north coast of Cuba in November 1492, and introduced corn to Europe upon his return to Spain (Goodman, 1988). Within two generations after the introduction of corn to Europe, corn became distributed throughout those regions of the world where it could be cultivated.

regime.and The original corn-growing areas did not include the Northcentraland region (U.S. corn belt) AG of the United States. The highly productive U.S. corn belt dent corn was derived after the colonization of North America. The European settlers accepted the local native American varieties and incorporated them Bayerwith other cropsproperty to provide protection publishingfood, feed, and fuel for their survival. The current U.S. ofcorn belt dent corn evolved fromcontents the gradual its mingling of those settlements that spread north andparties. westdata from Southeasterntherefore North America, and those settlements that spread south and west and/orfromor Northeastern North affiliates. America. property intellectualthird may its as the of and The corn types grown inis the northeast are calleregulatoryd northern flints; their owner.origin is not clear, a document but races from the highlandsany of Guatemalasuch have similar ear morphologyits (Goodman and reproductiondocument Brown, 1988). Northern flints areowner largely eight-rowedthis with cylindricalof ears, are early maturing, and are short-statured plants underwith tillers. of The southern dent corn grown in the rightsthe this southeast Uniteddocument Statesand/or appears to have originated from therights southeast coast of Mexico. to of fall use of Southern dent corn is characterized as having tall, latethe maturing, non-tillered, poorly rooted plantsThis with soft-texturedmay white distribution, kernelands on many-rowed, tapering ears. It seems the rights owner Tuxpeno race contributedsubject to the development of southern dents. The intentional and/or unintentional crossing between the earlythe north violateern flints and late southern dents led be copy eventually to the highly productive U.S.of corn belt dent corn that is used extensively and throughoutmay the worlddocument today.publication, It exploitation this The origin of corn anyhas been studied extensively and four main hypotheses have been permission suggested (OECD, 2003): prohibited

the be 1. Descent fromcommercial teosinte - corn originated by direct selection from teosinte; Furthermore,2. The anytripartite hypothesis: a) corn originated from pod corn, b) teosinte derived Consequently,from a crosswithout of corn and Tripsacum, and c) modern corn varieties evolved by corn intercrossing with teosinte or Tripsacum or both (Mangelsdorf, 1974); 3. The common origin hypothesis - corn, teosinte, and Tripsacum descended independently from a common, unknown ancestor; and, 4. The catastrophic sexual transmutation hypothesis – that modern corn originated from teosinte by an epigenetic sexual mutation causing ear development.

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Other suggestions have included Coix and species of the genus Manisuris in the tribe Andropogoneae as contributing to the genome of corn. The hypotheses have been tested by the study of crosses for genome commonality, fertility, variation, and segregation of morphological plant traits, by archeological evidence, and by the use of molecular genetic markers.

Evidence has been reported to support the different hypotheses, but it seems that the preponderance of evidence supports the hypothesis that corn descended from teosinte (Galinat, 1988). The teosinte genome is similar to corn, teosinte easily crosses with corn, and teosinte has several plant morphological traits similar to corn. Teosinte has a more weedy appearance and more tillers than modern corn varieties. The one major distinct difference between corn and teosinte is the female inflorescence, or ear. Modern corn regime.and varieties have one to three lateral branches that terminate in anand ear with 8 to 24 kernel AG rows of 50 seeds, and the ear is enclosed in modified leaves or husks. Teosinte also has lateral branches, but they terminate in two-rowed spikes of perhaps 12 fruit cases, with each fruit case having one seed enclosed byBayer an indurated property glume protection(Goodman,publishing 1988). of contents its II.C. Taxonomy parties.data therefore and/oror affiliates. Corn (Zea mays L.) is a memberproperty of the tribeintellectual Maydaethird , which is includedmay in the subfamily its Panicoideae of the grass family Gramineaeas . Table II-1 summarizes the taxonomic the of and classification of corn andis its close relatives. regulatory owner. a document any such its reproductiondocument The genera included in the tribe Maydaeowner include Zeathis and Tripsacumof in the Western Hemisphere, and Coix, Polytoca, Chionachneunder , Schlerachneof , and Trilobachne in Asia. rightsthe this Although somedocument researchersand/or have implicated the Asian generarights in the origin of corn, the to of fall use of evidence for them is not as extensive and convincingthe as for the genera located in the WesternThis Hemisphere. may distribution,and rights owner subject The genus Zea includes two sections: Luxuriantesthe violate and Zea. Corn (Zea mays L.) is a be copy separate species within the subgenus Zea,of along with three subspecies. All species within and the genusmay Zea , exceptdocument corn,publication, are different species of teosinte. Until recently, the teosinte It exploitation species were includedthis in the genus Euchlaena rather than the genus Zea. any permission The other genus included in theprohibited Maydae tribe is Tripsacum. Tripsacum includes 16 species with a basic set of 18 chromosomes (n = 18), and the different species of the be Tripsacum includecommercial multiples of 18 chromosomes ranging from 2n = 36 to 2n = 108 (Furthermore,Table II-1any). An additional species, Tripsacum hermaphrodita (Anthephora hermaphroditaConsequently,),without has been described in the literature but has not been formally included in this taxonomic classification. In addition to their occurrence in other regions of the Americas, five Tripasacum species are native to the U.S. (USDA-NRCS, 2006). Tripsacum dactyloides, or Eastern gamagrass, is the most widely distributed species and can be found in the Midwestern, Eastern and Southern regions of the U.S. The other species have a more limited geographical distribution and can be found in the following states and territories: Tripsacum floridanum in Florida, Tripsacum lanceolatum in Arizona and New Mexico, Tripsacum fasciculatum in Puerto Rico, and Tripsacum

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hermaphrodita in Florida, Hawaii, Puerto Rico and the Virgin Islands (USDA-NRCS, 2006). Twelve of 16 Tripsacum species are native to Mexico and Guatemala. Tripsacum australe and two other species are native to South America. The center of variation for Tripsacum is the western slopes of Mexico, the same area where teosinte is frequently found. The habitat preferences of Tripsacum are similar to those for teosinte: seasonally dry, summer rains, elevation of 1500 m, and limestone soils (Wilkes, 1972).

Five genera are included in the tribe Maydeae that originated in Asia. Except for Coix, the basic chromosome number is n = 10. Within Coix, n = 5 and n = 10 have been reported.

II.D. Genetics regime.and and AG Corn is genetically one of the best developed and best characterized of the higher plants. Because of the separation of male and female inflorescence, number of seeds produced on the female inflorescence, ease in handlinBayerg (growing property and hand protection pollipublishingnating), nature of the chromosomes, and low basic chromosomeof number (n = 10), corn has contentsbeen accessible its for study at all levels of genetics. parties.data therefore and/oror affiliates. Corn was one of the first cropproperty species includintellectualedthird in genetic laboratoriesmay to obtain a basic its understanding of mitosis, meiosis, chromoas some segregation, linkage and effects of the of and crossing-over, and transposableis elements. Becauseregulatory of the importance owner.of corn in the U.S. a document and world economies, and theany genetic such information obtained since 1900,its corn continues to reproductiondocument receive extensive study in modern geneticowner laboratories.this of under of rightsthe this Molecular geneticistsdocumentand/or have developed extensive genetic rightsmaps of corn to complement to of fall use of those developed by the early corn geneticists. Cornthe has been used in tissue culture research,This in extensive studies mayto relate distribution, molandecular markers to qualitative and quantitative rights owner traits, in sequencingsubject of genes, in study of transposable elements for gene tagging and generating genetic variability, and in genethe transformationviolate (Coe et al., 1988; Carlson, be copy 1988; Phillips et al., 1988; Walbot and Messing,of 1988). and may documentpublication, It exploitation 6 The corn genomethis is approximately 5×10 kb in size (OECD, 2003). It includes highly repeated sequences anythat constitute about 20% of the genome; these sequences are present permission in about ten superabundant sequenceprohibited type s. There are more than 1000 different moderately repetitive sequence families, collectively representing 40% of the genome. the be The remaining 40%commercial of the genome contains single-copy sequences, or more than 106 gene-sizeFurthermore, regions.any Consequently,without

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Table II-1. Taxonomic classification of corn and its close relatives

Family - Gramineae Subfamily - Panicoideae Tribe - Maydae Western Hemisphere: I. Genus - Zea A. Subgenus - Luxuriantes 1. Zea luxurians (2n = 20) 2. Zea perennis (2n = 40) 3. Zea diploperennis (2n = 20) B. Subgenus - Zea regime.and 1. Zea mays (2n = 20) and Subspecies AG 1. Z. mays parviglumis (2n = 20) 2. Z. mays huehuetenangensis (2n = 20) Bayer property protectionpublishing 3. Z. mays mexicanaof (Schrad.) (2n = 20) contents its parties.data therefore II. Genus – Tripsacum and/oror A. Section – Tripsacumaffiliates. B. Section – Fasciculata property intellectualthird may Species its Species 1. T. andersomii (2nas = 64) 1. T. jalapense (2n = 72) the of and 2. T. isaustrale (2n = 36) regulatory 2. T. lanceolatumowner. (2n = 72) a document Varietiesany such 3. T. fasciculatumits (2n = 36) reproductiondocument a) T. australeowner var. australe this4. T. maizarof (2n = 36, 72) b) T. australe var. hirstum of 5. T. pilosum (2n = 72) rights under this 3.and/or T. bravum the(2n = 36, 72) Varieties document to fall of rights 4. T. cundinamarceof (2n = 36)use a) T. pilosum var. guatemalense 5. T. dactyloides (2n = 72) the b) T. pilosum var. pilosum This may distribution,and Varietiesrights owner subjecta) T. dactyloides var. hispidum the be b) T. dactyloides var. dactyloidesviolate copy of c) T. dactyloides var.and meridonale may documentd)publication, T. dactyloides var. mexicanum It exploitation this 6. T. floridanum (2n = 36) 7.any T. intermedium (2n = 72 8. T. manisuroidespermission (2n = 72) prohibited 9. T. latifolium (2n = 36) the commercial10. T. percuvianum be (2n = 72, 90, 108) 11. T. zopilotense (2n = 36, 72) Furthermore, any Asia: Consequently, I.without Genera— Chionachne (2n = 20) Schlerachne (2n = 20) Coix (2n = 10, 20) Trilobachne (2n = 20) Polytoca (2n = 20) Tribe—Andropogoneae I. Genus - Manisuris

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II.E. Life Cycle

Corn is an annual plant and the duration of its life cycle depends on the cultivars and on the environments in which the cultivars are grown (Hanway, 1966). Corn cannot survive temperatures below 0°C for more than six to eight hours after the growing point is above ground (five- to seven-leaf stage). Damage from freezing temperatures, however, depends on the extent of temperatures below 0°C, soil condition, amount of residue, the duration of freezing temperatures, wind movement, relative humidity, and stage of plant development. Light frosts in the late spring of temperate areas can cause leaf burning, but the extent of the injury usually is not great enough to cause permanent damage, although the corn crop will have a ragged appearance because the leaf areas damagedand by and regime. frost persist until maturity. The completion of theAG life cycle of corn, therefore, is dictated by the duration of the average number of frost-free days.

The number of frost-free days dictates thatBayer corn with propertydifferences in thepublishing length of their life protectioncontents cycles can be grown in north-to-south directionsof of temperate areas. In the United States, its corn with relative maturities of 80 days or less are grownparties. datain the extreme northerntherefore areas, and/oror and corn with relative maturities of more than 125 days are grown in the southern areas. affiliates. Corn having relative maturitiepropertys of 100 tointellectual 115 thirddays is typically grownmay in the U.S. corn its belt. Relative maturities, however, areas not parallel lines east-to-west because they are the of and dependent on prevailingis weat her patterns, topogrregulatoryaphy, large bodies owner.of water, and soil a document types (Troyer, 1994). any such its reproductiondocument owner this of under of Another measure used to rightsjudgethe the relative maturity ofthis corn is the number of growing documentand/or rights degree units (GDU) requiredto offrom emergencefall useto maturity. of Based on the GDU required to mature, corn is assigned to areas that have, on the average,the less than 1850 GDU in the extreme Thisnorthern areas of the Unitedmay distribution, Statesand to corn that requires more than 2750 GDU in rights owner more southern areas.subject Assume a 115-day maturity hybrid is grown in central Iowa. The the violate average last befrost copydate is May 1 and the average first frost date is October 5, resulting in an expected 158 frost-free days. If averagof e emergence is May 15 and average flowering and is July may15, 60 daysdocument are publication,required from emergence to flowering. Corn requires 50 to 60 It exploitation days to attain thisphysiological maturity. If physiological maturity occurs 55 days after flowering, physiologicalany maturity will occur on or about September 10, or 115 days from permission emergence to physiological maturity.prohibited the be If one considerscommercial the central U.S. corn belt as an example, the following time frame for eachFurthermore, stage ofany corn development could be as follows: Consequently,without Planting date: May 1 ± 10 days Date of emergence: May 10 ± 4 days Date of flower: July 20 ± 10 days Physiological maturity: September 10 ± 5 days Harvest maturity: October 10 ± 10 days

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These estimated time frames could vary within the same year among locations and among years at the same location, depending on the environmental conditions experienced from planting to harvesting.

II.F. Hybridization

Hybridization is a fundamental concept used in the breeding, production, and growing of corn in the United States. Corn evolved as an open-pollinated (cross-fertilizing) crop species, and until the 20th century, only open-pollinated corn varieties were grown. Because corn is essentially 100% cross-pollinated, the corn varieties were a collection of heterozygous and heterogeneous individuals (genotypes). Varieties were developed by simple mass selection by the indigenous natives prior to the arrival of Columbus. Their regime.and methods of selection were simple by present-day standards,and but they obviously were AG effective in developing races, varieties, and strains to satisfy their food, fuel, feed, and cultural needs. Hybridization occurred between varieties as cultures moved within the Western Hemisphere, releasing genetic variabilityBayer to developropertyp other protection uniquepublishing varieties. of contents its The fundamental concepts for development of hybridparties. datacorn were definedtherefore by 1920 (Sprague, 1946). Basic studies on the genetic compositionand/or of ora corn variety were affiliates. conducted to determine the propertyeffects of intellectualself pollinatingthird (or inbreeding,may which is the its opposite of outcrossing) within a cornas variety (Shull, 1908). Because corn naturally the of and cross-fertilizes, the geneticis composition of eachregulatory plant is not known. owner.Continuous selfing a document of individuals for seven to anyten generationssuch resulted in pure lines (orits inbred lines) within reproductiondocument which every plant had similar traits.owner The correct interpretationthis of what occurred during inbreeding was based on Mendelian genetics:under theof heterozygous loci were eliminated by rightsthe this inbreeding to documenthomozygousand/or loci of either one of the two allelesrights at each locus. The fixation to of fall use of of alleles in pure lines caused a general reduction inthe vigor and productivity. Recent techniquesThis such as marker-assistedmay distribution, breedingand have reduced the time and number of rights owner generations requiredsubject to produce pure inbreds (Youseff and Juvik, 2001). the violate be copy It was found upon crossing two pureof lines that vigor was restored. If no selection and occurredmay during inbreeding,documentpublication, the average performance (e.g., grain yield) of all possible It exploitation crosses was similarthis to performance of the original variety in which inbreeding was initiated. Some crosses,any however, were better than the original open-pollinated variety permission and could be reproduced from theprohibited cross of the pure-line parents of the cross. Hence, the concept of hybrid corn was determined: self to develop pure lines, cross the pure lines to the be produce hybrids,commercial evaluate hybrids to determine the best hybrid, and use pure-line parents toFurthermore, reproduceany the superior hybrid and distribute it for use by the growers (Shull, 1909). Consequently,without Hybridization is used in many phases of corn breeding because of the expression of heterosis. Hybridization is used to produce breeding populations (e.g., F2) to develop inbred lines for use in hybrids, to produce the crosses of superior lines for distribution to growers. Hybridization is easily accomplished either by hand-pollination or by wind- pollination in large crossing fields (male and female inbred lines) to produce large quantities of high-quality hybrid seed.

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II.G. Pollination

II.G.1. Outcrossing with Wild Zea Species

Annual teosinte (Zea mays subsp. mexicana) and corn are wind-pollinated, self- compatible, and are highly variable, interfertile species (Wilkes, 1972 and 1989). Corn and teosinte are genetically compatible, and in areas of Mexico and Guatemala they freely hybridize when they are in proximity to each other and other conditions are favorable. Teosinte exists primarily as a weed around the margins of corn fields, and the frequency of hybrids between teosinte and corn has been studied. A frequency of one F1 hybrid (corn × teosinte) for every 500-corn plants has been reported for the Chalco region and of the Valley of Mexico (Wilkes, 1972). The F1 hybrid of teosinteand by cornregime. is robust and fertile and is capable of backcrossing to corn. IntercrossingAG and gene exchange between teosinte and corn occurs freely, and, accompanied by selection, teosinte had a significant role in the evolution of corn. Recently, Evans and Kermicle (2001) have shown that Bayer property protectionpublishing although corn can introgress into teosinte,of there is incompatibility betweencontents some corn populations and certain types of teosinte, resulting in low fitness of itssome hybrids that parties.data therefore prevents a high rate of introgression. and/oror affiliates. property intellectualthird may Although corn easily crosses withits teosinte, teosinte is not present in the U.S. corn belt. as The natural distribution of theteosinte of is limitedand to the seasonally dry, subtropical zone with is regulatory owner. summer rain along the western escarpment ofa Mexico anddocument Guatemits ala and the Central Plateau of Mexico (Wilkesany, 1972; such Gonzalez andreproduction Corral, 1997). Dependent upon the this documentof human characterization of teosinte ownerwith its local environment, it may be considered a under of weed. However, it has beenrights notedthe that populations ofthis teosinte have been in decline for documentand/or rights several decades becauseto of inofcreased fall grazing anduse urbanizationof in Mexico (Wilkes, 1995). Except for special plantings, there are no reports ofthe teosinte occurring in the United This may distribution,and States. rights owner subject be the violate II.G.2. Outcrossingcopy with Tripsacum Speciesof document and may publication,exploitation TripsacumIt evolved by polyploidy, whereas corn and teosinte have undergone this introgressive hybridizatany ion at the diploid level (2n = 20). The diploid forms of Tripsacum (2n = 36) are permissionmorphologically distinct and allopathic in their distribution prohibited (Wilkes, 1989). Tripsacum species are perennials and seem to be more closely related to the the genus Manisuriscommercial than beto either corn or teosinte (Goodman, 1976). Tripsacum received greater interest in the evolution of corn after Mangelsdorf and Reeves (1931) Furthermore,any successfullyConsequently, crossedwithout corn and Tripsacum dactyloides (2n = 36). The cross by Mangelsdorf and Reeves (1931) was made with the diploid Tripsacum dactyloides (2n = 36) as the male parent. Silks of the female corn parent were cut to permit successful pollination. The cross had 28 chromosomes and was male sterile. Five other Tripsacum species have been crossed with corn, and Galinat (1988) has mapped more than 50 homologous loci on the chromosomes of corn and Tripsacum.

In contrast with corn and teosinte, which can be easily hybridized, both in the wild and by controlled pollinations, special techniques are required to hybridize corn and Tripsacum.

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With the exception of Tripsacum floridanum, it is difficult to cross Tripsacum with corn, and the offspring of the cross show varying levels of sterility. Tripsacum-corn hybrids have not been observed in the field, and Tripsacum-teosinte hybrids have not been produced (Wilkes, 1972).

In recent years additional research has been conducted on the hybridization of corn with Tripsacum species. Eubanks (1995, 1998) has developed a method for transferring Tripsacum genes into corn. In this method two wild relatives of corn, Tripsacum and diploid perennial teosinte (Zea diploperennis), were crossed to produce a hybrid, which is called tripsacorn and can be used to generate corn-tripsacorn hybrids. The use of tripsacorn is intended to confer resistance to pests and disease, drought tolerance, and improved uniformity. Eubanks (2000) has claimed that traits such as apomixis, regime.and totipotency, perennialism, adaptation to adverse soil conditionsand and to a carbon dioxide AG enriched atmosphere can be transmitted to corn by such techniques. While these discoveries show promise, additional research needs to be conducted to determine their widespread applicability. Bayer property protectionpublishing of contents its In experiments with Tripsacum dactyloides or Easternparties. gamagrass,data DeWaldtherefore and others at the USDA successfully obtained a true Tripsacum cytoplasmand/or withor a corn nuclear affiliates. background (DeWald et al., 1999).property In this intellectual expethirdriment Eastern gamagrassmay was used as the its female parent and corn as the maleas or pollen donor. The Tripsacum-derived the of and mitochondrial chondromeis and chloroplast plastoregulatoryme in these hybridsowner. contribute to the a document seed qualities of the plants,any but thesuch nuclear genome appeared to be itstotally corn in origin. reproductiondocument These results suggest that any possibilityowner of corn contributingthis genetiof c material to Eastern gamagrass through random pollen flowunder in agricu lturalof situations is extremely remote, and rightsthe this if this were todocument occur,and/or the resultant genome would be lackingrights in most or all of the corn to of fall use of chromosomal complement. the This may distribution,and rights owner II.G.3. Outcrossingsubject with Cultivated Zea Varieties be the violate copy of Corn is wind-pollinated, and the distancesand that viable pollen can travel depend on may documentpublication, prevailingIt wind patterns, humidity,exploitation and temperat ure. Occasionally it has been found that corn pollen canthis travel any up to 3.2 km (2 miles) by wind under favorable conditions. All corn will interpollinate, except for certain popcorn varieties and hybrids that have one of permission the dent-sterile gametophyte factorsprohibited (Gas , Ga, and ga allelic series on chromosome 4). the Pollen of a specificcommercial hybrid becan be carried by wind to pollinate other dent corn, sweet corn, and popcorn if the popcorn does not carry the dent-sterile gametophyte factor. Corn pollen,Furthermore, therefore,any moves freely within an area, lands on silks of the same cultivar or differentConsequently, cultivars,without germinates almost immediately after pollination, and completes fertilization within 24 hours. Although there may be some minor differences in the rate of pollen germination and pollen tube elongation on some genotypes, corn pollen is promiscuous. It is estimated each corn plant can shed more than 10 million pollen grains.

Certification standards for distances between different corn genotypes have been established to assist in the production of hybrid corn having desired levels of purity. A specific isolation field to produce commercial seed will be located so that the seed parent

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is no less than 660 feet (200 m) from other corn of a similar type (i.e., if seed parent is a yellow, dent corn it should be isolated at least 660 ft from other yellow, dent corn). The distance of 660 ft can be modified because of size of field, number of border rows, and different maturity dates of flower, provided no receptive silks are available at the time pollen is being shed from the contaminating field.

II.H. Weediness of Corn

Modern-day corn cannot survive outside of cultivation (Gould and Shaw, 1968). One does not find volunteer corn growing in fence rows, ditches, and roadsides as a weed. Although corn from the previous crop year can overwinter and germinate the following year, it cannot persist as a weed. The appearance of corn in soybean fields following the regime.and corn crop from the previous year is a common occurrence. Measuresand often are taken to AG eliminate either the plants with a hoe or to use herbicides to kill the corn plants in soybean fields, but the plants that remain and produce seed usually do not persist in the following years. Bayer property protectionpublishing of contents its It is difficult for corn to survive as a weed because parties.of pastdata selecti on in thetherefore evolution of corn. In contrast with weedy plants, corn has a polystichous femaleand/or orinflorescence (or ear) affiliates. on a stiff central spike (or cob)property enclosed intellectual with thirdhusks (modified leaves).may Consequently, its seed dispersal of individual kernels doesas not occur naturally because of the structure of the of and the ears of corn. Individualis kernels of corn, however,regulatory can be distributedowner. during grain a document harvest and transportation toany storage such facilities. In neither instance (naturalits or mechanical reproductiondocument harvesting) does corn become a troublesomeowner weed. thisCorn cannotof su rvive without human assistance and is not capable of survivingunder as a weed.of rightsthe this documentand/or rights to of fall use of II.I. Characteristics of the Recipient Corn Material the This may distribution,and rights owner The corn germplasmsubject that was utilized as the initial recipient of the transgenes in MON 89034 is the proprietary inbred line, LH172the (E ggerlingviolate 1994). This inbred line was used be copy because it responds well to transformationof with Agrobacterium and tissue regeneration. and may documentpublication, It exploitation The inbred linethis LH172 was developed from the cross of (LH82 x LH122) x LH82. LH172 is characterizedany as a non-Stiff-Stalk, yellow dent corn. Its parental line LH82 is permission considered to represent a uniqueprohibited heterotic group that typically is crossed to Stiff Stalk lines to produce commercial F hybrids. The progenitors of LH82 include Pioneer 3558, the be1 W153R (Wisconsincommercial Agricultural Experiment Station release), and ‘Krug’ (open- pollinatedFurthermore, variety).any Parental line LH122 was derived from Pioneer 3535. Consequently,without II.J. Corn as a Test System in this Petition

In developing the data in support of this petition, appropriate test and control materials were developed, and where feasible, conventional reference corn materials were used to establish a range of responses expected for commercial corn in the U.S. In general, the background of the test material was matched with that of the control material so that the effect of the genetic insert could be assessed in an unbiased manner. The transgenic

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(MON 89034) and non-transgenic corn lines were used as test and control (and/or reference) materials, respectively. Further descriptions of the test, control, and reference materials are provided in the methods and materials sections for each trial or study.

II.K. References

Carlson, W.C. 1988. The Cytogenetics of Corn. pp. 259-343. In Corn and Corn Improvement, 3rd edition. Sprague, G.G. and J.W. Dudley (eds.). American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Madison, Wisconsin.

Coe, E.H., Jr., M.G. Neulffer and D.A. Hoisington. 1988. The Genetics of Corn.and Pages 81-258 in Corn and Corn Improvement, 3rd Ed. Sprague,and G.G. andregime. J.W. Dudley (eds.). American Society of Agronomy, AGCrop Science Society of America, and Soil Science Society of America, Madison, Wisconsin. DeWald, C.L., P. Sims, Y. Li, and V.A. Sokolov.Bayer 1999.property A novel protection cytoplasmpublishing for maize. Maize Genetics Conference Abstracts.of 41:114 contents its parties.data therefore Eggerling, D.G. 1994. Inbred corn line LH172. U.S. patent 5,276,266.and/oror affiliates. Eubanks, M. 1995. A cross propertybetween twointellectual maithirdze relatives: Tripsacummay dactyloides and its Zea diploperennis (Poaceae). Econ.as Bot. 49:172-182. the of and Eubanks, M. 1998. Methodis and materials forregulatory conferring Tripsacumowner. genes in maize. a document U.S. patent 5,750,828.any such its reproductiondocument owner this of Eubanks, M. 2000. Novel genetic materialsunder for oftransmission into maize. International application, publishedrights underthe the Patent Cooperationthis Treaty (PCT) WO00/7434. documentand/or rights to of fall use of Evans, M.M.S. and J.L. Kermicle. 2001. Teosinte crossing barrier1, a locus governing the hybridizationThis of Teosintemay with distribution, maize.and Theor. Apppl. Genet. 103:259-265. rights owner FAOSTAT. 2006.subject Online database (faostat.fao.org/faostat) of Food and Agriculture Organizationbe (FAO) of the United theNations. violate Accessed on June 16, 2006. copy of Galinat, W.C. 1988. The Origin of Corn.and Pa ges 1-31 in Corn and Corn Improvement. may documentpublication, It Sprague, G.F. and J.W.exploitation Dudley (eds.). American Society of Agronomy, Crop Sciencethis Society any of America, and Soil Science Society of America, Madison, Wisconsin. permission prohibited Gonzalez, J. and J. Corral. 1997. Teosinte Distribution in Mexico. pp. 18-39. In the be Proceedingscommercial of a Forum: Gene Flow Among Maize Landraces, Improved Maize Furthermore,Varieties,any and Teosinte: Implications for Transgenic Maize. Serratos, J.A., M.C. Consequently,Willcox,without and F. Castillo (eds.). INIFAP, CIMMYT and CNBA, Mexico. Goodman, M.M. 1976. Maize. pp. 128-136. In Evolution of Crop Plants. N.W. Simmonds (ed.). Longman Group Ltd., London. Goodman, M.M. 1988. The history and evolution of maize. CRC Critical Rev. Plant Sci. 7:197-220 Goodman, M.M. and W.L. Brown. 1988. Races of Corn. Pages 33-79 in Corn and Corn Improvement. Sprague, G.F. and J.W. Dudley (eds.). American Society of

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Agronomy, Crop Science Society of America, and Soil Society of America. Madison, Wisconsin. Gould, F.W. and R.B. Shaw 1968. Grasses and Man. P. 382. Grass Systematics. McGraw Hill, New York. Hanway, J.J. 1966. How a Corn Plant Develops. Spec. Rep. No. 48. Iowa State Univ. Coop. Extension Service, Ames, Iowa. Mangelsdorf , P.C. and R.G. Reeves. 1931. Hybridization of maize, Tripsacum and Euchlaena. J. Hered. 22:329-343. Mangelsdorf, P.C. 1974. Corn: its Origin, Evolution, and Improvement. Harvard University. Press. Cambridge, Massachusetts. and Morris, M.L. 1998. Overview of the World Maize Economy.and Pages 13-34regime. in Maize Seed Industries in Developing CountriesAG. M.L. Morris (ed.). Lynne Rienner Publishers, Inc. and CIMMYT, Int. 99. OECD. 2003. Consensus document on theBayer biology propertyof Zea mays subsp.publishing mays (maize). protectioncontents Available online at www.olis.oecd.org/olis/2003doc.nsf/LinkTo/env-jm-of its mono(2003)11. Organization for Economicparties. Co-dataoperation and Development.therefore and/oror Accessed on March 6, 2006. affiliates. property intellectualthird may Phillips, R.L., D.A. Somers, and K.A.its Hibberd. 1988. Cell Tissue Culture and In Vitro as Manipulation. Pagesthe 345-387 of in Cornand and Corn Improvement, 3rd ed. Sprague, is regulatory owner. G.F. and J.W. Dudley (eds.). Americana Societydocument of Agronomy, Crop Science any such its Society of America, and Soil Science Societyreproduction of America,document Madison, Wisconsin. owner this of Shull, G. H. 1908. The composition underof a fi eld ofof maize. Am. Breeders’ Assoc. Rep. rightsthe this 4:296-301.document and/or rights to of fall use of Shull, G.H. 1909. A pure line method of corn breeding.the Am. Breeders Assoc. Rep. This may distribution,and 5:51-59 rights owner subject Sprague, G. F. 1946. The experimental basis for hybrid maize. Biolog. Rev. 21:101- be the violate 120. copy of Troyer, A.F. 1994.document Breeding Early Corn.and pp. 341-396. In Specialty Corns. Hallauer, may publication,exploitation It A.R. (ed.). CRC Press, Inc. Boca Raton, Florida. this any Tsaftaris, A.S. 1995. The Biology of Maize (Zea mays L.). Document X!/754/95 permission European Commission. prohibited the USDA-APHIS. commercial2006. Corn. be Available online at www.aphis.usda.gov/brs/corn.html. United States Department of Agriculture, Animal and Plant Health Inspection Furthermore,Service.any Accessed on March 6, 2006. Consequently,without USDA-NASS. 2006. Online database (www.nass.usda.gov) at the United States Department of Agriculture, National Agricultural Statistics Service (USDA- NASS). Accessed on June 26, 2006. USDA-NRCS. 2006. Online database (www.plants.usda.gov, keyword Tripsacum) at the United States Department of Agriculture, National Resources Conservation Service (USDA-NRCS). Accessed on July 10, 2006.

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Walbot, V. and J. Messing. 1988. Molecular Genetics of Corn. pp. 389-429. In Corn and Corn Improvement, 3rd edition. Sprague, G.F. and J.W. Dudley (eds.). American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Madison, Wisconsin. Wilkes, H.G. 1972. Maize and its wild relatives. Science 177:1071-1077. Wilkes, H.G. 1989. Maize: Domestication, Racial Evolution, and Spread. Pages 440- 455 in Foraging and Farming - The Evolution of Plant Exploration. Harris, D.R. and G.G. Hillman (eds.). Unwin Hyman, London. Wilkes, H.G. 1995. Teosinte in Mexico: Personal Retrospective and Assessment. Pages 10-17 in Proceedings of a Forum: Gene Flow Among Maize Landraces, Improved Maize Varieties, and Teosinte: Implications for Transgenicand Maize. and regime. Serratos, J.A., M.C. Wilcox and F. CastilloAG (eds.). INIFAP, CIMMYT and CNBA, Mexico. Yousef, G.G. and J.A. Juvik. 2001. Crop breeding, genetics and cytology. Crop Sci. Bayer property protectionpublishing 41:645-655. of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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III. Description of the Transformation System

III.A. Transformation System

MON 89034 was developed through Agrobacterium-mediated transformation of the corn LH172 to express the Bacillus thuringiensis insecticidal proteins Cry1A.105 and Cry2Ab2 using the plasmid vector PV-ZMIR245 (Figure III-1). Agrobacterium- mediated transformation is a well-documented process for the transfer and integration of exogenous DNA into a plant’s nuclear chromosome. MON 89034 was produced using the 2T-DNA transformation plasmid vector PV-ZMIR245 which contains two separate T-DNA’s. The first T-DNA, designated as T-DNA I, contains the cry1A.105 and the regime.and cry2Ab2 expression cassettes. The second T-DNA, designatedand as T-DNA II, contains the AG nptII expression cassette. During transformation, both T-DNAs were inserted into the genome. Traditional breeding was then used to isolate plants that only contain the T-DNA I (cry1A.105 and the cry2Ab2 expressionBayer cassettes) propertyand do notprotection containpublishing the T-DNA II (nptII expression cassette). This resultedof in the production of marker-free,contents lepidopteranits protected corn – MON 89034. parties.data therefore and/oror affiliates. The use of selectable marker genes,property such intellectualas nptIIthird, is essential to selectmay transformed cells its due to the relatively small number of targetas cells in which integration of the foreign DNA the of and occurs. Once the transgenicis cells are identified,regulatory the selectable markerowner. gene is no longer a document needed. For commercial development,any such it is desirable to produce marker-freeits plants. Co- reproductiondocument transformation of plants with Agrobacteriumowner tumefaciensthis 2T-DNAof binary vector system is an effective approach to generate marker-funderree plants.of This approach takes advantage of rightsthe this Agrobacteriumdocument-mediatedand/or transfer of two different T-DNAsrights bearing the gene of interest to of fall use of and the selectable marker gene, and their subsequent integrationthe at sufficiently unlinked sites in theThis primary tranformantsmay followed distribution,and by genetic segregation in the progenies. This rights owner 2T-DNA binarysubject vector approach has been successfully used in tobacco (Komari et al. 1996); soybean (Xing et al. 2000), barleythe (Matthews violate et al. 2001), corn (Miller et al. be copy 2002); and rice (Komari et al. 1996; Breitlerof et al. 2004). MON 89034 was developed and using suchmay 2T-DNA document vectorpublication, transfor mation and selection techniques. It exploitation this Freshly isolated immatureany corn embryos were used in the transformation (Ishida et al. permission 1996; Rout and Armstrong, 1997).prohibited Agrobacterium tumefaciens strain ABI, containing plasmid PV-ZMIR245 was induced to be virulent by the use of acetosyringone. Strain the be ABI also containscommercial a disarmed Ti plasmid that is incapable of inducing tumor formation becauseFurthermore, ofany the deletion of the phytohormone genes originally present in the AgrobacteriumConsequently, withoutTi plasmid. Following inoculation with Agrobacterium, the immature embryos were transferred to a co-culture medium for one to three days to ensure transformation of individual cells. This process of Agrobacterium-mediated transformation of corn involves the attachment of the bacterium to the corn cells, which leads to transfer of the region of DNA between the Left and Right Borders of the binary plasmid (i.e., the T-DNA) into the corn genomic DNA. Each T-DNA was integrated into the plant genome at separate loci.

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Following the incubation period on the co-culture medium, the immature embryos were transferred to selection medium containing carbenicillin to eliminate Agrobacterium, and paromomycin to eliminate cells that were not transformed, so that only cells containing T-DNA II and/or T-DNA I + T-DNA II survived. The resulting transformed cells were then subcultured several times on a selection medium and regenerated into plants according to the protocol described by Armstrong and Phillips (1988).

During subsequent breeding at the F1 generation the unlinked insertions of T-DNA I or T- DNA II were segregated. The plants containing only the insert that contains the cry1A.105 and cry2Ab2 gene cassettes were selected using molecular analysis, while the plants containing the nptII cassette (T-DNA II) were eliminated from subsequent breeding. The absence of the nptII gene and the NPTII protein was further confirmed by regime.and both Southern blot and ELISA analyses. Figure III-2 displaysand the process map of the AG major steps involved in the transformation, selection, and development of MON 89034.

III.B. References Bayer property protectionpublishing of contents its Armstrong, C.L. and R.L. Phillips. 1988. Genetic parties.and cytogeneticdata variationtherefore in plants regenerated from organogenic and friable, embryogenicand/or tissueor cultures of maize. affiliates. Crop Sci. 28:363-369. property intellectualthird may its as Breitler, J.C., D. Meynard,the J.V. of Boxtel, andM. Royer, F. Bonnot, L. Cambillau, and E. is regulatory owner. Guiderdoni. 2004. A novel two T-DNAa binary vectordocument allows efficient generation any such its of marker-free transgenic plants in three reproductionelite cultivarsdocument ofof rice (Oryza sativa L.). Trans. Res. 13:271-287. owner this under of this Ishida, Y., H. Saito,and/or S. Ohta,rights the Y. Hiei, T. Komari, and T. Kumashiro. 1996. High document to fall of rights efficiency transformationof of maize (Zeause mays L.) mediated by Agrobacterium the tumefaciens.This Nat. Biotechnol.may distribution, 14:745-750.and rights owner Komari, T., Y. Hiei,subject Y. Saito, N. Murai, and T. Kumashiro. 1996. Vectors carrying two separate T-DNAs for co-transformatithe violateon of higher plants mediated by be copy Agrobacterium tumefaciens andof segregation of transformants free from selection and markers.may Plantdocument J.publication, 10:165-174. It exploitation Matthews, P.R.,this M.B. any Wang, P.M. Waterhouse, S. Thornton, S.J. Fieg, F. Gubler, and J.V. Jacobsen. 2001. Marker gene elimination from transgenic barley, using co- permission transformation with adjacentprohibited ‘twin T-DNAs’ on a standard Agrobacterium the transformationcommercial vector. be Mol. Breeding 7:195-202. Miller, M., L. Tagliani, N. Wang, B. Berka, D. Bidney, and Z.Y. Zhao. 2002. High Furthermore,any Consequently,efficiencywithout transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system. Trans. Res. 11:381-396. Rout, J.R. and C.L. Armstrong. 1997. Factors affecting transfer and integration of T- DNA into corn (Zea mays L.) cells. Program Issue of Congress on In Vitro Biology, June 14-18, 1997. J. Soc. In Vitro Biol. 33(3):14A. Xing, A., Z. Zhang, S. Sato, P. Staswick, and T. Clemente. 2000. The use of two T-DNA binary system to derive marker-free transgenic soybeans. In Vitro Cell Dev Biol 36:456-463.

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aadA BstE II 458 B-Right Border

P-e35S OR-ori-PBR322 Nde I 16501 PflF I 16425 L-Cab CS -rop BstE II 2464 OR-ori V I-Ract1 and Ssp I 15077 regime. and B-Left Border AG Nco I 2940 T-DNA II P-35S PflF I 13802 CS-nptII Bayer property protectionpublishing of PV-ZMIR245 contents 17600 bp BstE II 4613 CS-cryits 1A.105 T-nos parties.data therefore and/or BstEor II 4894 affiliates. property intellectualthird may B-Right Border its as the of and T-Hsp17 is regulatory owner. B-Left Border a documentP-FMV any such its reproductiondocument of T-nosowner this I-Hsp70 T-DNA I Nco I 10511 under of Sspthis I 7921 and/orrights the document to of BstE IIfall 10267 of rights CS-cry2Ab2 use the This may distribution,and rights ownerTS -SSU-CTP subject PflF I 8536 the violateNco I 8589 be copy of and may documentpublication, It exploitation this any permission Figure III-1. Plasmid map of prohibitedvector PV-ZMIR245

the be A circular map ofcommercial the plasmid vector PV-ZMIR245 used to develop corn MON 89034 is shown.Furthermore, PV-ZMIR245any contains two T-DNA regions designated as T-DNA I and T-DNA II. Consequently,The geneticwithout elements and restriction sites used in Southern blot analyses (with positions relative to the size of the plasmid vector) are shown on the exterior of the map.

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Assembly of plasmid vector PV-ZMIR245 in E. coli and transfer into Agrobacterium tumefaciens strain ABI

Transformation of the immature embryos of LH172 tissue with Agrobacterium tumefaciens ABI containing PV-ZMIR245

Initial selection of transformed cells that contain either T-DNA II orregime. and and T-DNA I + T-DNA II on a medium containingAG paromomycin

Bayer property protectionpublishing of contents Regeneration of transformed corn plants its parties.data therefore and/oror affiliates. property intellectualthird may its as Separation of unlinkedthe ofDNA insertands of T-DNA I and T-DNA II is regulatory owner. through segregation during traditionala breedingdocument any such its reproductiondocument owner this of under of rightsthe this documentand/or rights Screening of transformedto of fallplants for theuse presence of of T-DNA I (cry1A.105 and cry2Ab2 gene cassettes) and thethe absence of the This may distribution,and rightsT-DNA II (the nptII geneowner cassette) subject the violate be copy of document and mayEvaluation of selectedpublication,exploitation plants for agronomic performance and It this field efficacy any permission prohibited

the be commercial Selection of MON 89034 as the lead commercial Furthermore,any candidate for further evaluation Consequently,without

Figure III-2. Process map for transformation, selection, regeneration and evaluation of MON 89034

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IV. Donor Genes and Regulatory Sequences

This section describes the donor genes and regulatory sequences used in the development of MON 89034 and the deduced amino acid sequences of the Cry1A.105 and Cry2Ab2 proteins produced in MON 89034.

IV.A. Vector PV-ZMIR245

The plasmid vector PV-ZMIR245 (Figure III-1) was used for the transformation of corn cells to produce MON 89034. It was constructed at Monsanto’s research laboratories in and St. Louis, Missouri, using molecular biology techniques. PV-ZMIR245and regime.is a 2T-DNA transformation plasmid vector which contains twoAG separate T-DNAs.

The first T-DNA, designated as T-DNA I, contains two expression cassettes – cry1A.105 Bayer property protectionpublishing and cry2Ab2 expression cassettes. An expressionof cassette, or gene cassette,contents is defined as a coding sequence and the sequences necessary for proper expressionits of the coding parties.data therefore sequence (e.g., promoter, intron, and 3' end sequence). The and/orfirst orcassette in T-DNA I contains the cry1A.105 coding sequenceaffiliates. under the regulation of the e35S promoter, Ract1 property intellectualthird may intron, and the Hsp17 3' end sequence.its The second cassette in T-DNA I contains the as coding sequence for a chloroplastthe of transit andpeptide (SSU-CTP) and the cry2Ab2 coding is regulatory owner. sequence under the regulation of the FMV promoter,a the Hsp70document intron, and the nos 3' end any such its sequence. reproductiondocument of owner this under of The second T-DNA, designatedrights the as T-DNA II, contains thisthe nptII expression cassette. The documentand/or rights nptII coding sequence isto under of the fallregulation useof the ofCaMV 35S promoter and nos 3' end sequence. The nptII coding sequence encodes thethe ne omycin phosphotransferase II This may distribution,and protein which confers therights bact erial cells resistanceowner to kanamycin and was used during the initial selection subjectprocess. Traditional breeding was used to isolate plants that contain the be the violate cry1A.105 and thecopy cry2Ab2 expressionof cassettes (T-DNA I) but do not contain the nptII expression cassette (T-DNA II), thereforeand producing marker-free, lepidopteran-protected may documentpublication, MONIt 89034 (see Section IIIexploitation). The specific genetic elements and origins of the various components usedthis to any construct plasmid vector PV-ZMIR245 are provided in Table IV-1. The following sections provide further descriptions on these genetic elements. permission prohibited IV.B. T-DNA I the commercial be

IV.B.1.Furthermore, Theany cry1A .105 Coding Sequence and Cry1A.105 Protein Consequently,without

The cry1A.105 coding sequence encodes the 133 kDa Cry1A.105 insecticidal protein that provides protection against feeding damage by lepidopteran insect pests. The Cry1A.105 is a modified Bt Cry1A protein with amino acid sequence identity to Cry1Ab, Cry1Ac and Cry1F proteins of 90.0%, 93.6% and 76.7%, respectively (see Section VI). The deduced amino acid sequence of the Cry1A.105 protein produced in MON 89034 is presented in Figure IV-1.

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Table IV-1. Summary of the genetic elements in plasmid vector PV-ZMIR245

Genetic Location in Element Plasmid Function (Reference) Vector Backbone Intervening Sequence 1-257 Sequences used in DNA cloning Bacterial promoter, coding sequence, and terminator for an aminoglycoside-modifying enzyme, 3'(9)-O- nucleotidyltransferase from the transposon Tn7 (Flingand et al., 1985) (GenBank accessionand X03043). Theregime. aadA aadA 258-1146 confers resistanceAG to streptomycin and spectinomycin. Intervening Sequence 1147-1261 SequencesBayer used in DNAproperty cloning protection publishing of contents T-DNA I its parties.data therefore DNA region from Agrobacteriumand/or tumefaciensor B1-Right containing the 24 bp right border sequence used for affiliates. may 1262-1618property transfer intellectualof thethird T-DNA (Depicker et al., 1982) Border its as Intervening the of and is regulatory owner. Sequence 1619-1728 Sequences useda in DNA documentcloning its any suchThe promoter andreproduction 9 bp leader for the cauliflower mosaic this documentof virusowner (CaMV) 35S RNA (Odell et al., 1985) containing 2 under of this P -e35S and/or1729-2349rights the the duplicated enhancer region (Kay et al., 1987) document to of fall of rights Intervening use the SequenceThis 2350-2375may Sequences distribution,and used in DNA cloning rights owner subject The 5' untranslated leader of the wheat chlorophyll 3 L -Cab be 2376-2436 a/b-bindingthe proteinviolate (Lamppa et al., 1985) copy of Intervening and may documentpublication, SequenceIt 2437-2452exploitation Sequences used in DNA cloning 4 I -Ract1 this any2453-2932 Intron from the rice actin gene (McElroy et al., 1991) Intervening permission prohibited Sequence 2933-2941 Sequences used in DNA cloning the commercial be Coding sequence for Bacillus thuringiensis Cry1A.105 Furthermore,CS5-cry1A.105any 2942-6475 protein (Monsanto unpublished data) Consequently,without Table IV-1 continues on next page.

1 B – border region 2 P - promoter 3 L - leader 4 I - intron 5 CS – coding sequence. The sequence length listed in this table for cry1A.105 includes one stop codon which accounts for 3 bp.

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Table IV-1 (continued). Summary of the genetic elements in plasmid vector PV- ZMIR245

Genetic Location in Element Plasmid Function (Reference) Intervening Sequence 6476-6506 Sequences used in DNA cloning The 3' nontranslated region of the coding sequence for wheat heat shock protein 17.3, which ends transcription T1-Hsp17 6507-6716 and directs polyadenylation (McElwain and Spiker, 1989) Intervening and Sequence 6717-6783 Sequences used in DNA cloningand regime. AG P-FMV 6784-7347 The figwort mosaic virus 35S promoter (Rogers, 2000) Intervening Bayer property protectionpublishing of contents Sequence 7348-7369 Sequences used in DNA cloning its parties.data therefore The first intron from the maizeand/or heat orshock protein 70 I-Hsp70 7370-8173 geneaffiliates. (Brown and Santino, 1995) property intellectualthird may its Intervening as the of and Sequence 8174-8189is Sequences usedregulatory in DNA cloning owner. a document any suchThe DNA region containing the targetingits sequence for reproductiondocument theowner transit peptide regionthis of maizeof ribulose 1,5- bisphosphateunder carboxylaseof this small subunit and the first TS2-SSU-CTP and/or8190-8590rights the intron (Matsuoka et al., 1987) document to fall of rights of Coding sequenceuse for Cry2Ab2 protein from Bacillus the This maythuringiensis distribution,and (Widner and Whitely, 1989; Donovan, rights 1991). This ownercoding sequence uses a modified codon subject CS3-cry2Ab2 8591-10498 usage. be the violate Intervening copy10499- of Sequence document10524 Sequencesand used in DNA cloning may publication,exploitation It The 3' transcript termination sequence of the nopaline this any synthase (nos) gene from Agrobacterium tumefaciens 10525-permissionwhich terminates transcription and directs T-nos 10777 prohibitedpolyadenylation (Bevan et al., 1983) the be Intervening commercial10778- Furthermore,Sequence any 10844 Sequences used in DNA cloning Consequently,without DNA region from Agrobacterium tumefaciens 10845- containing the 25 pb left border sequence used for B-Left Border 11286 transfer of the T-DNA (Barker et al., 1983) Table IV-1 continues on next page.

1 T – transcript termination sequence 2 TS – targeting sequence 3 CS – coding sequence. The sequence length listed in this table for cry2Ab2 includes two stop codons which account for 6 bp.

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Table IV-1 (continued). Summary of the genetic elements in plasmid vector PV- ZMIR245

Genetic Location in Element Plasmid Function (Reference) Vector Backbone Intervening 11287- Sequence 12489 Sequences used in DNA cloning

T-DNA II and DNA region from Agrobacteriumand tumefaciensregime. B-Right 12490- containing the 24AG bp right border sequence used for Border 12846 transfer of the T-DNA (Depicker et al., 1982) Intervening 12847- Bayer property publishing protectioncontents Sequence 12971 Sequencesof used in DNA cloning its The 3' termination sequenceparties.data of the nopaline thereforesynthase and/oror (nos) coding sequence from Agrobacterium tumefaciens affiliates. 12972- propertywhich terminatesintellectualthird transcription and directsmay its T-nos 13224 polyadenylationas (Bevan et al., 1983) the of and is regulatory owner. Intervening 13225- a document any such its Sequence 13255 Sequences used reproductionin DNA cloningdocument of Codingowner sequence forthis neomycin phosphotransferase II under of 13256-rightsthe protein that confers resistancethis to neomycin and documentand/or rights CS-nptII 14050to of kanamycinfall (Beckuse etof al., 1982) the InterveningThis 14051- may distribution,and Sequence 14083rights Sequences usedowner in DNA cloning subject be 14084- The promoterthe violateand 31 pb leader for the cauliflower mosaic P-35S copy 14407 virus (CaMV)of 35S RNA (Odell et al., 1985) document and Interveningmay 14408-publication, It exploitation Sequence 14457 Sequences used in DNA cloning this any DNA region from Agrobacterium tumefaciens permission 14458- prohibitedcontaining the left border sequence used for transfer of B-Left Border 14899the the T-DNA (Barker et al., 1983) commercial be Vector Backbone Furthermore,any InterveningConsequently, without 14900- Sequence 14985 Sequences used in DNA cloning 14986- Origin of replication for Agrobacterium derived from the OR1-ori V1 15382 broad host range plasmid RK2 (Stalker et al., 1981)

Table IV-1 continues on next page.

1 OR-origin of replication

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Table IV-1 (continued). Summary of the genetic elements in plasmid vector PV- ZMIR245

Genetic Location in Element Plasmid Function (Reference) Intervening 15383- Sequence 16119 Sequences used in DNA cloning Coding sequence for repressor of primer protein for 16120- maintenance of plasmid copy number in E. coli (Giza CS-rop 16311 and Huang, 1989) regime.and Intervening 16312- and Sequence 16738 Sequences used AGin DNA cloning 16739- Origin of replication from pBR322 for maintenance of OR-ori-PBR322 17327 plasmid inBayer E. coli (Sutcliffe,property 1978) protection publishing of contents Intervening 17328- its parties.data therefore Sequence 17600 Sequences used in DNA cloningand/or or affiliates. property intellectualthird may its as IV.B.2. The cry1A.105 Regulatorythe of Sequencesand is regulatory owner. a document any such its reproductiondocument The expression cassette for the codingowner sequen ce of thethis Cry1A.105 of protein consists of the promoter (P-e35S) and leader for the cauliflowerunder mosaicof virus (CaMV) 35S RNA (Odell rightsthe this et al., 1985) documentwith aand/or duplicated enhancer region (Kay et rightsal., 1987). The cassette also to of fall use of contains the 5' untranslated leader of the wheat chlorophyll a/b/ binding protein (L-Cab) the (LamppaThis et al., 1985), the intronmay fromdistribution, andthe rice actin gene (I-Ract1) (McElroy et al., rights owner 1991), the cry1A.105subject coding sequence that was optimized for expression in monocots, and the 3’ nontranslatedbe region of the codingthe sequenceviolate for wheat heat shock protein 17.3 (T-Hsp17), whichcopy terminates transcriptionof and provides the signal for mRNA and polyadenylationmay (McElwaindocumentpublication, and Spiker, 1989). It exploitation this IV.B.3. The cry2Ab2any Coding Sequence and Cry2Ab2 Protein permission prohibited The Cry2Ab2 protein thepresent in MON 89034 is a member of the Cry2Ab class of commercial be proteins that share >95% amino sequence homology (Crickmore et al., 1998). It is a variantFurthermore, of theany wild-type Cry2Ab2 protein isolated from Bacillus thuringiensis subsp. kurstakiConsequently,. The deducedwithout amino acid sequence of the Cry2Ab2 protein, together with the chloroplast transit peptide (CTP), produced in MON 89034 is shown in Figure IV-2.

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1 MDNNPNINEC IPYNCLSNPE VEVLGGERIE TGYTPIDISL SLTQFLLSEF 51 VPGAGFVLGL VDIIWGIFGP SQWDAFLVQI EQLINQRIEE FARNQAISRL 101 EGLSNLYQIY AESFREWEAD PTNPALREEM RIQFNDMNSA LTTAIPLFAV 151 QNYQVPLLSV YVQAANLHLS VLRDVSVFGQ RWGFDAATIN SRYNDLTRLI 201 GNYTDHAVRW YNTGLERVWG PDSRDWIRYN QFRRELTLTV LDIVSLFPNY 251 DSRTYPIRTV SQLTREIYTN PVLENFDGSF RGSAQGIEGS IRSPHLMDIL 301 NSITIYTDAH RGEYYWSGHQ IMASPVGFSG PEFTFPLYGT MGNAAPQQRI 351 VAQLGQGVYR TLSSTLYRRP FNIGINNQQL SVLDGTEFAY GTSSNLPSAV 401 YRKSGTVDSL DEIPPQNNNV PPRQGFSHRL SHVSMFRSGF SNSSVSIIRA 451 PMFSWIHRSA EFNNIIASDS ITQIPLVKAH TLQSGTTVVR GPGFTGGDIL 501 RRTSGGPFAY TIVNINGQLP QRYRARIRYA STTNLRIYVT VAGERIFAGQ 551 FNKTMDTGDP LTFQSFSYAT INTAFTFPMS QSSFTVGADT FSSGNEVYID 601 RFELIPVTAT LEAEYNLERA QKAVNALFTS TNQLGLKTNV TDYHIDQVSN and 651 LVTYLSDEFC LDEKRELSEK VKHAKRLSDE RNLLQDSNFK DINRQPERGWand regime. 701 GGSTGITIQG GDDVFKENYV TLSGTFDECY PTYLYQKIDEAG SKLKAFTRYQ 751 LRGYIEDSQD LEIYSIRYNA KHETVNVPGT GSLWPLSAQS PIGKCGEPNR 801 CAPHLEWNPD LDCSCRDGEK CAHHSHHFSL DIDVGCTDLN EDLGVWVIFK 851 IKTQDGHARL GNLEFLEEKP LVGEALARVKBayer RAEKKWRDKR property EKLEWETNIVprotectionpublishing 901 YKEAKESVDA LFVNSQYDQL QADTNIAMIHof AADKRVHSIR EAYLPELSVIcontents 951 PGVNAAIFEE LEGRIFTAFS LYDARNVIKN GDFNNGLSCW NVKGHVDVEEits parties.data therefore 1001 QNNQRSVLVV PEWEAEVSQE VRVCPGRGYI LRVTAYKEGY GEGCVTIHEIand/oror 1051 ENNTDELKFS NCVEEEIYPN NTVTCNDYTV NQEEYGGAYT SRNRGYNEAP affiliates. 1101 SVPADYASVY EEKSYTDGRRproperty ENPCEFNRGY intellectual thirdRDYTPLPVGY VTKELEYFPEmay its 1151 TDKVWIEIGE TEGTFIVDSV ELLLMEEas the of and is regulatory owner. Figure IV-1. Deduced amino acid sequencea of the Cry1A.105document protein produced in any such its reproductiondocument MON 89034 owner this of

under of this and/orrights the document fall of rights to of use 001 MAPTVMMASS ATAVAPFQGL KSTASLPVAR RSSRSLGNVSthe NGGRIRCMQV WPAYGNKKFE This may distribution,and 061 IRTLSYLPPL STGGRCMQArights M DNSVLNSGRT ownerTICDAYNVAA HDPFSFQHKS LDTVQKEWTE 121 WKKNNHSLYLsubject DPIVGTVASF LLKKVGSLVG KRILSELRNL IFPSGSTNLM QDILRETEKF 181 LNQRLNTDTLbe ARVNAELTGL QANVEEFNRQthe VDNFLNPNRNviolate AVPLSITSSV NTMQQLFLNR 241 LPQFQMQGYQcopy LLLLPLFAQA ANLHLSFIRDof VILNADEWGI SAATLRTYRD YLKNYTRDYS 301 NYCINTYQSA FKGLNTRLHD MLEFRTYMFLand NVFEYVSIWS LFKYQSLLVS SGANLYASGS may documentpublication, 361 GPQQTQSFTSIt QDWPFLYSLFexploitation QVNSNYVLNG FSGARLSNTF PNIVGLPGST TTHALLAARV 421 NYSGGISSGDthis IGASPFNQNFany NCSTFLPPLL TPFVRSWLDS GSDREGVATV TNWQTESFET 481 TLGLRSGAFT ARGNSNYFPD YFIRNISGVP LVVRNEDLRR PLHYNEIRNI ASPSGTPGGA 521 RAYMVSVHNR KNNIHAVHENpermission GSMIHLAPND YTGFTISPIH ATQVNNQTRT FISEKFGNQG prohibited 681 DSLRFEQNNT TARYTLRGNG NSYNLYLRVS SIGNSTIRVT INGRVYTATN VNTTTNNDGV the 701 NDNGARFSDIcommercial NIGNVVASSN be SDVPLDINVT LNSGTQFDLM NIMLVPTNIS PLY

FigureFurthermore, IV-2.any Deduced amino acid sequence of the chloroplast transit peptide and the Cry2Ab2Consequently, proteinwithout produced in MON 89034 The chloroplast transit peptide (CTP) is underlined. Accumulation of the Cry2Ab2 protein is targeted to the chloroplasts using CTP. The CTP is typically cleaved from the mature protein on uptake into the chloroplasts and is then rapidly degraded (see Section VI.B.1 for more detail).

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IV.B.4. The cry2Ab2 Regulatory Sequences

The cry2Ab2 gene expression cassette that produces the Cry2Ab2 protein consists of the 35S promoter from figwort mosaic virus (P-FMV) (Rogers, 2000), the first intron from the corn heat shock protein 70 gene (I-Hsp 70) (Brown and Santino, 1995). The cassette also contains a cry2Ab2 coding sequence with a modified codon usage (CS-cry2Ab2) (Widner and Whitely, 1989; Donovan, 1991) fused to a chloroplast transit peptide region of corn ribulose 1,5-biphosphate carboxylase small subunit including the first intron (TS- SSU-CTP) (Matsuoka et al., 1987). The 3’ nontranslated region of the nopaline synthase (T-nos) coding region from Agrobacterium tumefaciens T-DNA terminates transcription and and directs polyadenylation (Bevan et al., 1983). and regime. AG IV.B.5. T-DNA Borders

Bayer property protectionpublishing Plasmid vector PV-ZMIR245 contains ofsequences that are necessary forcontents transfer of T- its DNA into the plant cell. These sequences are termedparties. dataas the Right andtherefore Left Border regions. The Right and Left Border regions each contains a borderand/or sequenceor that is a 24- affiliates. 26 bp sequence that defines theproperty extent ofintellectual the thirdDNA that should be transferredmay into the its plant genome. They flank both T-DNAas I and T-DNA II, allowing for independent the of and transfer and integration isof each T-DNA into theregulatory plant genome duringowner. transformation.. a document The Right Borders present inany PV-ZMIR245 such are made of a 24 bp nucleotideits sequence that reproductiondocument was originally derived from plasmiownerd pTiT37 whichthis was isolatedof from A. tumefaciens (Depicker et al., 1982). The Left Borders presentof in PV-ZMIR245 are made of a 25 bp rights under this nucleotide sequenceand/or from the A.the tumefaciens plasmid pTi5955, a derivative of plasmid document to of fall of rights pTiA6 (Barker et al., 1983). use the This may distribution,and rights owner IV.C. T-DNA IIsubject be the violate IV.C.1. The nptIIcopy gene and NPTII proteinof and may documentpublication, It exploitation The nptII genes encodes the neomycin phosphotransferase II enzyme (NPTII) that this any inactivates certain aminoglycoside antibiotics such as kanamycin, neomycin and permission paromomycin. The use of selectableprohibited marker genes, such as nptII, is essential to select transformed cells underthe selective growth conditions. In the presence of paromomycin; commercial be cells transformed with nptII survive, while those that do not contain nptII are eliminated dueFurthermore, to the actionany of the paromomycin. The T-DNA II, and therefore the nptII gene, is segregatedConsequently, out atwithout the F1 generation.

IV.C.2. The nptII regulatory sequences

The nptII gene cassette that produces the NPTII protein consists of the promoter (P-e35S) from the the cauliflower mosaic virus (CaMV) 35S RNA (Odell et al., 1985). The cassette also containts the coding sequence for the NPTII protein (Beck et al., 1982), followed by the 3’ nontranslated region of the nopaline synthase (T-nos) sequence from

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Agrobacterium tumefaciens which terminates the transcription and directs polyadenylation (Bevan et al., 1983).

IV.C.3. T-DNA borders

The Right and Left T-DNA borders are described under Section IV.B.5.

IV.D. Genetic Elements outside the T-DNA Borders

The backbone region outside of the inserted DNA contains two origins of replication which are necessary for replication and maintenance of the plasmid in bacteria, as well as and a bacterial selectable marker gene, aadA, which encodes an aminoglycoside-modifyingand regime. enzyme that provides resistance to the action AGof the antibiotics spectinomycin and streptomycin. Detailed descriptions of all elements in the plasmid backbone region are presented in Table IV-1. Bayer property protectionpublishing of contents IV.E. References its parties.data therefore and/oror Barker, R.F., K.B. Idler, D.V. Thompson,affiliates. J.D. and Kemp. 1983. Nucleotide sequence of property intellectualthird may the T-DNA region from theits Agrobacterium tumefaciens Octopine Ti Plasmid as pTi15955. Plant Mol.the Biol.of 2:335-350.and is regulatory owner. a document Beck, E., G. Ludwig, E.A.any Auerswald, such B. Reiss, and H. Schaller.its 1982. Nucleotide reproductiondocument sequence and exact localizationowner of the neomycinthis phosphotransferaseof gene from under of transposon Tn5. Generights 19:327-336.the this documentand/or of rights Bevan, M., W.M. Barnes,to andof M.fall Chilton. use1983. Structure and transcription of the nopaline synthase gene region of T-DNA. Nucl.the Acids Res. 11:369-385. This may distribution,and rights owner Brown, S.M. andsubject C.G. Santino. 1995. Enhanced expression in plants. United States Patentbe No. 5,424,412. the violate copy of Crickmore, N., D.R. Ziegler, J. Feitelson, andE. Schnepf, J. Van Rie, D. Lereclus, J. Baum, may documentpublication, It and D.H. Dean. 1998.exploitation Revision of the nomenclature for the Bacillus thuringiensis pesticidalthis crystal any proteins. Microb. Mol. Biol. Rev. 62:807-813. permission Depicker, A., S. Stachel, P. Dhaese,prohibited P. Zambryski, and H.M. Goodman. 1982. Nopaline synthase: transcriptthe mapping and DNA sequence. J. Mol. Appl. Genet. 1:561-573. commercial be Donovan, W. P. 1991. CryIIB crystal protein gene from Bacillus thuringiensis. United Furthermore,any Consequently,States Patentwithout No. 5,073,632. Fling, M., J. Kopf, and C. Richards. 1985. Nucleotide sequence of the transposon Tn7 gene encoding an aminoglycoside-modifying enzyme, 3’(9)-O- nucleotidyltransferase. Nucleic Acids Res. 13:7095-7106. Giza, P.E. and R.C. Huang. 1989. A self-inducing runaway-replication plasmid expression system utilizing the Rop protein. Gene 78:73-84.

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Kay, R., A. Chan, M. Daly, and J. McPherson. 1987. Duplication of CaMV 35S promoter sequences created a strong enhancer for plant genes. Science 236:1299-1302. Lamppa G., G. Morelli, and N. Chua. 1985. Structure and developmental regulation of a wheat gene encoding the major chlorophyll a/b-binding polypeptide. Mol. Cell. Biol. 5:1370-1378. Matsuoka, M., Y. Kano-Murakami, Y. Tanaka, Y. Ozeki, and N. Yamamoto. 1987. Nucleotide sequence of the cDNA encoding the small subunit of ribulose-1,5- bisphosphate carboxylase from maize. J. Biochem. 102:673-676. McElroy, D., A.D. Blowers, B. Jenes, and R. Wu. 1991. Construction of expression vectors based on the rice actin 1 (Act1) 5’ region for use in monocotand transformation. Mol. Gen. Genet. 231:150-160. and regime. AG McElwain, E. and S. Spiker. 1989. A wheat cDNA clone which is homologous to the 17 kd heat-shock protein gene family of soybean. Nucl. Acids Res. 17:1764. Bayer property protectionpublishing Odell, J.T., F. Nagy, and N. Chua. 1985.of Identification of DNA sequencescontents required for its activity of the cauliflower mosaic virus 35S promoter.parties.data Nature 313:810-812.therefore and/oror Rogers, S.G. 2000. Promoter for transgenicaffiliates. plants. United States Patent No. 6,018,100. property intellectualthird may its Stalker, D.M., C.M. Thomas, and D.R.as Helinski. 1981. Nucleotide sequence of the region the of and of the origin of isreplica tion of the broadregulatory host range plasmidowner. RK2. Mol. Gen. a document Genetics 181:8-12. any such its reproductiondocument owner this of Sutcliffe, J.G. 1978. Complete nucleotideunder sequenceof of the Escherichia coli plasmid rightsthe this pBR322.document Symposiaand/or on Quantitative Biology 43:77-103.rights to of fall use of Widner, W.R. and H.R. Whitely. 1989. Two highly relatedthe insecticidal crystal proteins of This may distribution,and Bacillus thuringiensisrights subsp. kurstaki ownerpossess different host range specificities. J. Bacteriol.subject 171:965-974. be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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V. Genetic Analysis

Characterization of the DNA insert in MON 89034 was conducted by Southern blot analyses, PCR, and DNA sequencing. The results demonstrated that MON 89034 contains a single functional copy of cry1A.105 and cry2Ab2 expression cassettes. The complete insert includes: (1) the cry1A.105 coding sequence whole transcription is directed by a modified e35S promoter, the 5’ untranslated leader of the wheat chlorophyll a/b- binding protein (Cab), the intron sequence from rice actin gene (Ract1), and the transcriptional termination and polyadenylation sequence derived from the 3’ nontranslated region of the coding sequence for wheat heat shock protein 17.3 (Hsp17); and (2) the cry2Ab2 coding sequence whole transcription is directed by figwort mosaic virus 35S regime.and promoter (FMV), the first intron from the maize heat shock proteinand 70 gene (Hsp70), the AG DNA region containing the targeting sequence for the chlorophyll transit peptide of maize ribulose 1,5-bisphosphate carboxylase small subunit and the first intron (SSU-CTP), and the transcriptional termination and polyadenylationBayer sequence property derived protection frompublishing the 3’ termination sequence of the nopaline synthase (nos) ofcoding sequence from Agrobacteriumcontents tumefaciens . its This T-DNA (T-DNA I) was inserted into the corn genomeparties.data and results in thetherefore synthesis of Cry1A.105 and Cry2Ab2 proteins from the expression of the cry1A.105and/oror and cry2Ab2 genes. affiliates. The chlorophyll transit peptideproperty (CTP) is intellectualpresentthird to direct the Cry2Ab2may protein to corn its plastids. as the of and is regulatory owner. a document The PCR and DNA sequencingany analysissuch of the complete DNA insert itsand adjacent genomic reproductiondocument DNA in MON 89034 confirmed the ownerorganization of thethis genetic elementsof within the insert. PCR primers were designed to amplifyunder seven overlappingof this regions of DNA that span the entire length of the and/orinsert,rights and the the sequencing results of the amplified DNA fragments document to fall of rights confirmed that the sequence ofof the DNA insertuse in MON 89034 matched the designed, the correspondingThis sequences in PV-ZMIR245may distribution,and with one exception. This exception is that the rights owner e35S promoter thatsubject regulates expression of the cry1A.105 gene has been modified and that the Right Border sequence present in PV-ZMIR245 was replaced by a Left Border be the violate sequence in MONcopy 890343. This molecularof rearrangement can be explained by a recombination event which occurred, eitherand prior to or during the process of T-DNA may documentpublication, transferIt to the plant cell, betweenexploitation the DNA sequences near the 35S promoters in T-DNA I and T-DNA II this(Figure any V-1). Due to this recombination event, the reconstituted e35S 89 promoter in MON 89034 permission(referred to as modified e35S or e35S ) no longer has the duplicated enhancer elements comparedprohibited to the original e35S promoter in PV-ZMIR245. the Despite the deletioncommercial of the enhance be elements, the Cry1A.105 protein expression levels in MON 89034 are still sufficiently high under the regulation of the modified e35S promoter toFurthermore, deliver theany required efficacy against target insect pests. Consequently,without

Analysis of the stability of the integrated DNA demonstrated that a unique Southern blot fingerprint of MON 89034 was maintained in seven generations during conventional breeding, thereby confirming the stability of the insert. Additionally, T-DNA II analysis of multiple generations in the MON 89034 breeding history indicated that there were no detectable T-DNA II elements other than those which are common to T-DNA I, i.e., 35S promoter, nos 3´ end sequence, Left Border sequence. Furthermore, these generations

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were shown not to contain any detectable backbone sequence from plasmid PV- ZMIR245.

The following sections describe the molecular analysis experiments and results. Southern blot, PCR and DNA sequencing analyses were used to assess the following: 1) number of insertions of the integrated expression cassettes; 2) number of copies of the integrated expression cassettes; 3) intactness of the expression cassettes; 4) the presence or absence of plasmid backbone sequences; 5) the stability of the inserted DNA during conventional breeding; and 6) organization of the insert in MON 89034. Detailed materials and methods of Southern blot and PCR analyses are provided in Appendix A. Maps of plasmid vector PV-ZMIR245 annotated with the probes used in the Southern analyses are shown in Figures V-2 and V-3. The generations used in the studies are depicted in regime.and Figure-17. The overall results of the molecular analyses are summarizedand in a linear map AG (Figure V-4) which illustrates the organization of the insert, the corn genomic DNA flanking the insert, the restriction sites, and expected sizes of the DNA fragments after restriction digestion. The description of theBayer genetic elemenpropertyts ofprotection the DNApublishing insert in MON 89034 is provided in Table V-1. of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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B-Left Border P-35S CS-nptII T-nos B-Right Border A) and and regime. nptII cassette AG

Bayer property protectionpublishing of contents B) B-Right Border DER P-e35S LI cry1A.105 TP Icry2Ab2 TB-Left Border its parties.data therefore and/oror cry1A.105 cassette cry2Ab2 cassette affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its 89 reproductiondocument C) B-Left Border P-e35S LI cry1A.105owner TPthis Icry2Ab2of TB-Left Border under of rightsthe this documentand/orcry1A.105 cassette cry2Ab2rightscassette to of fall use of the This may distribution,and rights owner Figure V-1. Description of the recombinationsubject process that explains the modified 5’end of the insert the A) Illustration of the T-DNA II of plasmidbe PV-ZMIR245. violate copy of B) Illustration of the T-DNA I of plasmid PV-ZMIR245. and may documentpublication, C) Illustration of the modified T-DNAIt I in MON 89034. exploitation Abbreviations and symbols: DER = duplicatedthis enhancer any region; L = leader sequence; I = intron sequence; P = promoter; T = termination sequence. Detailed description of all the geneticpermission elements are described in Table V-1 and Figure V-4. The diagram illustrates a recombination event, which likely occurrprohibiteded prior to or during the process of T-DNA transfer to the plant cells, between the the DNA sequences near the 35S promoters in T-DNAcommercial I and T-DNA be II. Due to this recombination event, the reconstituted e35S promoter in MON 89034 (referred to as modified e35S or e35S89) no longer has the duplicated enhancer elements (DER) compared to the original e35S promoter in PV-ZMIR245. Despite the deletionFurthermore, of theany enhance elements, the Cry1A.105 protein expression levels in MON 89034 are still sufficiently high under the regulation of the modifiedConsequently, e35S promoterwithout to deliver the required efficacy against target insect pests.

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aadA BstE II 458

OR-ori-PBR322 B-Right Border P-e35S Nde I 16501 L-Cab

CS-rop BstE II 2464 OR-ori V I-Ract1 Ssp I 15077 B-Left Border 16 17 P-35S Nco I 2940 T-DNA II 15 1 2 13 CS-nptII 3 12 PV-ZMIR245 11 17600 bp BstE II 4613 CS-cry1A.105 T-nos 4 and 14 regime. andBstE II 4894 10 5 AG6 9 8 7 B-Right Border

Bayer propertyT-Hsp17 protectionpublishing B-Left Border P-FMV contentsT-DNA I of its parties.data therefore T-nos I-Hsp70 and/oror Ssp I 7921 Nco I 10511 affiliates. TS-SSU-CTP may BstEpropertyII 10267 intellectualthird its CS-cry2Ab2as Nco I 8589 the of and is regulatory owner. a document any such its reproductiondocument Probe DNA Probe Description ownerStart Position (bp)this End Positionof (bp) Total Length (~kb) Number under of rightsthe this 1 documentP-and/ore35S/ L-Cab 1714 of rights2447 0.7 2 I-Ract1to of fall use2427 2941 0.5 3 CS-cry1A.105 probe 1 2942 the 4923 2.0 4 This CS-cry1A.105 probemay 2 distribution,and 4726 6505 1.8 rights owner 5 subjectT-Hsp17 6490 6797 0.3 6 P-FMV 6755 7366 0.6 be the violate 7 copyI- Hsp70 of 7347 8179 0.8 8 TS-SSU-CTP/CS-cry2Ab2 probe 1 8173 9516 1.3 document and 9 mayTS- SSU-CTP/CS-publication,cry2Ab2exploitation probe 2 9296 10509 1.2 10 It T-nos 10525 10778 0.3 this 11 T-DNAany II probe 1 12458 13391 0.9 12 T-DNA II probe 2/CS-permissionnptII probe 13256 14050 0.8 13 T-DNA II probe 3 prohibited 13973 14916 0.9 14 Backbonethe 1be 11287 12489 1.2 15 commercialBackbone 2 14900 16511 1.6 16 Backbone 3 16289 136 1.4 Furthermore,17 any Backbone 4 48 1261 1.2 Consequently,without Figure V-2. Plasmid map of vector PV-ZMIR245 showing probes 1-17 used in Southern blot analyses A circular map of the plasmid vector PV-ZMIR245 used to develop corn MON 89034 is shown. The genetic elements and restriction sites used in Southern analyses (with positions relative to the size of the plasmid vector) are shown on the exterior of the map. The probes used in the Southern analyses are shown on the interior of the map. PV-ZMIR245 contains two T-DNA regions designated as T-DNA I and T-DNA II.

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aadA BstE II 458 B-Right Border

P-e35S OR-ori-PBR322 Nde I 16501 L-Cab CS-rop BstE II 2464 OR-ori V I-Ract1 Ssp I 15077 B-Left Border Nco I 2940 P-35S regime.and 18 and AG CS-nptII PV-ZMIR245 19 Bayer17600 bp BstE II 4613 property protectionpublishingCS-cry1A.10 5 T-nos of contents 20 BstEits II 4894 parties.data therefore and/oror 23 21 affiliates. B-Right Border property intellectualthird22 may its the as T-Hsp17 of and owner. B-Leftis Border regulatory P-FMV a document any such its reproductiondocument of T-nos owner this I-Hsp70 under of NcorightsI 10511the thisSsp I 7921 documentand/or of rights to BstEofII 10267fall use

CS-cry2Ab2 the This may distribution,and owner rights subject TS-SSU-CTP the violate be copy of Nco I 8589 and may documentpublication, It exploitation this Probe DNA anyProbe Description Start Position (bp) End Position (bp) Total Length (~kb) Number permission 18 T-DNA I probe 1 prohibited 1210 2753 1.5 19 T-DNAthe I probe be 2 2649 4676 2.0 20 commercialT-DNA I probe 3 4518 6505 2.0 21 T-DNA I probe 4 6371 8179 1.8 Furthermore,22 any T-DNA I probe 5 8004 9863 1.9 23Consequently, withoutT-DNA I probe 6 9780 11354 1.6

Figure V-3. Plasmid map of vector PV-ZMIR245 showing probes 18 – 23 used in Southern blot analyses A circular map of the plasmid vector PV-ZMIR245 used to develop corn MON 89034 is shown. The genetic elements and restriction sites used in Southern analyses (with positions relative to the size of the plasmid vector) are shown on the exterior of the map. The overlapping T-DNA I probes used in the Southern analyses are shown on the interior of the map.

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3' Genomic Flank DNA 5' Genomic Flank DNA BstE II 5196 Ssp I 8223 Nco I 3242 BstE II 4915 Nco I 8891 BstE II 10569 Nde I 452 Nde I 1531 BstE II 2766 Nco I 10813 Nco I 11920 Ssp I 3 and BstE II 12274 and regime. AG 1 12282

Bayer property publishing protectioncontents cry2Ab2 cry2Ab2 cry1A.105cry1A.105 of SSU-CTPSSU-CTP nosnos Hsp17Hsp17 Cab Cab FMVFMV e35Se35S Hsp70 Hsp70 Ract1Ract1 I-I- TS- TS- CS-CS- T-T- CS-CS- T-T- P-P- B-Left Border Border B-Left B-Left I-I- B-Left Border Border B-Left B-Left P-P- L-L- its parties.data therefore and/oror

affiliates. property intellectualthird may its as the of and regulatory owner. is ~8.2 kb document >4.3 kb Ssp I a its any such reproduction >2.8 kb ~2.1 kb ~0.3 kb documentof ~1.7 kb owner this ~5.4 kb BstE II under of rightsthe this documentand/or >11.4 kb of rights Nde I to of fall use

>3.2 kb ~5.6 kb the ~1.9 kb ~1.1 kb Nco I This may distribution,and rights owner subject the violate be copy of and Figure V-4. Schematic representationmay of thedocument inspublication,ert and genomic flanking sequences in MON 89034 It exploitation The linear DNA derived from T-DNA I ofthis vector any PV-ZMIR245 which was incorporated into MON 89034 is shown. Arrows indicate the end of the insert and the beginning of corn genomic flanking sequence. Identified on the map are genetic elements within the permission insert, as well as restriction sites with positions relative to prohibitedthe size of the linear map for enzymes used in the Southern blot analyses. A portion of Left Border sequence and a modified e35Sthe promoter sequence is present at the 5' insert-to-flank junction in MON 89034. commercial be Furthermore,any Consequently,without

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Table V-1. Summary of genetic elements in MON 89034

Location Genetic in Element Sequence Function (Reference) Sequence flanking the 5′ end of the insert 1-2060 Corn genomic DNA The 239 bp DNA region from the B-Left Border region B1-Left Border 2061-2299 remaining after integration Intervening regime.and Sequence 2300-2349 Sequences used in DNA cloning and AG Modified e35S promoter and 9 bp leader resulting from a P-e35S89 2350-2651 recombination between the e35S and 35S promoters Bayer property protectionpublishing Intervening of contents its Sequence 2652-2677 Sequences used in DNA parties.cloningdata therefore and/oror The 5' untranslated leader of the wheat chlorophyll affiliates. L2-Cab 2678-2738 a/b-bindingproperty proteinintellectual third(Lamppa et al. 1985) may its as Intervening the of and is regulatory owner. Sequence 2739-2754 Sequences used ina DNA cloningdocument any such its I3-Ract1 2755-3234 Intron from the rice actinreproduction gene (McElroydocument et al. 1991) owner this of Intervening under of rightsthe this Sequence document3235-3243and/or Sequences used in DNA cloningrights to of fall use of Coding sequence for the Bacillusthe thuringiensis Cry1A.105 4 This may distribution,and CS -cry1A.105 3244-6777rights protein (Monsantoowner unpublished data) subject Intervening be the violate Sequence 6778-6808copy Sequences ofused in DNA cloning The 3' nontranslatedand region of the coding sequence for may documentpublication, It exploitationwheat heat shock protein 17.3, which ends transcription 5 T -Hsp17 this6809-7018 any and directs polyadenylation (McElwain and Spiker, 1989) Intervening permission Sequence 7019-7085 Sequencesprohibited used in DNA cloning the commercial be Table V-1 continues on next page. Furthermore, any 1 B – borderConsequently, region without 2 L - leader 3 I - intron 4 CS – coding sequence of cry1A.105, including one stop codon which accounts for 3 bp. 5 T – transcript termination sequence

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Table V-1 (continued). Summary of genetic elements in MON 89034

Genetic Location in Element Sequence Function (Reference)

P1-FMV 7086-7649 The figwort mosaic virus 35S promoter (Rogers, 2000) Intervening Sequence 7650-7671 Sequences used in DNA cloning The first intron from the maize heat shock protein 70 I-Hsp70 7672-8475 gene (Brown and Santino, 1995) and Intervening and regime. Sequence 8476-8491 Sequences used in DNAAG cloning DNA region containing the targeting sequence for the transit peptide region of maize ribulose 1,5- Bayer property publishing bisphosphate carboxylase small subunitprotection and thecontents first 2 of TS -SSU-CTP 8492-8892 intron (Matsuoka et al., 1987) its parties.data therefore Coding sequence for a Cry2Ab2 proteinand/oror from Bacillus thuringiensisaffiliates. (Widner and Whitely, 1989; Donovan, property intellectualthird may 1991).its This coding sequence uses a modified codon 3 as CS -cry2Ab2 8893-10800the usage.of and is regulatory owner. a document Intervening any such its reproductiondocument Sequence 10801-10826 Sequencesowner used in DNAthis cloning of The 3' nontranslatedunder ofregion of the nopaline synthase rightsthe this documentand/or (nos) coding sequence offrom Agrobacteriumrights tumefaciens to ofwhich fall terminates use transcription and directs T-nos 10827-11079 polyadenylation (Bevan etthe al. 1983) This may distribution,and rights owner Intervening subject Sequence be 11080-11146 Sequences usedthe inviolate DNA cloning copy of The 230 bp andDNA region from the Left Border region B-Left Bordermay 11147-11377documentpublication, remaining after integration It exploitation Sequence this any flanking the 3′ permission end of the prohibited insert 11378-12282 Corn genomic DNA the be commercial 1 P – promoter 2 Furthermore,any TS – targetingConsequently, sequencewithout 3 CS – Coding sequence for Cry2Ab2, including two stop codons which account for 6 bp.

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V.A. Insert and Copy Number

The insert number (the number of integration sites of T-DNA I in the corn genome) was evaluated by digesting the test and control DNA with Nde I, a restriction enzyme that does not cleave within T-DNA I. This enzyme generates a restriction fragment containing T-DNA I and adjacent, plant genomic DNA. The number of restriction fragments detected indicates the number of inserts present in MON 89034. The number of copies of the T-DNA I integrated at a single locus was determined by digesting test and control genomic DNA samples with Ssp I, which cleaves once within the insert. If MON 89034 contains one copy of T-DNA I, probing with T-DNA I should result in two bands, each representing a portion of the T-DNA I along with adjacent, corn plant regime.and genomic DNA. The blot was examined with overlapping T-DNAand I probes spanning the AG entire inserted DNA sequence (probes 18 – 23 in Figure V-3). The results of this analysis are shown in Figure V-5. It is noted that the long run (overnight runs) enabled greater separation of the higher molecular weBayeright restriction property fragments protection publishingwhile the short run (runs for a few hours) allowed the smallerof molecular weight restriction fragmentscontents to its be retained on the gel. parties.data therefore and/oror affiliates. Genomic DNA isolated from convepropertyntional cornintellectual digestedthird with Nde I (lanesmay 2 and 6) or its Ssp I (lanes 4 and 8) produced several hybridias zation signals. This is not unexpected the of and because several genetic elementsis comprising T-DNAregulatory I were originally derivedowner. from corn. a document These hybridization signals resultany fromsuch the probes hybridizing to endogenousits targets reproductiondocument residing in the corn genome and are not ownerspecifi c to the insertedthis DNA.of These signals were produced in all lanes, including those contaiunderning the conventionalof corn control DNA, and rightsthe this therefore they aredocument considand/orered to be endogenous backgroundrights bands. Plasmid PV- to of fall use of ZMIR245 DNA mixed with conventional corn control DNAthe and digested with Ssp I (lanes 9 andThis 10) produced the expectedmay distribution, bandsand at approximately 10.4 and 7.2 kb in rights owner addition to the endogenoussubject background hybridization produced by the conventional corn control DNA (lane 8). MON 89034 DNA digestedthe violate with Nde I (lanes 1 and 5) produced a be copy single unique band of approximately 13 kbof in addition to the endogenous background and hybridizationmay observed documentin thepublication, conventional corn control DNA (lanes 2 and 6). This result It exploitation confirms that MONthis 89034 contains one insert located on an approximately 13.0 kb Nde I restriction fragment. any permission prohibited The MON 89034 DNA digested with Ssp I (lanes 3 and 7) produced two bands in the be addition to the endogenouscommercial background hybridization observed in the conventional corn controlFurthermore, DNA (lanesany 4 and 8). The approximately 8.2 kb band is the expected size for the border fragmentConsequently, containingwithout the 5' end of the inserted DNA (T-DNA I) along with the adjacent genomic DNA flanking the 5' end of the insert. The approximately 7.4 kb band represents the 3' border fragment containing the 3' end of the inserted DNA along with the adjacent genomic DNA flanking the 3' end of the insert which was expected to be greater than 4.3 kb (refer to Figure V-4).

These results indicate that MON 89034 contains one copy of T-DNA I that resides at a single locus of integration on an approximately 13.0 kb Nde I restriction fragment.

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regime.and 10 and3.1 AG 8.1 2.0 7.1 1.6 6.1 Bayer property protectionpublishing of contents 5.1 its parties.data 1.0 therefore and/oror 4.1 affiliates. property intellectualthird 0.5 may its the as of and owner. 3.1 is regulatory a document any such its reproductiondocument owner this of under of rightsthe this documentand/or of rights 2.0 to of fall use the This may distribution,and rights owner subject Figure V-5. Southern blot analysis of MONthe 89034 violate – insert and copy number be copy analysis of and may documentpublication, The long Itrun enabled greater separationexploitation of the higher molecular weight restriction fragments while the short run allowed the smaller molecular weight restriction fragments to be retained on this any the gel. The gel side arrow symbols denote the sizes of DNA (kb) based on the reference DNA permission size markers on the ethidium bromide prohibitedstained gel. The arrows on the left side of the gel are for the long run lanes, and the arrows on the right side of the gel are for the short run lanes. The blot the 32be was examined with overlappingcommercial P-labeled probes that spanned the T-DNA I sequence (probes 18 – 23 in Figure V-3). Each lane contains approximately 10 µg of digested genomic DNA Furthermore,any isolated Consequently,from grain. withoutLane designations are as follows: Lane 1: MON 89034 (Nde I) 9: Conventional corn spiked with 2: Conventional corn (Nde I) PV-ZMIR245 (Ssp I) [0.5 3: MON 89034 (Ssp I) copy] 4: Conventional corn (Ssp I) 10: Conventional corn spiked 5: MON 89034 (Nde I) with PV-ZMIR245 (Ssp I) [1.0 6: Conventional corn (Nde I) copy] 7: MON 89034 (Ssp I) 8: Conventional corn (Ssp I)

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V.B. Intactness of cry1A.105 and cry2Ab2 Expression Cassettes

The presence of all the elements of the inserted cry1A.105 and cry2Ab2 expression cassettes was assessed by digestion of the test DNA with the restriction enzyme Ssp I, Nco I, or BstE II.

Digestion with Ssp I releases two border fragments with the expected size of approximately 8.2 and greater than 4.3 kb (refer to Figure V-4). The approximately 8.2 kb fragment contains genomic DNA flanking the 5' end of the insert, Left Border sequence, modified e35S promoter sequence, Cab leader, Ract1 intron, cry1A.105 coding sequence, Hsp17 3' end sequence, FMV promoter, and a portion of the Hsp70 intron. The regime.and greater than 4.3 kb fragment contains the remaining portion ofand the Hsp70 intron, SSU- AG CTP targeting sequence, cry2Ab2 coding sequence, nos 3' end sequence, Left Border sequence, and genomic DNA flanking the 3' end of the inserted DNA. Bayer property protectionpublishing Digestion of the test substance with Ncoof I releases two internal restrictioncontents fragments and its two border fragments (refer to Figure V-4). The 5'parties. borderdata fragme nt is expectedtherefore to be greater than 3.2 kb and contains genomic DNA flanking the 5'and/or end ofor the insert, the Left affiliates. Border sequence, modified e35Sproperty promoter intellectual sequence,third the Cab leader,may and the Ract1 its intron. The approximately 5.6 kb internalas fragment contains the cry1A.105 coding the of and sequence, Hsp17 3' endis sequence, FMV promoter,regulatory Hsp70 intron, owner.and the SSU-CTP a document targeting sequence. The approximatelyany such 1.9 kb internal fragment itscontains the cry2Ab2 reproductiondocument coding sequence. The 3' border fragmentowner is expectedthis to be approf oximately 1.1 kb and contains the nos 3' end sequence, a secondunder Left ofBorder sequence, and genomic DNA rightsthe this flanking the 3'document end ofand/or the inserted DNA. rights to of fall use of the DigestionThis of the test substancemay with distribution, BstEand II generates two border fragments and three rights owner internal fragmentssubject (refer to Figure V-4). The 5' border fragment is expected to be greater than 2.8 kb and contains genomic DNA theflanki violateng the 5' end of the insert, Left Border be copy sequence, modified e35S promoter sequence,of and the Cab leader sequence. The 3' border and fragmentmay is expecteddocument topublication, be approximately 1.7 kb and contains a portion of the cry2Ab2 It exploitation coding sequence,this the nos 3' end, Left Border sequence, and genomic DNA flanking the 3' end of the inserted DNA.any Plasmid PV-ZMIR245 DNA was combined with conventional permission corn control DNA and digested prohibitedwith Nco I or BstE II (BstE II was used for the T-nos and T-DNA II Southern blots) and loaded on the gel to serve as a positive hybridization the be control. commercial Furthermore,any IndividualConsequently, Southernwithout blots were examined with the following probes: e35S promoter including the Cab leader, the Ract1 intron, the cry1A.105 coding sequence, the Hsp17 3' end sequence, the FMV promoter, the Hsp70 intron, the SSU-CTP targeting sequence/cry2Ab2 coding sequence, and the nos 3' end sequence (probes 1-10 in Figure V-2).

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V.B.1. e35S Promoter/Cab Leader

Figure V-6 shows the Southern blot examined with the probe of e35S promoter including the Cab leader (probe 1 in Figure V-2). Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) showed no detectable hybridization bands, as expected for the negative control. Conventional corn control DNA spiked with plasmid PV-ZMIR245 DNA digested with Nco I (lanes 9 and 10) produced the expected size band at approximately 10.0 kb.

MON 89034 DNA digested with Ssp I (lanes 1 and 5) produced the expected single and unique band of approximately 8.2 kb. MON 89034 DNA digestedand with regime.Nco I (lanes 3 and 7) produced the single unique band of approximatelyAG 5.4 kb. This is consistent with the expected band being greater than 3.2 kb. No unexpected bands were detected, indicating that MON 89034 contains no additional, detectable e35S promoter and Cab Bayer property protectionpublishing leader elements other than those associatedof with the cry1A.105 cassette. contents its parties.data therefore V.B.2. Ract 1 Intron and/oror affiliates. property intellectualthird may its Figure V-7 shows the Southern blot asexamined with Ract 1 intron probe (probe 2 in the of and Figure V-2). Conventionalis corn control DNA digestedregulatory with Ssp I (lanesowner. 2 and 6) or Nco a document I (lanes 4 and 8) showed noany de tectablesuch hybridization bands, as expected,its for the negative reproductiondocument control. owner this of of rights under this Conventional corn controland/or DNAthe spiked with plasmid PV-ZMIR245 DNA digested with document to of fall of rights Nco I (lanes 9 and 10) produced the expecteduse size band at approximately 10 kb. The the migrationThis of the approximatelymay 10 kbdistribution, fragmentand is slightly higher than indicated by the rights owner size marker band.subject The altered migrations may be due to the difference in salt concentrationsbe between the corn DNA thesample violate and the reference DNA size markers (Sambrook and Russell,copy 2001). of document and may publication,exploitation MONIt 89034 DNA digested with Ssp I (lanes 1 and 5) produced the expected single this unique band of approximatelyany 8.2 kb. MON 89034 DNA digested with Nco I (lanes 3 and 7) produced the single permissionunique band of approximately 5.4 kb. This is consistent with prohibited the expected band being greater than 3.2 kb. No unexpected bands were detected, the indicating that MONcommercial 89034 be contains no additional, detectable Ract1 intron elements other than those associated with the cry1A.105 cassette. Furthermore,any Consequently,without

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40 20 15 40 10 8.1 20 7.1 6.1 15 regime.and and 5.1 AG 4.1 10 3.1 8.1 7.1 Bayer property protectionpublishing of 2.0 contents 6.1 its parties.data 1.6 therefore and/or 5.1 or affiliates. property intellectualthird 1.0 may 4.1 its as the of and is regulatory owner. a document0.5 3.1 any such its reproductiondocument owner this of under of this and/orrights the document to fall of rights 2.0 of use the This may distribution,and rights owner subject Figure V-6. be Southern blot analysis of MONthe 89034violate – e35S promoter/Cab leader copy of The long run enabled greater separation of theand hi gher molecular weight restriction fragments may documentpublication, while Itthe short run allowed theexploitation smaller molecular weight restriction fragments to be retained on the gel. The gelthis side arrow symbols denote the sizes of DNA (kb) based on the reference DNA size markers on the ethidiumany bromide stained gel. The arrows on the left side of the gel are for the long run lanes, and the arrowspermission on the right side of the gel are for the short run lanes. The blot 32 prohibited was examined with a P-labeledthe probe that spanned the e35S promoter and Cab leader region (probe 1 in Figurecommercial V-2). Each be lane contains approximately 10 µg of digested genomic DNA isolated from grain. Lane designations are as follows: LaneFurthermore, 1: MONany 89034 (Ssp I) Consequently,without 2: Conventional corn (Ssp I) 3: MON 89034 (Nco I) 4: Conventional corn (Nco I) 5: MON 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy]

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1 2 3 4 5 6 7 8 9 10

40 20 15

40 10 8.1 7.1 20 6.1 15 5.1 4.1 10 regime.and 3.1and 8.1 AG 7.1 2.0 6.1 1.6 Bayer property protectionpublishing 5.1 of contents 1.0 data its therefore 4.1 parties. and/oror affiliates. 0.5 property intellectualthird may its 3.1 as the of and is regulatory owner. a document any such its reproductiondocument owner this of 2.0 under of rightsthe this documentand/or of rights 1.6 to of fall use the This may distribution,and rights owner Figure V-7. Southernsubject blot analysis of MON 89034 – Ract1 intron be the violate The long run enabledcopy greater separation ofof the higher molecular weight restriction fragments while the short run allowed the smaller molecularand weight restriction fragments to be retained on the gel. may The gel sidedocument arrowpublication, symbols denote the sizes of DNA (kb) based on the reference DNA It exploitation size markers on thisthe ethidium bromide stained gel. The arrows on the left side of the gel are for the long run lanes, andany the arrows on the right side of the gel are for the short run lanes. The blot was examined with a 32P-labeledpermission probe that spanned the Ract1 intron (probe 2 in Figure V-2). prohibited Each lane contains approximatelythe 10 µg of digested genomic DNA isolated from grain. Lane designations are ascommercial follows: be Lane 1: MON 89034 (Ssp I) Furthermore, 2: Conventionalany corn (Ssp I) without Consequently, 3: MON 89034 (Nco I) 4: Conventional corn (Nco I) 5: MON 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy]

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V.B.3. cry1A.105 Coding Sequence

Figure V-8 shows the Southern blot examined with overlapping probes spanning the cry1A.105 coding sequence (probes 3 and 4 in Figure V-2). Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) showed no detectable hybridization bands, as expected for the negative control. Conventional DNA spiked with plasmid PV-ZMIR245 DNA digested with Nco I (lanes 9 and 10) produced the expected size band at approximately 5.6 kb. The migration of the approximately 5.6 kb fragment is slightly higher than indicated by the reference DNA size markers. The altered migrations may be due to the difference in salt concentrations between the corn and DNA sample and the reference DNA size markers (Sambrook andand Russell, regime.2001). AG MON 89034 DNA digested with Ssp I (lanes 1 and 5) produced the expected single unique band of approximately 8.2 kb. MON 89034 DNA digested with Nco I (lanes 3 Bayer property protectionpublishing and 7) produced the expected singleof unique band of approximatelycontents 5.6 kb. No unexpected bands were detected, indicating that MON 89034 containsits no additional, parties.data therefore detectable cry1A.105 elements other than those associated withand/or the cry1A.105or cassette. affiliates. property intellectualthird may V.B.4. Hsp17 3' End Sequence its as the of and is regulatory owner. a document Figure V-9 shows the Southernany blotsuch examined with Hsp17 3' end sequenceits probe (probe reproductiondocument 5 in Figure V-2). Conventional cornowner control DNA digestedthis withof Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) showed no detectable hybridizationof bands, as expected for the rights under this negative control. Conventionaland/or the corn control DNA spiking with plasmid PV-ZMIR245 document to of fall of rights DNA digested with Nco I (lanes 9 and 10)use produced the expected size band at the approximatelyThis 5.6 kb. may distribution,and rights owner subject MON 89034 DNA digested with Ssp I the(lanes violate 1 and 5) produced the expected single be copy unique band of approximately 8.2 kb. of MON 89034 DNA digested with Nco I (lanes 3 and and 7) mayproduced documentthe publication,expect ed single unique band of approximately 5.6 kb. No It exploitation unexpected bandsthis were detected, indicating that MON 89034 contains no additional, detectable Hsp17 3'any end elements other than those associated with the cry1A.105 cassette. permission prohibited the commercial be Furthermore,any Consequently,without

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8.1 regime.and and 7.1 AG 2.0

6.1 1.6

5.1 Bayer property protection1.0publishing of contents 4.1 its parties.data 0.5 therefore and/oror affiliates. property intellectualthird may 3.1 its as the of and is regulatory owner. a document any such its reproductiondocument owner this of 2.0 under of rightsthe this documentand/or of rights 1.6to of fall use the This may distribution,and rights owner Figure V-8. Southernsubject blot analysis of MON 89034 – cry1A.105 coding sequence be the violate The long run enabledcopy greater separation ofof the higher molecular weight restriction fragments while the short run allowed the smaller molecularand weight restriction fragments to be retained on the gel. may The gel sidedocument arrowpublication, symbols denote the sizes of DNA (kb) based on the reference DNA It exploitation size markers on thisthe ethidium bromide stained gel. The arrows on the left side of the gel are for the long run lanes, andany the arrows on the right side of the gel are for the short run lanes. The blot was examined with overlappingpermission 32P-labeled probes that spanned the cry1A.105 coding sequence prohibited (probes 3 and 4 in Figurethe V-2 ). Each lane contains approximately 10 µg of digested genomic DNA isolated fromcommercial grain. Lane be designations are as follows: Lane 1: MON 89034 (Ssp I) Furthermore, 2: Conventionalany corn (Ssp I) without Consequently, 3: MON 89034 (Nco I) 4: Conventional corn (Nco I) 5: MON 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy]

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1 2 3 4 5 6 7 8 9 10

40 20 15 40 10 20 8.1 7.1 15 6.1 5.1 and and regime. 10 4.1 AG 8.1 3.1 7.1 Bayer property 2.0 publishing 6.1 protectioncontents of 1.6 its 5.1 parties.data therefore and/oror 1.0 4.1 affiliates. property intellectualthird may its as 0.5 the of and is regulatory owner. 3.1 a document any such its reproductiondocument owner this of under of rightsthe this documentand/or2.0 rights to of fall use of the This may distribution,and rights owner Figure V-9. Southernsubject blot analysis of MON 89034 – Hsp17 3' end sequence be the violate The long run enabledcopy greater separation ofof the higher molecular weight restriction fragments while the short run allowed the smaller molecularand weight restriction fragments to be retained on the gel. may The gel sidedocument arrowpublication, symbols denote the sizes of DNA (kb) based on the reference DNA It exploitation size markers on thisthe ethidium bromide stained gel. The arrows on the left side of the gel are for the long run lanes, andany the arrows on the right side of the gel are for the short run lanes. The blot was examined with a 32P-labeledpermission probe that spanned the Hsp17 3' end sequence (probe 5 in prohibited Figure V-2). Each lane thecontains approximately 10 µg of digested genomic DNA isolated from grain. Lane designationscommercial are as be follows: Lane 1: MON 89034 (Ssp I) Furthermore, 2: Conventionalany corn (Ssp I) without Consequently, 3: MON 89034 (Nco I) 4: Conventional corn (Nco I) 5: MON 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy]

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V.B.5. FMV Promoter

Figure V-10 shows the Southern blot examined with FMV promoter probe (probe 6 in Figure V-2). Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) showed no detectable hybridization bands, as expected for the negative control.

Conventional corn control DNA spiked with plasmid PV-ZMIR245 DNA digested with Nco I (lanes 9 and 10) produced the expected size band at approximately 5.6 kb. The migration of the approximately 5.6 kb fragment is slightly higher than indicated by the reference DNA size markers. The altered migrations may be due to the difference in salt and concentrations between the corn DNA sample and the referenceand DNAregime. size markers (Sambrook and Russell, 2001). AG

MON 89034 DNA digested with Ssp I (lanes 1 and 5) produced the expected single Bayer property protectionpublishing unique band of approximately 8.2 kb. ofMON 89034 DNA digested withcontents Nco I (lanes 3 and 7) produced the expected single unique band of approximatelyits 5.6 kb. No parties.data therefore unexpected bands were detected, indicating that MON 89034and/or containsor no additional, detectable FMV elements other than thoseaffiliates. associated with the cry2Ab2 cassette. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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1 2 3 4 5 6 7 8 9 10

40 20 15

40 10 8.1 20 7.1 6.1 15 5.1 regime.and and4.1 10 AG

8.1 3.1 7.1 Bayer property protectionpublishing 6.1 of 2.0 contents 1.6 its 5.1 parties.data therefore and/oror affiliates. 4.1 property intellectualthird 1.0 may its as the of and is regulatory 0.5 owner. 3.1 a document any such its reproductiondocument owner this of under of this and/orrights the document2.0 rights to of fall use of the This may distribution,and rights owner Figure V-10. Southernsubject blot analysis of MON 89034 – FMV promoter be the violate The long run enabledcopy greater separation ofof the higher molecular weight restriction fragments while the short run allowed the smaller molecularand weight restriction fragments to be retained on the gel. may The gel sidedocument arrowpublication, symbols denote the sizes of DNA (kb) based on the reference DNA It exploitation size markers on thisthe ethidium bromide stained gel. The arrows on the left side of the gel are for the long run lanes, andany the arrows on the right side of the gel are for the short run lanes. The blot was examined with a 32P-labeledpermission probe that spanned the FMV promoter (probe 6 in Figure V-2). prohibited Each lane contains approximatelythe 10 µg of digested genomic DNA isolated from grain. Lane designations are ascommercial follows: be Lane 1: MON 89034 (Ssp I) Furthermore, 2: Conventionalany corn (Ssp I) without Consequently, 3: MON 89034 (Nco I) 4: Conventional corn (Nco I) 5: MON 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy]

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V.B.6. Hsp70 Intron

Figure V-11 shows the Southern blot examined with Hsp70 intron probe (probe 7 in Figure V-2). Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) produced several hybridization signals. This is not unexpected because the Hsp70 intron was originally derived from corn. These hybridization signals result from the probes hybridizing to endogenous sequences residing in the corn genome and are not specific to the inserted DNA. These signals were produced in both test and control corn lanes, and therefore the bands are considered to be endogenous background.

and Conventional corn control DNA spiked with plasmid PV-ZMIR245and DNAregime. digested with Nco I (lanes 9 and 10) produced the expected AGsize band at approximately 5.6 kb in addition to the endogenous bands. The migration of the approximately 5.6 kb fragment is slightly higher than indicated by the reference DNA size markers. The altered migrations Bayer property protectionpublishing may be due to the difference in salt concentrof ations between the corn DNAcontents sample and the reference DNA size markers (Sambrook and Russell, 2001). its parties.data therefore and/oror MON 89034 DNA digested with Sspaffiliates. I (lanes 1 and 5) produced two expected bands of property intellectualthird may approximately 8.2 and 7.4 kb in additionits to the endogenous bands. The approximately as 8.2 kb band is the expectedthe sizeof for the andborder fragment containing the 5' end of the is regulatory owner. inserted DNA (T-DNA I) along with the adjacea nt genomicdocument DNA flankingits the 5' end of the insert (refer to Figure V-4any). suchThe approximatelyreproduction 7.4 kb band represents the 3' border this documentof fragment containing the 3' end of ownerthe inserted DNA along with the adjacent genomic under of DNA flanking the 3' end ofrights the the insert, which was expectedthis to be >4.3 kb. MON 89034 documentand/or rights DNA digested with Ncoto I (lanesof 3fall and 7) produceduse of the expected single unique band of approximately 5.6 kb in addition to the endogenous bands.the No unexpected bands were This may distribution,and detected, indicating thatrights MON 89034 containsowner no additional, detectable Hsp70 intron elements other thansubject those associated with the cry2Ab2 cassette. be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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1 2 3 4 5 6 7 8 9 10

40 20 15 40 10 20 8.1 15 7.1 6.1 5.1 regime.and and 10 AG 4.1

8.1 3.1 7.1 Bayer property protectionpublishing 2.0 contents 6.1 of 1.6 its parties.data therefore 5.1 and/oror affiliates. 1.0 property intellectualthird may 4.1 its as the of and is regulatory 0.5 owner. a document any such its 3.1 reproductiondocument owner this of under of this and/orrights the document to of fall of rights 2.0 use the This may distribution,and rights owner Figure V-11. Southernsubject blot analysis of MON 89034 – Hsp70 intron be the violate The long run enabledcopy greater separation ofof the higher molecular weight restriction fragments while the short run allowed the smaller molecularand weight restriction fragments to be retained on the gel. may The gel sidedocument arrowpublication, symbols denote the sizes of DNA (kb) based on the reference DNA It exploitation size markers on thisthe ethidium bromide stained gel. The arrows on the left side of the gel are for the long run lanes, andany the arrows on the right side of the gel are for the short run lanes. The blot was examined with a 32P-labeledpermission probe that spanned the Hsp70 intron (probe 7 in Figure V-2). prohibited Each lane contains approximatelythe 10 µg of digested genomic DNA isolated from grain. Lane designations are ascommercial follows: be Lane 1: MON 89034 (Ssp I) Furthermore, 2: Conventionalany corn (Ssp I) without Consequently, 3: MON 89034 (Nco I) 4: Conventional corn (Nco I) 5: MON 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy]

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V.B.7. SSU-CTP Targeting Sequence / cry2Ab2 Coding Sequence

Figure V-12 shows the Southern blot examined with overlapping probes spanning the SSU-CTP targeting sequence / cry2Ab2 coding sequence (probes 8 and 9 in Figure V-2). Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) produced several hybridization signals. This is not unexpected because the SSU-CTP targeting sequence was originally derived from corn. These hybridization signals result from the probe hybridizing to endogenous targets residing in the corn genome and are not specific to the inserted DNA. These signals were produced with both test and control, therefore they are considered to be endogenous background. Endogenous bands were not and detected in the long runs of the Nco I digests (lane 4) because theyand ran off theregime. gel. AG Conventional corn control DNA spiked with plasmid PV-ZMIR245 DNA digested with Nco I (lanes 9 and 10) produced the expected size bands at approximately 1.9 and 5.6 kb Bayer property protectionpublishing in addition to the endogenous bands. of contents its parties.data therefore MON 89034 DNA digested with Ssp I (lanes 1 and 5) producedand/or oneor expected band of approximately 7.4 kb in addition to affiliates.the endogenous bands. The approximately 7.4 kb property intellectualthird may band is consistent with the expectedits band of >4.3 kb (refer to Figure V-4). MON 89034 as DNA digested with Nco theI (lanes of 3 andand 7) produced two bands in addition to the is regulatory owner. endogenous bands that are consistent with thea expected documentsizes of approximatelyits 5.6 and 1.9 kb. The migration of theany approximately such 5.6 reproductionand 1.9 kb fragments is slightly higher this documentof than indicated by the reference DNAowner size markers in the long run (lane 3) but run under of concurrently with the bandsrights producedthe by PV-ZMIR245this in the short run (lane 7, 9, and documentand/or rights 10). The altered migrationsto mayof befall due to theuse difference of in salt concentrations between the corn DNA sample and the reference DNA size themarkers (Sambrook and Russell, This may distribution,and 2001). No unexpectedrights bands were detected,owner indicating that MON 89034 contains no additional, detectablesubject SSU-CTP/cry2Ab2 elements other than those associated with the be the violate cry2Ab2 cassette.copy of and may documentpublication, V.B.8.It nos 3' End Sequence exploitation this any Figure V-13 shows the Southernpermission blot examined with nos 3' end sequence probe (probe prohibited 10 in Figure V-2). Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or the BstE II (lanes 4commercial and 8) showed be no detectable hybridization bands, as expected for the negative control. Conventional corn control DNA spiked with plasmid PV-ZMIR245 Furthermore,any DNAConsequently, digested withoutwith BstE II (lanes 9 and 10) produced the expected size band at approximately 7.8 kb.

MON 89034 DNA digested with Ssp I (lanes 1 and 5) produced a single, unique band of approximately 7.4 kb that is consistent with the expected band of >4.3 kb (refer to Figure V-4). MON 89034 DNA digested with BstE II (lanes 3 and 7) produced the expected single unique band of approximately 1.7 kb. No unexpected bands were detected indicating that MON 89034 contains no additional nos 3' elements other than those associated with the cry2Ab2 cassette.

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LONG RUN SHORT RUN

1 2 3 4 5 6 7 8 9 10

40 20 15 10 40 8.1 7.1 20 6.1 15 5.1 4.1 10 3.1 regime.and 8.1 and AG 7.1 2.0 6.1 1.6 5.1 Bayer property publishing protection1.0 of contents 4.1 its parties.data therefore and/or0.5 or affiliates. property intellectualthird may 3.1 its as the of and is regulatory owner. a document any such its 2.0 reproductiondocument owner this of under of this 1.6 rightsthe documentand/or rights to of fall use of the Figure V-12.This Southern blot analysismay distribution, of andMON 89034 – SSU-CTP targeting rights owner sequence/cry2Ab2subject coding sequence be the violate The long run enabledcopy greater separation ofof the higher molecular weight restriction fragments while the short run allowed the smaller molecularand weight restriction fragments to be retained on the gel. may The gel sidedocument arrowpublication, symbols denote the sizes of DNA (kb) based on the reference DNA It exploitation size markers on thisthe ethidium bromide stained gel. The arrows on the left side of the gel are for the long run lanes, andany the arrows on the right side of the gel are for the short run lanes. The blot was examined with overlappingpermission 32P-labeled probes that spanned the SSU-CTP targeting sequence prohibited and cry2Ab2 coding sequence (probes 8 and 9 in Figure V-2). Each lane contains approximately the 10 µg of digested commercialgenomic DNA be isolated from grain. Lane designations are as follows: Lane 1: MON 89034 (Ssp I) Furthermore, 2: Conventionalany corn (Ssp I) Consequently, 3: MONwithout 89034 ( Nco I) 4: Conventional corn (Nco I) 5: MON 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy]

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LONG RUN SHORT RUN

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40 20 15 40 10 20 8.1 7.1 15 6.1 5.1 regime.and 10 4.1and 8.1 AG 3.1 7.1

6.1 2.0 Bayer property protectionpublishing 1.6 contents 5.1 of its parties.data therefore 4.1 1.0and/oror affiliates. property intellectualthird may its 3.1 as 0.5 the of and is regulatory owner. a document any such its reproductiondocument owner this of 2.0 under of rightsthe this documentand/or1.6 rights to of fall use of the This may distribution,and rights owner Figure V-13. Southernsubject blot analysis of MON 89034 – nos 3' end sequence be the violate The long run enabledcopy greater separation ofof the higher molecular weight restriction fragments while the short run allowed the smaller molecularand weight restriction fragments to be retained on may documentpublication, the gel.It The gel side arrow symbolsexploitation denote the sizes of DNA (kb) based on the reference DNA size markers on thisthe ethidiumany bromide stained gel. The arrows on the left side of the gel are for the long run lanes, and the arrows on the right side of the gel are for the short run lanes. The blot 32 permission was examined with a P-labeled probeprohibited that spanned the nos 3' end sequence (probe 10 in Figure V-2). Each lane containsthe approximately be 10 µg of digested genomic DNA isolated from grain. Lane designationscommercial are as follows: LaneFurthermore, 1: MONany 89034 (Ssp I) Consequently, 2: Conventionalwithout corn (Ssp I) 3: MON 89034 (BstE II) 4: Conventional corn (BstE II) 5: MON 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (BstE II) 8: Conventional corn (BstE II) 9: Conventional corn spiked with PV-ZMIR245 (BstE II) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (BstE II) [1.0 copy]

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V.C. Analysis of the Presence or Absence of the Plasmid Backbone

Figure V-14 shows the Southern blot analysis to determine the presence or absence of plasmid PV-ZMIR245 backbone. Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) examined with overlapping probes spanning the vector backbone of PV-ZMIR245 (detected with probes 14 – 17 of Figure V-2) showed no detectable hybridization bands, as expected for the negative control.

Conventional corn control DNA spiked with plasmid PV-ZMIR245 DNA digested with Nco I (lanes 9 and 10) produced the expected size band of approximately 10 kb.

regime.and MON 89034 DNA digested with Ssp I (lanes 1 and 5) or Nco andI (lanes 3 and 7) produced AG no detectable hybridization bands, indicating that MON 89034 contains no PV-ZMIR245 backbone elements. Bayer property protectionpublishing V.D. Southern Blot Analysis to Confirmof the Absence of T-DNA II contents its parties.data therefore V.D.1. Analysis of the Presence or Absence of the nptII Codingand/or Sequenceor affiliates. property intellectualthird may its as Southern blot analysis wasthe usedof to confirmand that the nptII sequence necessary for is regulatory owner. transformation is no longer present in MON 89034.a Thisdocument is importantits to ensure that only desired proteins are producedany insuch MON 89034. reproduction Figure V-15 shows the Southern blot this documentof analysis used to determine the presenceowner or absence of the nptII coding sequence. under of Conventional corn control rightsDNAthe digested with Ssp I (lanesthis 2 and 6) or Nco I (lanes 4 and documentand/or rights 8) examined with the nptIIto codingof fallsequence probeuse (probeof 12 in Figure V-2) showed no detectable hybridization bands, as expected for the negativethe control. This may distribution,and rights owner Conventional cornsubject control DNA spike with plasmid PV-ZMIR245 DNA digested with be the violate Nco I (lanes 9 andcopy 10) produced the expectedof size band of approximately 10 kb. and may documentpublication, MONIt 89034 DNA digested withexploitation Ssp I (lanes 1 and 5) or Nco I (lanes 3 and 7) produced no detectable hybridizationthis any bands, indicating that MON 89034 contains no nptII coding sequence. permission prohibited the commercial be Furthermore,any Consequently,without

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1 2 3 4 5 6 7 8 9 10

9.4

23 6.6

4.4

and 9.4 regime. and AG 2.3

6.6 Bayer property protectionpublishing of contents its 4.4 parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of 2.3 under of rightsthe this documentand/or rights to of fall use of the Figure V-14.This Southern blot analysismay distribution, of MONand 89034 – PV-ZMIR245 backbone rights owner sequence subject be the violate The long run enabledcopy greater separation ofof the higher molecular weight restriction fragments while the short run allowed the smaller molecularand weight restriction fragments to be retained on the gel. may The gel sidedocument arrowpublication, symbols denote the sizes of DNA (kb) based on the reference DNA It exploitation size markers on thisthe ethidium bromide stained gel. The arrows on the left side of the gel are for the long run lanes, andany the arrows on the right side of the gel are for the short run lanes. The blot was examined with 32P-labeledpermission probes that spanned the PV-ZMIR245 backbone sequence (probes prohibited 14-17 in Figure V-2). theEach lane contains approximately 10 µg of digested genomic DNA isolated from grain.commercial Lane designations be are as follows: Lane 1: MON 89034 (Ssp I) Furthermore, 2: Conventionalany corn (Ssp I) without Consequently, 3: MON 89034 (Nco I) 4: Conventional corn (Nco I) 5: MON 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy]

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LONG RUN SHORT RUN

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40 20 15 40 10 8.1 20 7.1 15 6.1 5.1 regime.and 4.1and 10 AG

8.1 3.1 7.1 Bayer property2.0 protectionpublishing 6.1 of contents 1.6 its 5.1 parties.data therefore and/oror 1.0 4.1 affiliates. property intellectualthird may its the as 0.5 of and owner. 3.1 is regulatory a document any such its reproductiondocument owner this of under of 2.0 rightsthe this documentand/or rights to of fall use of 1.6 the This may distribution,and rights owner Figure V-15. Southernsubject blot analysis of MON 89034 – nptII coding sequence be the violate The long run enabledcopy greater separation ofof the higher molecular weight restriction fragments while the short run allowed the smaller molecularand weight restriction fragments to be retained on the gel. may The gel sidedocument arrowpublication, symbols denote the sizes of DNA (kb) based on the reference DNA It exploitation size markers on thisthe ethidium bromide stained gel. The arrows on the left side of the gel are for the long run lanes, andany the arrows on the right side of the gel are for the short run lanes. The blot was examined with a 32P-labeledpermission probe that spanned the npt II coding sequence (probe 12 in prohibited Figure V-2). Each lane thecontains approximately 10 µg of digested genomic DNA isolated from grain. Lane designationscommercial are as be follows: Lane 1: MON 89034 (Ssp I) Furthermore, 2: Conventionalany corn (Ssp I) without Consequently, 3: MON 89034 (Nco I) 4: Conventional corn (Nco I) 5: MON 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy]

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V.D.2. Analysis of the Presence or Absence of the Overall T-DNA II Region

This analysis further confirms the absence of the nptII coding sequence and demonstrates the absence of any additional T-DNA II sequences except for those elements that are shared with the T-DNA I expression cassettes. The analysis confirmed that the F1 plants that contained T-DNA II elements were segregated out and that the plants selected for further development contained only T-DNA I.

Figure V-16 shows the Southern blot analysis to determine the presence or absence of T- DNA II. Conventional corn control DNA digested with Ssp I (lanes 2 and 6) and BstE II and (lanes 4 and 8) examined with overlapping probe spanning T-DNAand II (probesregime. 11, 12, and 13 in Figure V-2) showed no hybridization bands.AG

Conventional corn control DNA spiked with plasmid PV-ZMIR245 DNA digested with Bayer property protectionpublishing BstE II (lanes 9 and 10) produced the twoof expected size bands at approximatelycontents 7.8 and 2.0 kb. It should be noted that the overlapping probe spanning T-DNAits II contains the parties.data therefore 35S promoter, nos 3' end sequence, and Left Border sequencesand/or whichor are also contained in T DNA I. Therefore, the T-DNA IIaffiliates. probe is expected to hybridize to fragments which property intellectualthird may are common to T-DNA I. its as the of and is regulatory owner. MON 89034 DNA digested with Ssp I (lanesa 1 anddocument 5) producedits two bands of approximately 8.2 and 7.4any kb. such The 8.2 kb bandreproduction is consistent with the 8.2 kb band this documentof detected with the e35S promoter/Cabowner leader region probe (refer to Figure V-6, lanes 1 or under of 5). The 7.4 kb band is consistentrightsthe with the 7.4 kb band detectedthis with nos 3’ end sequence documentand/or rights probe (refer to Figure V-13to , lanesof 1fall or 5). use of the This may distribution,and MON 89034 DNA digestedrights with BstE II owner(lanes 3 and 7) produced two bands of approximately 4.2subject and 1.7 kb. The approximately 4.2 kb band is consistent with the >2.8 be the violate kb expected bandcopy for T-DNA I digestedof with BstE II (refer to Figure V-4), and the approximately 1.7 kb band is consistent andwith the T-DNA I specific band observed in may documentpublication, FigureIt V-13 (lanes 3 and 7).exploitation No unexpected bands were detected, indicating that MON 89034 containsthis no anyadditional, detectable T-DNA II elements other than those that are common to T-DNA I. permission prohibited the commercial be Furthermore,any Consequently,without

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LONG RUN SHORT RUN

1 2 3 4 5 6 7 8 9 10

40 20 40 15 20 10 15 8.1 7.1 6.1 regime.and 10 5.1and AG 4.1 8.1 7.1 3.1 Bayer 6.1 property protectionpublishing of contents 2.0 5.1 its parties.data therefore 1.6and/oror 4.1 affiliates. property intellectualthird may its 1.0 the as of and owner. 3.1 is regulatory a document any such 0.5 its reproductiondocument owner this of under of this 2.0 rightsthe documentand/or rights to of fall use of 1.6 the This may distribution,and rights owner Figure V-16. Southernsubject blot analysis of MON 89034 – T-DNA II be the violate The long run enabledcopy greater separation ofof the higher molecular weight restriction fragments while the short run allowed the smaller molecularand weight restriction fragments to be retained on the gel. may The gel sidedocument arrowpublication, symbols denote the sizes of DNA (kb) based on the reference DNA It exploitation size markers on thisthe ethidium bromide stained gel. The arrows on the left side of the gel are for the long run lanes, andany the arrows on the right side of the gel are for the short run lanes. The blot was examined with overlappingpermission 32P-labeled probes that spanned the T-DNA II sequence (probes prohibited 11-13 in Figure V-2). theEach lane contains approximately 10 µg of digested genomic DNA isolated from grain.commercial Lane designations be are as follows: Lane 1: MON 89034 (Ssp I) Furthermore, 2: Conventionalany corn (Ssp I) without Consequently, 3: MON 89034 (BstE II) 4: Conventional corn (BstE II) 5: MON 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (BstE II) 8: Conventional corn (BstE II) 9: Conventional corn spiked with PV-ZMIR245 (BstE II) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (BstE II) [1.0 copy]

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V.E. Insert Stability across Generations of MON 89034

In order to demonstrate the stability of MON 89034 across generations, additional Southern blot analyses were performed using DNA samples obtained from multiple generations in the MON 89034 breeding history. For reference, the breeding history of MON 89034 is presented in Figure V-17. The specific generations tested are indicated in the legends of Figures V-18, V-19, and V-20. In these analyses, DNA samples were digested with the restriction enzyme Ssp I which cleaves once within the inserted DNA and in both the 5' and 3' genomic flanking sequences of MON 89034. This produces two DNA fragments of approximately 8.2 and >4.3 kb (refer to Figure V-4). The approximately 8.2 kb fragment contains genomic DNA flanking the 5' end of the insert, regime.and Left Border sequence, modified e35S promoter sequence, Caband leader, Ract1 intron, AG cry1A.105 coding sequence, Hsp17 3' end sequence, FMV promoter, and a portion of the Hsp70 intron. The >4.3 kb fragment contains the remaining portion of the Hsp70 intron, SSU-CTP targeting sequence, cry2Ab2 codingBayer sequence,property nos protection 3' endpublishing sequence, Left Border sequence, and genomic DNA flankingof the 3' end of the inserted contentsDNA (refer to its Figure V-4). parties.data therefore and/oror affiliates. Plasmid PV-ZMIR245 DNA wasproperty spiked intellectualinto thethird conventional corn controlmay DNA which its served as a positive hybridization control.as All samples were subjected to the same the of and digestion and probing procedure.is Individual Southernregulatory blots were examinedowner. with three a document probe sets: (1) The stabilityany of thesuch MON 89034 insert across generationsits was confirmed reproductiondocument using overlapping T-DNA I probesowner spanning the entirethis insertedof DNA sequence (probes 18 – 23 in Figure V-3); (2) The absenceunder of the ofnptII selectable marker and unique T- rightsthe this DNA II geneticdocument elementsand/or was confirmed using overlappingrights probe s spanning T-DNA II to of fall use of (probes 11, 12, and 13 in Figure V-2); and (3) The theabsence of plasmid PV-ZMIR245 backboneThis sequence across generationsmay distribution, wasand confirmed using overlapping probes spanning rights owner the vector backbonesubject of PV-ZMIR245 (probes 14 – 17 in Figure V-2). A second conventional corn control (referred to as conventionalthe violate corn A) was used in these Southern be copy blots to ensure that the genetic backgroundsof of all the generations were accurately and represented.may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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R0 x LH172

LH172 BC0 F1 ⊗ LH172 BC0 F2 ⊗ Generations used for segregation analyses LH172 BC0 F 3 and and regime. ⊗ AG LH172 BC0 F4

⊗ Bayer property protectionpublishing of contents LH172 BC0 F5 its parties.data therefore ⊗ and/oror 1 affiliates. TI: BC1:F1 x RP property intellectualthird may LH172its BC0 F as 6 the of and is ⊗ regulatory owner. a document any such its x LH198 reproductiondocument owner this of LH172 BC0 F under 7 of this and/orrights the document [LH172rights BC0 F6 x LH198]F1H to of fall use of x LH198 the This may distribution,and ⊗ rights owner subject the be violate LH172 BC0 F6 x F2H1 copy of [LH172 BC0 F7 x LH198]F1H document and may publication,exploitation It this any R0 = transformed plant; F(#)permission = filial generation; ⊗ = self-pollination; BC(#) = backcross prohibited generation; RP = recurrent parent; H = hybrid; TI = Trait Integration the commercial be Figure V-17. Breeding history of MON 89034 Furthermore,any TheConsequently, LH172 BC0without F6 x F 2H1 generation was used for all molecular analyses. Generations used for molecular stability analysis are shown in bold in the breeding tree. ______1 Recurrent parent (RP) that was used in the analyses is referred to as conventional corn A

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V.E.1. Stability of the T-DNA I Insert across Generations of MON 89034

Figure V-18 shows the Southern blot analysis examined with overlapping T-DNA I probes spanning the entire inserted DNA sequence (probes 18-23 in Figure V-3). Conventional corn control DNA digested with Ssp I (lane 7 and 9) showed several hybridization bands. These bands are difficult to observe in the blot of Figure V-18, but are visible upon longer exposure. This is not unexpected since several genetic elements comprising T-DNA I were originally derived from corn.

Conventional corn control DNA containing plasmid PV-ZMIR245 DNA digested with and Ssp I (lanes 10 and 11) produced the two expected size bands andat approximately regime. 10.4 and 7.2 kb in addition to the expected endogenousAG hybridization (Figure V-18). The migration of the approximately 10.4 kb fragment is slightly higher than indicated by the reference DNA markers in the lanes 10 and 11. The altered migration may be due to the Bayer property protectionpublishing difference in salt concentration betweenof the test DNA samples and the referencecontents DNA markers (Sambrook and Russell, 2001). its parties.data therefore and/oror DNA extracted from seven MON 89034affiliates. generations digested with Ssp I (lanes 1, 2, 3, 4, property intellectualthird may 5, 6, and 8) each produced two bandsits of approximately 8.2 and 7.4 kb in addition to the as expected endogenous hybridizationthe of (Figureand V-18 ). The approximately 8.2 kb band is the is regulatory owner. expected size for the 5' border fragment and thea approximatelydocument 7.4its kb band is consistent with the expected band sizeany of such>4.3 kb for thereproduction 3' border fragme nt. These bands are this documentof consistent with the bands detected inowner Figure V-5 (lanes 3 and 7). under of rightsthe this documentand/or rights Two faint unexpected bandsto ofof approximatelyfall use 15.6 andof 12.0 kb were observed in lanes 3 and 4 of Figure V-18. These bands are likely the resultthe of partial digestion because they This may distribution,and are not seen in prior orrights subs equent generationsowner that were produced by self pollination. The presence ofsubject the 15.6 kb band is consistent with the possibility that internal Ssp I site be the violate was not digested.copy As a result, a combinaof tion of the 7.4 and 8.2 kb bands produced 15.6 kb band. The 12.0 kb band is consistent withand the partial digestion of the Ssp I site in the may documentpublication, 5' flankingIt genomic sequenceexploitation which would result in an approximately 11.8 kb band. this any No additional unexpected bands were detected, indicating that the single copy of T-DNA permission I in MON 89034 is stable acrossprohibited the generations tested. the commercial be Furthermore,any Consequently,without

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1 2 3 4 5 6 7 8 9 10 11

40 20 15

10 8.1 7.1 6.1 5.1 4.1

regime.and 3.1 and AG

2.0 Bayer property protectionpublishing 1.6 of contents its parties.data therefore and/oror 1.0 affiliates. property intellectualthird may its as 0.5 the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or of rights to of fall use the This may distribution,and Figure V-18. Insert stabilityrights of MON 89034owner – T-DNA I subject 32 The blot was beexamined with overlapping P-labeledthe violate probes that spanned the T-DNA I sequence (probes 18 – 23 in copyFigure V-3). The gel sideof arrow symbols denote the sizes of DNA (kb) based on the reference DNA size markers on the ethidiand um bromide stained gel. Each lane contains may documentpublication, approximatelyIt 10 µg of digestedexploitation genomic DNA isolated from grain. Lane designations are as follows: this any Lane 1: MON 89034 (LH172 BC0 F xF , Ssp I) permission6 2H1 2: MON 89034 (LH172 BC0prohibited F3, Ssp I) 3: MON 89034 (LH172the BC0 F , Ssp I) commercial be 4 4: MON 89034 (LH172 BC0 F5, Ssp I) Furthermore, 5: MONany 89034 (LH172 BC0 F6, Ssp I) Consequently, 6: MONwithout 89034 ([LH172 BC0 F7 x LH198]F1H, Ssp I) 7: Conventional corn (Ssp I) 8: MON 89034 ((TI: BC1: F1 x RP, Ssp I) 9: Conventional corn A1 (Ssp I) 10: Conventional corn spiked with PV-ZMIR245 (Ssp I) [1.0 copy] 11: Conventional corn spiked with PV-ZMIR245 (Ssp I) [0.5 copy]

1 Monsanto proprietary conventional corn hybrid

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V.E.2. Confirmation of the Absence of T-DNA II in Multiple Generations of MON 89034

Figure V-19 shows the Southern blot analysis examined with three overlapping probes spanning T-DNA II (probes 11, 12, and 13 in Figure V-2). Conventional corn control DNA digested with Ssp I (lane 7 and 9) showed no detectable hybridization bands. Conventional corn control DNA spiked with plasmid PV-ZMIR245 DNA digested with Ssp I (lanes 10 and 11) produced the two expected size bands at approximately 10 and 7.2 kb. The migration of the approximately 10 kb fragment is slightly higher than indicated by the reference DNA size markers in the lanes 10 and 11. The altered migration may be and due to the difference in salt concentration between the testand DNA samplesregime. and the reference DNA size markers (Sambrook and Russell,AG 2001). The overlapping probes spanning T-DNA II contains the 35S promoter, nos 3' end, and the Left Border which are also contained on T-DNA I. Therefore, the T-DNA II probe is expected to hybridize to Bayer property protectionpublishing fragments which are common to T-DNAof I. contents its parties.data therefore DNA extracted from seven generations of MON 89034 digestedand/or withor Ssp I (lanes 1, 2, 3, 4, 5, 6, and 8) each produced two bandsaffiliates. of approximately 8.2 and 7.4 kb (Figure V-19), property intellectualthird may which are consistent with those observedits with the overlapping T-DNA I probes. The as approximately 8.2 kb bandthe is ofthe expectedand size for the 5' border fragment and the is regulatory owner. approximately 7.4 kb band is consistent with athe expecteddocument band sizeits of >4.3 kb for the 3' border fragment. On longerany exposures, such two faintreproduction unexpected bands of approximately this documentof 15.6 and 12.0 kb were observed in ownerlanes 3 and 4 (not obvious in Figure V-19). These under of bands are consistent with rightsthe bandsthe observed in Figurethis V-18 (lanes 3 and 4) and are documentand/or rights likely the result of partialto digestion. of fall use of the This may distribution,and No additional unexpectedrights bands were detected,owner indicating that different generations of MON 89034 dosubject not contain any additional detectable T-DNA II elements other than be the violate those which are commoncopy to T DNA I. of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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4.1

regime.and 3.1 and AG

2.0 Bayer 1.6 property protectionpublishing of contents its parties.data therefore 1.0 and/oror affiliates. property intellectualthird may its as 0.5 the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or of rights Figure V-19. T-DNA IIto analysis of infall multip le usegenerations of MON 89034 – T-DNA II the The blot wasThis examined with overlappingmay distribution, 32P-labeledand probes that spanned the T-DNA II sequence rights owner (probes 11-13 in Figuresubject V-2 ). The gel side arrow symbols denote the sizes of DNA (kb) based on the referencebe DNA size markers on the ethidiumthe violatebromide stained gel. Each lane contains approximately 10 µcopyg of digested genomic DNAof isolated from grain. Lane designations are as follows: document and may publication,exploitation Lane It 1: MON 89034 (LH172BC0F6xF2H1, Ssp I) this 2: MON 89034any (LH172 BC0 F3, Ssp I) 3: MON 89034 (LH172 BC0 F , Ssp I) permission4 4: MON 89034 (LH172 BC0prohibited F5, Ssp I) 5: MON 89034 (LH172the BC0 F , Ssp I) commercial be 6 6: MON 89034 ([LH172 BC0 F7 x LH198]F1H, Ssp I) Furthermore, 7: Conventionalany corn (Ssp I) Consequently, 8: MONwithout 89034 ((TI: BC1: F1 x RP, Ssp I) 9: Conventional corn A1 (Ssp I) 10: Conventional corn spiked with PV-ZMIR245 (Ssp I) [1.0 copy] 11: Conventional corn spiked with PV-ZMIR245 (Ssp I) [0.5 copy]

1 Monsanto proprietary conventional corn hybrid

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V.E.3. Confirmation of the Absence of Plasmid PV-ZMIR245 Backbone Sequence in Multiple Generations of MON 89034

Figure V-20 shows the Southern blot analysis examined with four overlapping probes spanning the PV-ZMIR245 backbone sequence (probes 14-17 in Figure V-2). Conventional corn control DNA obtained from LH172, digested with Ssp I (lane 7 and 9) showed no detectable hybridization bands. Although difficult to observe in Figure V-20, overexposures of Southern blots showed that conventional corn A control DNA, digested with Ssp I (lane 7 and 9), produced two faint hybridization bands at 6.0 and 3.5 kb. These are likely the result of endogenous hybridization to corn genetic elements specific to this background. Conventional control DNA spiked with plasmid PV-ZMIR245and and and regime. digested with Ssp I (lanes 10 and 11) producedAG the two expected size bands at approximately 10.4 and 7.2 kb.

DNA extracted from seven generations of BayerMON 89034property digested protection with publishingSsp I (lanes 1, 2, 3, of contents 4, 5, 6, and 8) produced no hybridization bands. This indicates that MON 89034 does not its parties.data therefore contain any PV-ZMIR245 backbone elements across the differentand/or generationsor tested.

affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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1 2 3 4 5 6 7 8 9 10 11

9.4

6.6

4.4

regime.and and AG 2.3 2.0 Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its 0.5 as the of and is regulatory owner. a document any such its reproductiondocument owner this of of rights under this and/or the document to fall of rights Figure V-20. Backbone analysisof in multiple usegenerations of MON 89034 – PV- the ZMIR245This backbone sequencemay distribution,and rights owner The blot was examinedsubject with overlapping 32P-labeled probes that spanned the PV-ZMIR245 backbone sequence (probes 14-17 in Figure theV-2 ). violate The gel side arrow symbols denote the sizes be copy of DNA (kb) based on the reference DNAof size markers on the ethidium bromide stained gel. Each lane contains approximatelydocument 10 µg of digestedand genomic DNA isolated from grain. Lane may publication,exploitation designationsIt are as follows: this Lane 1: MON 89034any (LH172BC0F 6xF2H1, Ssp I) 2: MON 89034 (LH172permission BC0 F3, Ssp I) 3: MON 89034 (LH172 BC0prohibited F4, Ssp I) the 4: MON commercial89034 (LH172 be BC0 F5, Ssp I) 5: MON 89034 (LH172 BC0 F6, Ssp I) Furthermore, 6: MONany 89034 ([LH172 BC0 F7 x LH198]F1H, Ssp I) Consequently, 7: Conventionalwithout corn (Ssp I) 8: MON 89034 ((TI: BC1: F1 x RP, Ssp I) 9: Conventional corn A1 (Ssp I) 10: Conventional corn spiked with PV-ZMIR245 (Ssp I) [1.0 copy] 11: Conventional corn spiked with PV-ZMIR245 (Ssp I) [0.5 copy]

1 Monsanto proprietary conventional corn hybrid

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V.F. Organization and Sequence of the Insert and Adjacent Genomic DNA in MON 89034

The organization of the elements within the insert in MON 89034 was confirmed by DNA sequencing analyses. PCR primers were designed to amplify seven overlapping regions of DNA that span the entire length of the insert (9317 bp), and the amplified DNA fragments were subjected to DNA sequencing analyses (for more details refer to Appendix A). The results confirmed that the sequence of the DNA insert in MON 89034 matched the designed, corresponding sequences in PV-ZMIR245 with one exception. This exception is that the e35S promoter that regulates expression of the cry1A.105 gene has been modified and that the Right Border sequence present in PV-ZMIR245and was replaced by a Left Border sequence in MON 89034. This molecularand rearrangementregime. can be explained by a recombination event which AGoccurred, either prior to or during the process of T-DNA transfer to the plant cell, between the DNA sequences near the 35S promoters in T-DNA I and T-DNA II (FigureBayer V-1 ). Dueproperty to this protection recombinationpublishing event, the reconstituted e35S promoter in MON 89034of (referred to as modified e35Scontents or e35S 89) no its longer has the duplicated enhancer elements comparedparties. to datathe original e35Stherefore promoter in PV-ZMIR245. Despite the deletion of the enhance elements,and/or theor Cry1A.105 protein affiliates. expression levels in MON 89034property are stillintellectual suffithirdciently high under themay regulation of the its modified e35S promoter to deliver the requiredas efficacy against target insect pests. the of and is regulatory owner. a document V.G. Inheritance of the Lepidoany pteransuch Protection Trait in MON 89034its reproductiondocument owner this of Significance of the segregation pattern (positives/negatives)of was assessed by Chi square rights under this analysis over four generationsand/or ofthe MON 89034 to determine the heritability and stability document to of fall of rights of the trait (cry1A.105 and the cry2Ab2 genes,use and Cry1A.105 and Cry2Ab2 proteins) the (Table V-2This). The confirmationmay of thedistribution, presenceand of the gene and stability of the trait was based on one of severalrights assays: 1) ELISA to detectowner Cry2Ab2 protein; 2) ELISA to detect subject ® Cry1A.105 protein;be 3) GeneCheck immunoassaysthe violate to detect Cry2Ab2 protein (Cry2A QuickStix Lateralcopy Flow test strips, Envirologixof Inc., Portland, MN); and 4) PCR assay to and detect themay presence documentof thepublication, cry genes. It exploitation this As described earlier,any MON 89034 was developed using a 2T-DNA vector transformation system that employed twopermission separate T- DNAs. One T-DNA (T-DNA I) contained the cry1A.105 and cry2Ab2 genes prohibitedwhile the other T-DNA (T-DNA II) contained the nptII the be gene. F1 plants werecommercial generated in a LH172 germplasm by making crosses of the R0 plant with the LH172 inbred. From the population of produced F plants, selections were made Furthermore,any 1 for Consequently,the absencewithout of nptII gene, and the plants were screened for copy number of the cry1A.105 and cry2Ab2 inserted cassettes using Southern blot analysis. Plants selected in the F1 generation were either used to make BC1F1 seed, or were self-pollinated to generate F2 seed (Figure V-17). The overall goal in the F1 population of plants was to select single copy, marker–free plants. A final plant was selected from the F1 generation, designated as event MON 89034, and progeny derived from this plant showed the expected patterns for genetic segregation (Table V-2).

® GeneCheck is a trademark of Envirologix Inc.

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The Chi-square analysis is based on testing the observed segregation ratio of the Cry proteins to the ratio that is expected according to Mendelian principles as shown below:

Generation Expected Comment Ratioa LH172 BC0F1 n.a. screened for copy number and absence of nptII (segregation data not shown) LH172 BC0F2 3:1 positive:negative (product of self pollination) LH172 BC0F3 1:0 positive:negative (homozygous plant selection) LH172 BC0F4 1:0 positive:negative (homozygous plant selection) b LH172 BC1F1 1:1 positive:negative (product of backcrossing) c LH172 BC1F2 3:1 positive:negative (product of self pollination)regime. and LH172 BC1F c 3:1 positive:negative (product of andself pollination) 2 AG a n.a. = not applicable. b To confirm segregation, LH172 BC0F1 plants were backcrossed to the recurrent parent (LH172) to produce this generation (not shown on the breedingBayer tree,property Figure V-17 ). publishing c protection To confirm segregation, The LH172 BC1Fof1 plants were selfed to produce two differentcontents plant populations of this generation (not shown on the breeding tree, Figure V-17). its parties.data therefore and/oror affiliates. The Chi-square test was computedproperty as: intellectualthird may its as the χof2 = Σ [ (| o and– e | – 0.5)2 / e] is regulatory owner. a document any such its reproductiondocument where o = observed frequency of theowner genotype , e = expectedthis frequencyof of the genotype, and 0.5 = Yates correction factor for analysis with one degree of freedom (Little and under of this Hills, 1978). and/orrights the document fall of rights to of use the 2 Results ofThis the Chi-square test aremay summarized distribution,and in Table V-2. All χ values indicated no rights owner significant differencessubject between observed and expected genetic ratios across all tested generations of MON 89034. These results are consistent with molecular characterization be the violate data indicating a singlecopy insertion site forof the cry1A.105 and cry2Ab2 expression cassettes. and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table V-2. Segregation analyses of MON 89034

Number of Observed Observed Expected Expected Chi- Generation Probability plants Positives a Negatives Positives a Negatives Square

LH172 BC0F2 11 7 4 8.25 2.75 0.2727 >0.05

LH172 BC0F3 24 24 0 24 0 Fixed + n.a.

LH172 BC0F4 30 30 0 30 0 Fixed + n.a.

LH172 BC1F1 28 13 15 14 14 0.0357 >0.05

LH172 BC1F2 24 20 4 18 6 0.5 >0.05 LH172 BC1F 24 17 7 18 6 0.0556and >0.05 2 and regime. a The confirmation of the trait was based on one of severalAG assays: 1) ELISA to detect the Cry2Ab2 protein; 2) ELISA to detect the Cry1A.105 protein; 3) PCR assay to detect presence of the cry genes; and, 4) GeneCheck immunoassays to detect the Cry2Ab2 protein. Bayer property protectionpublishing of contents its parties.data therefore V.H. Conclusions for the Genetic Characterization of MONand/or 89034or affiliates. property intellectualthird may MON 89034 was produced by Agrobacterium-its mediated transformation of corn with the PV-ZMIR245, which is thea binary vectoras containing 2T-DNAs. The first T-DNA, of and owner. designated as T-DNA I,is contains the cry1A.105regulatory and the cry2Ab2 expression cassettes. a documentits The second T-DNA, designatedany such as T-DNA II, contains the nptII expression cassette. reproductiondocument of During transformation, both T-DNAsowner were insertedthis into the genome. Traditional under of breeding was used to isolaterights the plants that only containthis the cry1A.105 and cry2Ab2 documentand/or rights expression cassettes (T-DNAto ofI) andfall do not containuse theof nptII expression cassette (T-DNA II), thereby, producing marker-free corn MON 89034. the This may distribution,and rights owner Molecular characterizationsubject of MON 89034 by Southern blot analyses demonstrated that the the DNA insertedbe into the corn genome is prviolateesent at a single locus and contains one copy of functional copy of the cry1A.105 and cry2Ab2and expression cassettes. All expression may documentpublication, elementsIt are shown to be presexploitationent in the inserted DNA as expected with the exception of a modified e35S thispromoter any regulating the cry1A.105 gene, and there are no other elements, either full length or partial, present other other than those associated with the intended permission insert. No backbone plasmid DNAprohibited or nptII sequences were detected. the commercial be PCR and DNA sequence analyses provided the complete DNA sequence of the insert and confirmedFurthermore, theany organization of the elements within the insert. The sequence of the DNA insertConsequently, in MON 89034without matched the designed, corresponding sequences of T-DNA I in PV- ZMIR245 with one exception in that the e35S promoter that regulates expression of the cry1A.105 gene has been modified and that the Right Border sequence present in PV- ZMIR245 was replaced by a Left Border sequence in MON 89034. This molecular rearrangement can be explained by a recombination event which occurred, either prior to or during the process of T-DNA transfer to the plant cell, between the DNA sequences near the 35S promoters in T-DNA I and T-DNA II. Due to this recombination event, the reconstituted e35S promoter in MON 89034 (referred to as modified e35S or e35S89) no

Monsanto Company 06-CR-166U Page 96 of 262 CBI Deleted Version longer has the duplicated enhancer elements compared to the originally-designed e35S promoter in PV-ZMIR245. Despite this modification, the Cry1A.105 protein expression levels in MON 89034 are still sufficiently high under the regulation of the modified e35S promoter to deliver the required efficacy against target insect pests.

The stability of the integrated DNA was demonstrated by the fact that the Southern blot fingerprint of MON 89034 was maintained in the tested generations of the breeding history. Additionally, T-DNA II analysis of multiple generations from the MON 89034 breeding history indicated that there were no T-DNA II elements present other than those (i.e., 35S promoter, nos 3´ end sequence, Left Border sequence) which are common to T- DNA I. Furthermore, these generations have been shown not to contain any backbone sequence from plasmid PV-ZMIR245. This stability was further confirmed by the fact regime.and that the inheritance of the lepidopteran protection trait in MONand 89034 follows Mendelian AG segregation principle.

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V.I. References

Bevan, M., W.M. Barnes, and M. Chilton. 1983. Structure and transcription of the nopaline synthase gene region of T-DNA. Nucl. Acids Res. 11:369-385. Brown, S.M. and C.G. Santino. 1995. Enhanced expression in plants. United States Patent No. 5,424,412. Donovan, W.P. 1991. CryIIB crystal protein gene from Bacillus thuringiensis. United States Patent No. 5,073,632. Lamppa G., G. Morelli, and N. Chua. 1985. Structure and developmental regulation of a wheat gene encoding the major chlorophyll a/b-binding polypeptide. Mol.and Cell. and regime. Biol. 5:1370-1378. AG Little, T.M. and F.J. Hills. 1978. Agricultural Experimentation: Design and Analysis. John Wiley and Sons, New York. Bayer property protectionpublishing Matsuoka, M., Y. Kano-Murakami, Y.of Tanaka, Y. Ozeki, and N. Yamamoto.contents 1987. its Nucleotide sequence of the cDNA encodingparties. the datasmall subunit oftherefore ribulose-1,5- bisphosphate carboxylase from maize. J. Biochem. 102:673-676.and/oror affiliates. McElroy, D., A.D. Blowers, propertyB. Jenes, andintellectual R.third Wu. 1991. Constructionmay of expression its vectors based on the rice actinas 1 (Act1) 5’ region for use in monocot the of and transformation. Mol.is Gen. Genet. 231:150-160.regulatory owner. a document any such its McElwain, E. and S. Spiker. 1989. A wheat cDNAreproduction clone whichdocument is homologous to the 17 owner this of kd heat-shock protein gene familyunder of soybean. of Nucl. Acids Res. 17:1764. rightsthe this Rogers, S.G. 2000.document Promoterand/or for transgenic plants. United Statesrights Patent No. 6,018,100. to of fall use of Sambrook, J., and D. Russell. 2001. “Molecular Cloning”the third edition, Cold Spring This may distribution,and Harbor Laboratoryrights Press. Chapter 5, Protocolowner 1: Agarose gel electrophoresis. subject Widner, W.R. and H.R. Whitely. 1989. Two highly related insecticidal crystal proteins of be the violate Bacillus thuringiensiscopy subsp. kurstakiof possess different host range specificities. J. Bacteriol. 171:965-974. and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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VI. Characterization of the Introduced Cry1A.105 and Cry2Ab2 Proteins

This section summarizes the evaluation of the Cry1A.105 and Cry2Ab2 proteins produced in MON 89034 including: (a) equivalence of the in planta-produced proteins to the recombinant E. coli produced proteins used in protein safety studies; (b) the expression levels of the proteins determined in corn tissues; and (c) a summary of the food and feed safety assessment of the Cry1A.105 and Cry2Ab2 proteins.

VI.A. The Cry1A.105 Protein

VI.A.1. Identity and Function of the Cry1A.105 Protein and and regime. AG Cry1A.105 is a full-length, insecticidal protein consisting of 1177 amino acids with a molecular weight (MW) of 133 kDa. It is a chimeric protein that consists of domains I 1 Bayer property protectionpublishing and II from Cry1Ab or Cry1Ac , domainof III from Cry1F, and the C-terminalcontents domain from Cry1Ac (Figure VI-1). Cry1Ac, Cry1Ab and Cry1F are all wellits known and well parties.data therefore characterized insecticidal proteins derived from the soil bacteriumand/or Bacillusor thuringiensis (Bt). Cry1A.105 was designed usingaffiliates. domain exch ange strategy to achieve high levels of property intellectualthird may activity against target lepidopteranits insect pests. The domains I and II of Cry1A.105 are as 100% identical to the respectivethe of domainsand of Cry1Ab or Cry1Ac. The domain III of is regulatory owner. Cry1A.105 is 99% identical to the domain aIII of Cry1F.document The its C-terminal region of Cry1A.105 is 100% identicalany tosuch that of Cry1Ac.reproduction The overall amino acid sequence this documentof identity of Cry1A.105 to Cry1Ac, ownerCry1Ab, and Cry1F is 93.6%, 90.0%, and 76.7 %, under of respectively (Table VI-1).rights Accordingthe to an accepted phylogramthis (Crickmore et al., 1998) documentand/or rights for Bt crystal proteins, toCry1A.105 of fallcan be clustereduse ofwith Cry1Ac due to high degree of homology ( , 2004; Figure VI-2). the This may distribution,and rights owner Domain exchangesubject is a well known occurring mechanism in nature, resulting in Bt protein be the violate diversities, whichcopy has been described extensivof ely in the literature (De Maagd et al.; 2001; 2003; Masson et al., 2002). Domain exchangeand strategy with modern molecular biological may documentpublication, tools Ithas been used to switchexploitation the functional domains of Cry1 proteins to develop microbial biopesticidesthis any with improved specificity to lepidopteran insect pests. Microbial pesticides that contain Cry1Ac/Cry1F chimeric protein have been used for control of permission lepidopteran pests since 1997 (Baum,prohibited 1998; 1999), and a biotechnology-derived cotton the expressing a chimericcommercial protein be consisting of domains or sequences from Cry1F, Cry1C, and Cry1Ab has been commercialized (Gao et al., 2006). Furthermore,any TheConsequently, general mechanismwithout of insecticidal activity of Cry proteins is well understood (Gill et al., 1992; Schnepf et al., 1998; Zhuang and Gill, 2003). Cry proteins are comprised of several functional domains that have highly conserved regions among the classes. For example, the amino acid sequence of Cry1A proteins is highly conserved in domains I, II and III. These functional domains have been shown to determine the activity and specificity of the Cry proteins. Domain I is involved in membrane insertion and pore

1 Cry1Ab and Cry1Ac shares 100% amino acid sequence identity in domains I and II.

Monsanto Company 06-CR-166U Page 99 of 262 CBI Deleted Version formation. Domain II is involved in specific receptor recognition and binding. Domain III has been suggested to maintain the structural integrity of the protein molecule (Li et al., 1991) and also to contribute to specificity (De Maagd et al., 2000; 2001). The C- terminal domain is implicated in crystal formation which does not directly contribute to the insecticidal activity (De Maagd et al., 2001). C-terminal domain is cleaved upon entry into the insect midgut or by certain proteases in vitro. Domains I, II, and III are retained during the formation of active toxins (from the protoxins) in insect guts. Only those insects with specific receptors are affected and no toxicity is observed in species that lack these receptors (Crickmore et al., 1998; De Maagd et al. 2001).

As with other Cry1A proteins, Cry1A.105 is active against major lepidopteran insect pests. The spectrum of activity includes corn borers from the genera Ostrinia and regime.and Diatraea (such as European corn borer, Asian corn borer, sugarcaneand borer, southwestern AG corn borer), armyworms (Spodoptera spp. including fall armyworm), earworms (Helicoverpa spp. including corn earworm), and cutworms (e.g., Agrotis ipsilon, black cutworm). Bayer property protectionpublishing of contents its VI.A.2. Characterization of Cry1A.105 Protein Producedparties.data in MON 89034therefore and/oror affiliates. property intellectualthird may The expression levels of Cry1A.105its protein in different tissue of MON 89034 are as relatively low. Therefore,the it was of necessaryand to produce the protein in a high-expressing, is regulatory owner. recombinant microorganism in order to obtaia n sufficientdocument quantitiesits of the protein for safety studies. A recombinantany Cry1A.105such proteinreproduction was produced in Escherichia coli, the this documentof sequence of which was engineered toowner match that of Cry1A.105 protein produced in MON under of 89034. The equivalence ofrights thethe physicochemi cal characteristicsthis and functional activity documentand/or rights between the MON 89034-producedto of fall and E.use coli -producedof Cry1A.105 protein was confirmed by a panel of analytical techniques, includingthe sodium dodecyl sulfate This may distribution,and polyacrylamide gel electrophoresisrights (SDS-PAGE),owner Western blot analysis, matrix assisted laser desorption/ionizationsubject time-of-flight mass spectrometry (MALDI-TOF MS), be the violate glycosylation analysis,copy and assay of biologicalof activity. The details of the materials, methods, and results are described in Appendixand B, while the conclusions are summarized may documentpublication, as follows.It exploitation this any The Cry1A.105 protein isolated from MON 89034 was purified and characterized, and permission results confirmed the equivaleprohibitednce of MON 89034-produced and E. coli-produced the Cry1A.105 protein.commercial SDS-PAGE be demonstrated that the MON 89034-produced Cry1A.105 co-migrated to the same position on the gel as the E. coli-produced Cry1A.105Furthermore, anyprotein, indicating the protein from both sources was equivalent in molecular weight.Consequently, On thewithout basis of Western blot analysis with a polyclonal antibody against Cry1A.105, the electrophoretic mobility and immunoreactivity of the MON 89034- produced Cry1A.105 protein were shown to be equivalent to those of the E. coli- produced Cry1A.105 protein. The intactness of the N-terminus for the MON 89034- produced Cry1A.105 protein was confirmed with an antibody which is specific to the N- terminal peptide. Tryptic peptide mapping by MALDI-TOF MS yielded peptide masses consistent with the expected tryptic peptides generated in silico based on the predicted trypsin cleavage sites in the Cry1A.105 sequence. In addition, the MON 89034- and the

Monsanto Company 06-CR-166U Page 100 of 262 CBI Deleted Version

E. coli-produced Cry1A.105 protein was found to be equivalent based on functional activities and the lack of glycosylation. Taken together, these data provide a detailed characterization of the Cry1A.105 protein isolated from MON 89034 and were used in establishing its equivalence to the E. coli-produced Cry1A.105 protein.

Domain I Domain II Domain III C-Terminal Cry1Ab

Cry1Ac

regime.and and AG Cry1F

Bayer property protectionpublishing of contents its parties.data therefore and/oror Cry1Ab or Cry1Ac Cry1F Cry1Ac affiliates. property intellectualthird may 100% 100%its 99% 100% Cry1A.105 as the of and is regulatory owner. a document any such its reproductiondocument Figure VI-1. Schematic representationowner of the originthis of Cry1A.105of protein domains under of Different color and patterns rightsare usedthe to differentiate the originthis of domains. For simplicity, the documentand/or rights lengths of domains in this toillustration of arefall not in proportionuse of to the lengths of amino acid sequence of the respective domains. the This may distribution,and rights owner subject the violate be copy Table VI-1. Amino acid sequence identityof between the Cry1A.105 and Cry1Ac, document and Cry1Ab,may and Cry1F proteinspublication,exploitation It this any Amino acid identity to Cry1A.105 (%) Domain permission Cry1Acprohibited Cry1Ab Cry1F the be commercialI 100 100 57 Furthermore,any II 100 100 37 Consequently,withoutIII 57 46 99 C-terminal 100 92 93 Overall 93.6 90 76.7

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regime.and and } AG

Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. Figure VI-2. Phylogram showingproperty Cry1A intellectual.105third and Cry1Ac proteins mayin the same its cluster based on amino acid sequenceas identity the of and is regulatory owner. a document any such its reproductiondocument VI.B. The Cry2Ab2 Protein owner this of under of rightsthe this VI.B.1. Identitydocument andand/or Function of the Cry2Ab2 Protein rights to of fall use of the This may distribution,and The Cry2Ab2 is a proteinrights derived from Bacillusowner thuringiensis subsp. kurstaki (Btk). Btk has been used assubject an active ingredient in many commercial microbial pesticide products ® ® the violate such as DiPelbe andcopy Cutlass (EPA, 2005). Cry2Ab protein has been listed as one of the ® of ® proteins in Cutlass , and Cry2Aa as one andof the proteins in DiPel (Betz et al., 2000). may documentpublication, Cry2AbIt and Cry2Aa share 88%exploitation amino acid sequence identity (Dankocsik et al., 1990; Widner and Whiteley,this any 1989).

permission Like the Cry2Ab2 protein producedprohibited in the biotechnology-derived cotton Bollgard® II, the the Cry2Ab2 proteincommercial produced in be M ON 89034 is a slight variant of the wild-type Cry2Ab2 protein. The Cry2Ab2 produced in MON 89034 is identical to the Cry2Ab2 produced in BollgardFurthermore, II anycotton, and they differ from the wild-type Cry2Ab2 of Bt by only one amino acidConsequently, (Figure VI-3without ). Accumulation of the Cry2Ab2 protein in corn MON 89034 is targeted to the chloroplasts using a chloroplast transit peptide (CTP). CTPs facilitate the intracellular transport of proteins from the cytoplasm and the plastids (Bruce, 2000). CTPs are typically cleaved from the mature protein on uptake into the chloroplasts, and then rapidly degraded. To allow targeting of the Cry2Ab2 protein to the plastids in MON

® DiPel is a registered trademark of Abbott Laboratories. Cutlass is a registered trademark of Ecogen, Inc. Bollgard is a registered trademark of Monsanto Company.

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89034, the DNA sequence encoding the CTP region of corn ribulose 1,5-biosphosphate carboxylase small subunit was fused to the cry2Ab2 coding sequence.

The Cry2Ab2 protein expressed in cotton Bollgard® II is also targeted to plastids through a CTP. The attempts to determine the N-terminal sequence of the Cry2Ab2 in Bollgard® II (after the CTP was cleaved) indicated that the N-terminus was blocked, therefore, the exact excision site of the CTP is not known. Attempts to determine the N-terminal sequence of the full-length Cry2Ab2 protein produced by MON 89034 indicated that the protein was also blocked at the N-terminus. Consequently, the cleavage site in the CTP that is processed in the chloroplast could not be experimentally determined. The CTP used in MON 89034 has a preferred cleavage site (methionine) located three amino acids upstream from the start of the Cry2Ab2 protein sequence (Figure VI-3). The next regime.and preferred cleavage site would have resulted in a protein approximatelyand 2.8 kDa larger AG than what was actually detected. Since the MON 89034-produced Cry2Ab2 co-migrated to the similar position as the Cry2Ab2 produced by a recombinant E. coli, which has the three extra amino acids (Figure VI-3), veryBayer likely theproperty CTP in MONprotection publishing89034 was cleaved at the preferred cleavage site located threeof amino acids upstream from contentsthe start of the its Cry2Ab2 protein sequence. This resulted in the productionparties.data of a Cry2Ab2 proteintherefore of 637 amino acids (634 from Cry2Ab2 and 3 from the CTP) with a theoreticaland/oror molecular weight affiliates. of 71 kDa. property intellectualthird may its as the of and Cry2Ab2 protein is activeis against corn and regulatorycotton pests. The spectrumowner. of activity a document includes corn borers from anythe generasuch Ostrinia and Diatraea (suchits as European corn reproductiondocument borer, Asian corn borer, sugarcaneowner borer , southwesternthis cornof borer), armyworms (Spodoptera spp. including fall armyworm),under earwormsof (Helicoverpa spp. including corn rightsthe this earworm), cutwormsdocumentand/or (e.g., Agrotis ipsilon, black cutworm),rights tobacco budworm (Heliothis to of fall use of virescens) and pink bollworm (Pectinophora gossypiellathe). This may distribution,and rights owner subject be the violate copy of and 1 2 may documentMONpublication, 89034 M-Q-A -M-D - N-S-V-L-N It exploitation 1 2 this Recombinant E. coli M-Q-A -M-D - N-S-V-L-N B.any thuringiensis -M- - N-S-V-L-N permission prohibited 1 M-Q-A – predicted amino acids from the chloroplast transit peptide the (CTP)commercial be 2 D – an additional amino acid included for the ease of cloning Furthermore,any Consequently,without

Figure VI-3. Comparison of the putative N-terminal sequence of Cry2Ab2 protein produced in MON 89034, the recombinant E. coli, and Bacillus thuringiensis

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VI.B.2. Characterization of the Cry2Ab2 Protein Produced in MON 89034

Similar to the case for Cry1A.105, the expression level of Cry2Ab2 protein in the tissue of MON 89034 is relatively low. Therefore, it was necessary to express the protein in a high-expressing, recombinant host system in order to obtain sufficient quantities of the Cry2Ab2 protein for safety studies. Thus, a recombinant Cry2Ab2 protein was produced in E. coli, the sequence of which was engineered to match the Cry2Ab2 protein produced in MON 89034. The equivalence of the physicochemical characteristics and functional activity between the MON 89034-produced and E. coli-produced Cry2Ab2 protein was confirmed by a panel of analytical techniques, including SDS-PAGE, densitometry, and Western blot analysis, MALDI-TOF MS, N-terminal sequencing,and glycosylationregime. analysis, and functional bioassay with corn earworm (CEW)AG ( Helicoverpa zea). The details of the materials, methods, and results are described in Appendix C, while the conclusions are summarized as follows. Bayer property protectionpublishing of contents The Cry2Ab2 protein from MON 89034 was purified and characterized,its and the results parties.data therefore confirmed the equivalence between MON 89034- and E. coli-producedand/oror Cry2Ab2 protein. The apparent molecular weight was affiliates.estimated by SDS-PAGE. Since the MON 89034- property intellectualthird may derived Cry2Ab2 migrated comparablyits to the E. coli-produced protein on SDS-PAGE, as the apparent molecular weightthe ofof th e Cry2Ab2and protein from both MON 89034 and E. coli is regulatory owner. was determined to be equivalent. On thea basis ofdocument Westernits blot analysis, the electrophoretic mobility andany immunoreactiv such e propertiesreproduction of the MON 89034-produced this documentof Cry2Ab2 protein were demonstratedowner to be comparable to those of the E. coli-produced under of Cry2Ab2 protein for the antibodyrightsthe used. The N-terminusthis of the Cry2Ab2 derived from documentand/or rights MON 89034 was blockedto butof the peptidefall mappiuseng analysisof by MALDI-TOF MS yielded peptide masses consistent with the expected peptide massesthe based on the trypsin cleavage This may distribution,and sites in the sequence ofrights Cry2Ab2 protein, confownerirming the identity of the protein. In addition, the MONsubject 89034- and the E. coli-produced Cry2Ab2 protein was found to be be the violate equivalent based copyon functional activitiesof and the lack of glycosylation. Taken together, these data provide a detailed characterizationand of the Cry2Ab2 protein isolated from MON may documentpublication, 89034It and established its equivalenceexploitation to the E. coli-produced Cry2Ab2 protein. this any VI.C. Expression Levels of Cry1A.105 and Cry2Ab2 Proteins in MON 89034 permission prohibited the The levels of thecommercial Cry1A.105 be and Cry2Ab2 proteins in various tissues of MON 89034 were determined using enzyme-linked immunosorbent assays (ELISA). The materials andFurthermore, methodsany for the ELISA analysis, as well as a description of the tissue types, are providedConsequently, in Appendixwithout D. To produce the tissues for analysis, MON 89034 and conventional corn were planted at five field locations during the 2005 growing season. The sites were located in the major corn-growing regions of the United States. A randomized complete block design with three replications was used at all sites. Forage, stover, silk, pollen, and grain samples were collected at appropriate times of plant development. Leaf, root, and whole plant samples were collected multiple times over the season. The expression levels of Cry1A.105 and Cry2Ab2 proteins in these tissues are shown in Tables VI-2 to VI-4.

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The mean Cry1A.105 protein levels across all sites were 5.9 µg/g dwt in grain, 42 µg/g dwt in forage, 12 µg/g dwt in pollen, 520 µg/g dwt in leaves of plants at V2-V4 stage1, 120 µg/g dwt in leaves of plants at pre-VT stage, 12 µg/g dwt in forage root, and 50 µg/g dwt in stover. In tissues harvested throughout the growing season, mean Cry1A.105 protein levels across all sites varied from 72-520 µg/g dwt in leaf, 11-79 µg/g dwt in root, and 42-380 µg/g dwt in whole plant. In general, levels of the Cry1A.105 protein declined over the growing season.

The mean Cry2Ab2 protein levels across all sites were 1.3 µg/g dwt in grain, 38 µg/g dwt in forage, 0.64 µg/g dwt in pollen, 180 µg/g dwt in leaves of plants at V2-V4 stage, 160 µg/g dwt in leaves of plants at pre-VT stage, 21 µg/g dwt in forage root, and 62 µg/g dwt regime.and in stover. In tissues harvested throughout the growing season,and mean Cry2Ab2 protein AG levels across all sites varied from 130-180 µg/g dwt in leaf, 21-58 µg/g dwt in root, and 38-130 µg/g dwt in whole plant. In general, levels of the Cry2Ab2 protein declined over the growing season. Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may document 1 publication,exploitation CornIt growth stages are divided into vegetative (V) and reproductive (R) stages. Corn growth staging begins with VE, orthis emergence any of seedlings above ground. The second growth stage is V1, and is given to plants that display their first collared leaf. Subsequent stages run from V2, V3 ... V(n), each stage permission corresponding to the number of leavesprohibited displaying collars. V(n) refers to the last leaf stage, and will vary depending on the hybrid inthe consideration. be The final vegetative stage is VT, or tassling stage when last branch of the tassel commercialis completely visible and the silks have not yet emerged. VT begins approximately 2-3 days before silk emergence. Reproductive (R) stages range from R1 to R6. Stage R1 is the silking stage, whenFurthermore, silk is visibleany outside the husk. Stage R2 is the blister stage and occurs 10-14 days after silking. In this stageConsequently, kernels withoutare white and small, resembling blisters. Milking stage (R3) occurs 18-22 days after silking. Kernels are yellow and contain white fluid, due to accumulating starch. In the dough stage (R4), this fluid begins to thicken into a white paste within the kernels, and the shelled cob takes on a pinkish hue. Stage R5 is the dent stage, where kernels begin drying and exhibit a dent on their top. Around 55-65 days after silking the ears are physiologically mature (R6). In this stage, all kernels have attained their maximum dry weight. Black zones have formed internally at the base of kernels, indicating that kernel growth has ceased. Also, husks and leaves are no longer green at this stage, though stems may still be pigmented. Source: Iowa State University (maize.agron.iastate.edu/corngrows.html. Accessed on July 17, 2006.

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Table VI-2. Cry1A.105 and Cry2Ab2 protein expression levels in MON 89034 tissues

Tissue Growth Cry1A.105 Mean2 (SD), Cry2Ab2 Mean2 (SD), Type Stage Range, n=15 Range, n=15 µg/g fwt µg/g dwt µg/g fwt µg/g dwt

Young leaf 1 V2-V4 85 (21) 520 (130) 29 (6.8) 180 (59) 56 – 130 380 – 850 19 - 43 94 - 270

Pollen R1 6.4 (1.5) 12 (1.7) 0.34 (0.084) 0.64 (0.091) 3.8 – 8.8 8.5 - 16 0.21 – 0.47 0.49 -and 0.79 and regime. AG Silk R1 3.0 (0.57) 26 (3.9) 8.2 (3.6) 71 (35) 2.0 – 3.8 20 - 31 3.3 - 16 33 - 160 Bayer property publishing protectioncontents Forage R4-R6 14 (3.6) of 42 (9.4) 12 (4.0) 38 (14) its (early dent) 8.3 - 24 20 - 56 parties.data 6.5 - 18 therefore15 - 55 and/oror affiliates. Forage root R4-R6 2.2property (0.35) intellectual12 third(3.1) 4.1 (1.4) may 21 (5.9) its (early dent) 1.3 – 2.7 as 6.2 - 16 2.2 – 6.5 14 - 33 the of and is regulatory owner. a document Grain R6 any 5.1 (0.67)such 5.9 (0.77) 1.1 (0.31)its 1.3 (0.36) reproductiondocument 4.1 – 6.0owner 4.7 - 7.0 this 0.67 of– 1.8 0.77 – 2.1 of rights under this Stover R6 and/or 17 (4.4)the 50 (17) 22 (3.6) 62 (15) document to fall of rights (after harvest) 9.5of - 26 26use - 85 17 - 29 46 - 97 the This may distribution,and rights owner Senescent root R6 subject 2.2 (0.36) 11 (1.4) 5.3 (2.0) 26 (8.8) (after harvest) 1.7 – 3.1 9.4 - 15 2.4 – 9.1 13 - 43 be the violate copy of 1 Young leaf = overseason leaf 1 (OSL-1); n = numberand of samples; SD = standard deviation; fwt = may documentpublication, fresh weightIt tissue; dwt = dry weightexploitation tissue. 2 Limits of detection (LOD) and limits of quantitation (LOQ) in the various tissues were as follows: this any Cry1A.105 (µg/g fwt) Cry2Ab2 (µg/g fwt) permission Tissue LOD LOQprohibited LOD LOQ Forage 0.372the 0.44 0.191 0.44 commercial be Leaf 0.568 0.66 0.081 0.44 Furthermore,Pollen any 0.412 1.1 0.055 0.11 RootConsequently, without0.254 0.33 0.056 0.22 Silk 0.275 0.44 0.040 0.22 Grain 0.262 1.1 0.123 0.22

Whole plant and stover were analyzed in the same manner as forage, and senescent root was analyzed in the same manner as root.

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Table VI-3. Cry1A.105 proteins expression levels in overseason tissues of MON 89034

Plant growth stages1 and and regime. Over- V2-V4 V6-V8 V10-V12 AG pre-VT R4-R6 R6 Season (21-29 DAP) (28-43 DAP) (41-53 DAP) (56-68 DAP) (100-120 DAP) (130-160 DAP) Tissue Units µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g (n = 15) dwt fwt dwt fwt dwt Bayerfwt propertydwt protectionfwtpublishing dwt fwt dwt fwt of contents its parties. data therefore Leaf Mean2 (SD) 520 (130) 85 (21) 140 (36) 28 (8.7) 72 (14) 16 (4.3) 120 (77) and/or 30 or(20) N/A N/A N/A N/A affiliates. Range 380-850 56-130 80-200 12-45property 47-89 intellectual9.4-24third 27-240 6.3-59may N/A N/A N/A N/A its as Whole Mean2 (SD) 380 (90) 40 (5.7) 260 (52) 24the (3.7) of 100 (26)and 11 (2.4) 120 (29) 17 (3.7) 42 (9.4) 14 (3.6) 50 (17) 17 (4.4) is regulatory owner. plant Range 230-570 30-52 170-350 16-31 58-160 7.0-a 15 58-170document 9.3-22 20-56 8.3-24 26-85 9.5-26 any such its reproductiondocument owner this of Root Mean2 (SD) 79 (17) 8.9 (1.3) 48 (11) 5.8 (1.6) 45 (10) 6.4 (1.8) 42 (10) 6.7 (0.63) 12 (3.1) 2.2 (0.35) 11 (14) 2.2 (0.36) under of this Range 52-110 7.3-12 30-63 3.0-8.5rights the 26-64 4.4-10 30-63 5.6-8.1 6.2-16 1.3-2.7 9.4-15 1.7-3.1 documentand/or rights to of fall use of 1 Growth stages V2-V4, V6-V8, V10-V12 and pre-VT (tasseling) correspond to overseason tissue samples 1, 2, 3 and 4, respectively the (e.g., OSWP-1, etc.). The whole plantThis and root samples maycollected distribution, atand the R4-R6 (early dent) and R6 (after harvest) stages correspond to rights owner forage plant/root and stover/senescent root,subject respectively. N/A - not applicable; VT-vegetative stage at tasseling; DAP - days after planting; dwt - dry weight tissue; fwt - befresh weight tissue. the violate 2 The LODs and LOQs for Cry1A.105 can becopy found in Table VI-2of. SD = standard deviation. and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VI-4. Cry2Ab2 proteins expression levels in overseason tissues of MON 89034 regime.and Plant growth stages1 and Over- V2-V4 V6-V8 V10-V12 AG pre-VT R4-R6 R6 Season (21-29 DAP) (28-43 DAP) (41-53 DAP) (56-68 DAP) (100-120 DAP) (130-160 DAP) Tissue Units µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g Bayer property protectionpublishing (n = 15) dwt fwt dwt fwt dwt of fwt dwt fwt contentsdwt fwt dwt fwt its 2 parties.data therefore Leaf Mean (SD) 180 (59) 29 (6.8) 170 (34) 32 (5.3) 130 (34) 29 (5.4) 160 (44) and/or 37 or(12) N/A N/A N/A N/A Range 94-270 19-43 110-230 23-44 85-200affiliates. 23-41 48-210 11-56 N/A N/A N/A N/A property intellectualthird may its Whole Mean2 (SD) 130 (51) 13 (4.6) 79 (18) 7.5the (1.8) 40 (9.9)as 4.2 (0.94) 39 (16) 5.9 (2.6) 38 (14) 12 (4.0) 62 (15) 22 (3.6) of and regulatory owner. plant Range 52-230 5.2-21 45-110 is4.0-9.7 22-61 2.4-5.8 5.0-67 0.7-11 15-55 6.5-18 46-97 17-29 a documentits any such reproductiondocument of Root Mean2 (SD) 56 (17) 6.4 (1.6) 58 (18) 7.6 (4.2) 35owner (17) 5.0 (2.7) this 26 (7.7) 4.2 (1.2) 21 (5.9) 4.1 (1.4) 26 (8.8) 5.3 (2.0) under of Range 33-100 4.4-10 25-86 2.5-15rights the 15-74 2.2-12 this15-45 3.2-7.6 14-33 2.2-6.5 13-43 2.4-9.1 documentand/or of rights 1 Growth stages V2-V4, V6-V8, V10-V12 and pre-VTto (tasseling)of fall corresponduse to overseason tissue samples 1, 2, 3 and 4, respectively (e.g., OSWP-1, etc.). The whole plant and root samples collected at the R4-R6 (earlythe dent) and R6 (after harvest) stages correspond This may distribution,and to forage plant/root and stover/senescent root, respectively.rights N/A - not applicable;owner VT-vegetative stage at tasseling; DAP - days after subject planting; dwt - dry weight tissue; fwt - fresh weight tissue. the 2 be violate The LODs and LOQs for Cry1A.105 can becopy found in Table VI-2of. SD = standard deviation. and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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VI.D. Food and Feed Safety Assessment Summary of the Cry1A.105 and Cry2Ab2 Proteins

Numerous factors have been considered in the safety assessment of the Cry1A.105 and Cry2Ab2 proteins that are expressed in MON 89034. A comprehensive assessment of safety of these proteins was submitted to the EPA and FDA. The assessment leads to the following conclusions, which are consistent with the conclusions reached for other Cry proteins produced in a number of insect-protected crops currently on the market.

a) The donor organism, Bacillus thuringiensis, has been used commercially in the U.S. since 1958 to produce microbial-derived products with insecticidal activities. regime.and The extremely low mammalian toxicity of Bt-based insecticideand products has been AG demonstrated in numerous safety studies, and there are no confirmed cases of allergic reactions to Cry proteins in applicators of microbial-derived Bt products during the past 45 years of use. Bayer property protectionpublishing of contents b) Cry1A.105 is a chimeric Cry1A protein of Bt that is comprisedits of three N- parties.data therefore terminal domains (I, II, and III) and the C-terminal region.and/or Theor domains I and II affiliates. of Cry1A.105 are 100%property iden ticalintellectual to thirdthe respective domainsmay of Cry1Ab or Cry1Ac. The domain III itsof Cry1A.105 is 99% identical to the domain III of as Cry1F. The C-terminalthe ofregion of Cry1A.105and is 100% identical to that of Cry1Ac. is regulatory owner. a document The overall amino acidany sequencesuch identity of 93.6%, 90.0%, andits 76.7% to Cry1Ac, reproductiondocument Cry1Ab, and Cry1F proteins,owner respec tively. thisCry2Ab2 isof a Bt (subsp. kurstaki) protein. Bt strains producing Cry1Ac,under Cry1Ab,of thisCry1F, and Cry2A proteins have been used and/orfor decadesrights the as biopesticides, and Bt pesticides that contain document to fall of rights Cry1Ac/Cry1F chimericof protein have usebeen used since 1997. Bt corn and cotton the expressingThis Cry1Ac, Cry1Ab,may distribution, Cry1F,and or Cry2Ab2 proteins have been cultivated in rights owner large areassubject in the U.S. and other countries for up to a decade. There are no known reports of allergy or toxicity to Bt or to these Cry proteins. be the violate copy of c) A dietary safety assessment to evaluateand the risks to humans and animals from the may documentpublication, It Cry1A.105 and Cry2Ab2exploitation proteins present in the foods and feeds derived from MON 89034.this any Risks are quantified as a margin of exposure (MOE), which is defined as the ratio of the No Observable Effect Level (NOEL) from an acute permission mouse gavage study toprohibited estimates of the dietary intake of the respective Cry the protein. commercialMice acute beoral toxicity studies demonstrated that the two proteins are not acutely toxic and do not cause any adverse effects even at the higest dose Furthermore,levelsany test, which are 2072 and 2198 mg/kg body weight for Cry1A.105 and Consequently,Cry2Ab2without proteins, respectively. The dietary safety assessment showed that the MOEs for the overall U.S. population were greater than or equal to 199,000 and 981,000 for the Cry1A.105 and Cry2Ab2 proteins, respectively. For children aged 3-5 years old, an age group with the highest corn consumption, the MOEs were greater than or equal to 79,400 and 390,000 for the Cry1A.105 and Cry2Ab2 proteins, respectively. For poultry and livestock, the MOEs ranged between 1,930 – 13,500 and 2,160 – 47,600 for the Cry1A.105 and Cry2Ab2 proteins, respectively.

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d) Cry1A.105 and Cry2Ab2 proteins are rapidly digestible in simulated gastric fluids. Greater than 95% to 99% of the proteins were digested in simulated gastric fluids in less than 30 seconds. Proteins that are rapidly digestible in mammalian gastrointestinal systems are unlikely to be allergens when consumed. e) Cry1A.105 and Cry2Ab2 proteins do not share any amino acid sequence similarities with known allergens, gliadins, glutenins, or protein toxins which have adverse effects to mammals. This has been shown by extensive assessments with bioinformatic tools, such as FASTA sequence alignment tool and an eight- amino acid sliding window search. Using the guidance provided by the FDA, a conclusion of “no concern” is reached for the and donor organisms and the Cry1A.105 and Cry2Ab2 proteins. Theand food andregime. feed products containing MON 89034 or made of MON 89034AG are safe for human and animal consumption. The EPA has issued a time limted tolerance exemption for 3 years starting from July 17, 2007 for the two proteins while the review for a full registration is under Bayer property protectionpublishing review. of contents its parties.data therefore and/oror VI.E. References affiliates. property intellectualthird may its Baum, J.A. 1998. Transgenic Bacillusas thuringiensis. Phytoprotection 79 (suppl.):127- the of and 130. is regulatory owner. a document any such its reproductiondocument Baum, J.A., T.B. Johnson, and B.C.owner Carlton. 1999. thisBacillus thuringiensisof . Natural and recombinant biopesticide products. Pages 189-209 in Methods in Biotechnology: under of this Biopesticides:and/or Use rights and Delivery.the Vol. 5. Hall, F.R. and J.J. Menn (eds). Humana document fall of rights Press Inc., Totowa,to NJ.of use the This may distribution,and Betz, F.S., B.G. Hammond,rights and R.L. Fuchs.owner 2000. Safety and advantages of Bacillus thuringiensissubject-protected plants to control insect pests. Reg. Toxicol. Pharmacol. 32:156-173.be the violate copy of Bruce, B.D. 2000. Chloroplast transit peptide:and structure, function and evolution. Trends may documentpublication, It Cell Biol. 10.440-447.exploitation this any Crickmore, N., D.R. Ziegler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, permission and D.H. Dean. 1998. Revisionprohibited of the nomenclature for the Bacillus thuringiensis pesticidal crystalthe proteins. Microbiol. Mol. Biol. Rev. 62:807-813. commercial be 2004. Personal communication with Monsanto Company. Furthermore,any Dankocsik,Consequently, C.,without W.P. Donovan, and C.S. Jany. 1990. Activation of a cryptic crystal protein gene of Bacillus thuringiensis subspeceis kustaki by gene fusion and determiantion of the crstal protein insecticidal specificity. Mol. Microbiol. 4:2087-2094. De Maagd, R.A., A. Bravo, and N. Crickmore. 2001. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet. 17:193-199.

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De Maagd, R.A., A. Bravo, C. Berry, N. Crickmore, and H.E. Schnepf. 2003. Structure, diversity and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu. Rev. Genet. 37:409-433. De Maagd, R.A., M. Weemen-Hendriks, W. Stiekema, and D. Bosch. 2000. Bacillus thuringiensis delta-endotoxin Cry1C domain III can function as a specificity determinant for Spodoptera exigua in different, but not all, Cry1-Cry1C hybrids. Appl. Environ. Microbiol. 66:1559-1563. EPA. 2005. Biopesticide active ingredients and products containing them. Available at http://www.epa.gov/pesticides/biopesticides/product_lists/bppd-prods-12-16- 05.pdf. Accessed September 18, 2006. and Gao, Y., K.J. Fencil, X. Xu, D.A. Schwedler, J.R. Gilbert,and R.A. Herman.regime. 2006. Purification and characterization of a chimericAG Cry1F δ-endotoxin expressed in transgenic cotton plants. J. Agric. Food Chem. 54:829-835. Gill, S.S.; E.A. Cowles, and P.V. Pietrantonio.Bayer 1992.property The mode ofpublishing action of Bacillus protectioncontents thuringiensis endotoxins. Annu. ofRev. Entomol. 37:615-636. its parties.data therefore Li, J., J. Carroll, and D.J. Ellar. 1991. Crystal structure of insecand/orticidalor delta-endotoxin affiliates. from Bacillus thuringiensisproperty at 2.5 Åintellectual resolution.third Nature 353:815-821.may its as Masson, L., B.E. Tabashnik,the A. ofMazza, G. andPrefontaine, L. Potvin, R. Brousseau, and J-L. Schwartz. 2002. isMutagenic analysis of regulatorya conserved region ofowner. domain III in the a document Cry1Ac toxin of Bacillusany suchthuringiensis . Appl. Env. Microbiol.its 68:194-200. reproductiondocument owner this of Schnepf, E., N. Crickmore, J. Van Rie,under D. Ler eclus,of J. thisBaum, J. Feitelson, D.R. Zeigler, and D.H. Dean.and/or 1998.rights Bacillusthe thuringiensis and its pesticidal crystal proteins. document to fall of rights Microbiol. Mol. Biol. ofRev. 62:775-806.use the This may distribution,and Widner, W.R. and H.R.rights Whiteley. 1989. Twoowner highly related insecticidal crystal proteins of Bacillussubject thuringiensis subsp. kurstaki possess different host range specificities. J. Bacteriol.be 171:965-974. the violate copy of Zhuang, M., and S.S. Gill. 2003. Mode ofand action of Bacillus thuringiensis toxins. Pages may documentpublication, It 213-236 in Chemistryexploitation of Crop Protection, Progress and Prospects in Science and Regulation.this anyVoss, G. and G. Ramos (eds), Wiley-VCH, Weinheim. permission prohibited the commercial be Furthermore,any Consequently,without

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VII. Phenotypic, Agronomic, Ecological Interactions and Compositional Assessment

This section provides an assessment of the phenotypic and agronomic characteristics, the ecological interactions, and compositional analyses of MON 89034. Phenotypic and agronomic characteristics of MON 89034 were evaluated relative to an appropriate control to assess plant pest potential and meaningfully altered ecological impact. These assessments included 14 plant growth and development characteristics, five seed germination parameters, two pollen characteristics, and more than 70 observations for each of the plant-insect, plant-disease and plant responses to abiotic stressor interactions. Results from the phenotypic and agronomic assessments indicate that MON 89034 does regime.and not possess characteristics that would confer a plant pest risk orand increased ecological risk AG compared to conventional corn. Data on ecological interactions indicate that MON 89034 does not confer any increased susceptibility or tolerance to specific diseases, insects (except for the target lepidopteranBayer species), property or abiotic protection strepublishingssors. Data from compositional analyses demonstrated ofcompositional equivalence in contentsthe levels of its nutritional, anti-nutritional, and secondary metabolitesparties.data between MONtherefore 89034 and conventional corn grain and forage. Taken together, these dataand/or supportor a conclusion that affiliates. MON 89034 is not likely to poseproperty an increased intellectualthird plant pest risk or resultmay in meaningfully its altered ecological impact compared to asconventional corn. the of and is regulatory owner. a document VII.A. Phenotypic, Agronomic,any such and Ecological Interactions Assessmentits reproductiondocument owner this of under of VII.A.1. Phenotypic and Agronomicrightsthe Characteristics thisand Ecological Interactions documentand/or rights to of fall use of In the phenotypic, agronomic and ecological interactionsthe assessment of MON 89034, This may distribution,and data were collected to rightsevaluate specific ecologicalowner risks regarding altered pest potential based on requirementssubject of USDA-APHIS. The assessment encompasses five general data be the violate categories: 1) germination,copy dormancyof and emergence; 2) vegetative growth; 3) reproductive growth (including pollen characteristics);and 4) seed retention on the plant; and may documentpublication, 5) plantIt interactions with insects,exploitation diseases and abiotic stressors. An overview of the characteristics assessedthis any is presented in Table VII-1.

permission The phenotypic, agronomic andprohibited ecological interactions evaluations are based on a the combination of laboratorycommercial experiments be and field studies conducted by scientists who are familiar with the production and evaluation of corn. In each of these assessments, the regulatedFurthermore, article,any MON 89034 (referred to as test substance or material), was compared to an appropriateConsequently, conventiowithout nal corn (referred to as control substance or material) which has a genetic background similar to MON 89034 but does not possess the lepidopteran- protection trait. In addition, multiple commercial corn hybrids (referred to as reference substances or materials) were also employed to provide a range of baseline values that are common to the existing commercial corn hybrids for each measured phenotypic, agronomic, and ecological interaction characteristic. Further descriptions of the test, control and reference materials are provided in Appendices E and F.

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Table VII-1. Phenotypic, agronomic and ecological interaction characteristics measured for MON 89034

General Characteristics Evaluation characteristics measured timing Evaluation description Dormancy / Dormancy, After 4, 7, and Percent normally germinated, Germination Germination1 12 days abnormally germinated, viable hard (dormant), dead, and viable firm swollen seed Emergence Seedling vigor Stage V2 – V4 Rated on a 0-9 scale, where 0 = dead, and 9 = above average vigor Early stand count Stage V2 - V4 Number of emerged plants per plot and Vegetative Final stand count Pre-harvest Number ofand plants per plotregime. Growth Stay green Maturity AG Rated on a 0-9 scale, where 0 = entire plant is dried, and 9 = entire plant is green Ear height MaturityBayer Distanceproperty from the publishingsoil surface at the base protectioncontents of of the plant to the ear attachment node its Plant height Maturity parties. Distancedata from the soil surfacetherefore at the base of the plantand/or to theor flag leaf collar affiliates. Stalk lodged propertyPre-harvest intellectual third Number of plants permay plot broken below its plants as the ear Root lodgedthe plants of Pre-harvestand Number of plants per plot leaning at the is regulatory owner. a soil surfacedocument at >30 ° from the vertical any such its Reproductive Days to 50% Pollen shed reproduction Days fromdocument planting until 50% of the owner this of Growth pollen shed under of plants have begun to shed pollen Days to 50%rights the Silking Daysthis from planting until 50% of the documentand/or rights silking to of fall use ofplants have silks exposed Pollen viability1 Tasseling Viablethe and nonviable pollen based on This may distribution,and rights owner pollen grain staining characteristics subjectPollen Tasseling Diameter of viable pollen grains morphology1 the violate be copy Grain moisture Harvestof Moisture percentage of harvested shelled document and grain may publication,exploitation It Test weight Harvest Test weight of harvested shelled grain this (lb/bu)any Yield (bu/ac)permission Harvest Harvested shelled grain, adjusted to prohibited 15.5% moisture the Seed Retention commercial Dropped ears be Pre-harvest Number of mature ears dropped from plants Plant-Furthermore,any Insect, disease and Variable, from Qualitative assessment of each plot, with ecologicalConsequently, withoutabiotic stressors planting to rating on a 0-9 scale for plant-insect, interactions harvest plant-disease, and plant response to abiotic stressor interactions 1 These characteristics were measured in the laboratories.

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VII.A.2. Interpretation of Phenotypic, Agronomic and Ecological Interaction Data

The concept of familiarity is useful when evaluating the potential environmental impact of a biotechnology-derived plant. The concept of familiarity is based on the fact that the biotechnology-derived plant is developed from a conventional plant variety whose biological properties and plant pest potential are known to experts. Familiarity considers the biology of the crop, the introduced trait, the receiving environment and the interaction among these factors, and provides a basis for comparative risk assessment between a biotechnology-derived plant and its conventional counterpart. An expert’s knowledge and experience with a crop is the basis for selecting appropriate endpoints and estimating and the range of responses that would be considered familiar.and Thus, regime.assessment of phenotypic and agronomic characteristics and AGeco logical interactions can be used to compare the biotechnology-derived plant to the conventional counterpart, and a subset of the data (e.g., certain dormancy, lodging or pre-harvest seed loss characteristics) can be Bayer property protectionpublishing used to assess whether there is an enhancedof weediness potential. Based oncontents all of the data collected, an assessment can be made whether the biotechnology-deriveitsd plant is likely to parties.data therefore pose an increased plant pest potential or have a meaningfullyand/or alteredor ecological impact compared to the conventional counterpart.affiliates. property intellectualthird may its as Agronomists familiar withthe the of experimental and design, evaluation criteria, and expected is regulatory owner. outcomes were involved in all steps of data collection,a summadocumentrization and analyses. This any such its oversight ensured that measurements were takenreproduction properly,document dataof were consistent with expectations based on experience withowner the crop, and thethis experi mental and field sites were under of carefully monitored. Theserights the scientists were expectedthis to report any unexpected documentand/or rights observations or issues duringto ofth e coursefall of theuse studies. of The overall dataset was evaluated for evidence of biologically relevant changes and for anythe evidence of an unexpected plant This may distribution,and response, and the data wererights subsequently submittedowner for statistical analysis. subject be the violate Comparative plantcopy characteriz ation dataof between a biotechnology-derived crop and the control are considered in the context ofand contributions to increased pest/weediness may documentpublication, potential.It Characteristics forexploitation which no differe nces are detected support a conclusion of no increased thispest any potential of the biotechnology-derived crop compared to the conventional crop. Characteristics for which differences are detected are considered in permission the step-wise method (Figure prohibitedVII-1). Any detected difference for a characteristic is the considered in thecommercial context of bewhether or not the difference would increase pest/weediness potential of the biotechnology-derived crop. Ultimately, a weight of evidence approach consideringFurthermore, anyall characteristics and studies is used for the overall risk assessment of differencesConsequently, andwithout their significance in terms of increased pest potential. In detail, Figure VII-1 illustrates the stepwise assessment process employed:

• Steps 1 & 2. Combined-site and individual-site statistical analyses are conducted and evaluated on each measured characteristic. Differences detected in the individual-site analysis must be observed in the combined-site analysis to be considered further for potential adverse effects in terms of pest/weediness potential. A difference in the combined-site analysis is further assessed

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regardless of whether or not the difference is detected in the individual-site analysis. • Step 3. If a difference is detected in the combined-site analysis across multiple environments, then the test substance mean value is assessed relative to the range of values of the reference substances. • Step 4. If the test substance mean is outside the variation of the reference substances (e.g., reference range or tolerance interval), the test substance mean is considered in the context of known values common for the crop. • Step 5. If the test substance mean is outside the range of values common for the crop, the test substance is considered “non-familiar” for that characteristic. The and detected difference is then assessed for whether or notand it is adverseregime. in terms of pest/weediness potential. AG • Step 6. If an adverse effect (hazard) is identified, risk assessment on the difference is conducted. The Bayerrisk assessmentproperty considers protectionpublishing contributions to enhanced pest potential of the ofcrop itself, the impact of differencescontents detected in its other measured characteristics, and potentialparties. for,data and effects of traittherefore transfer to feral populations of the crop or a sexually compatible and/orspecies.or affiliates. property intellectualthird may its as Step 1 the of and is regulatory owner. Differences detected in the combined-sitea document and individual-siteany such analyses are evaluated* its reproductiondocument of owner this No adverse changes in Step 2 under of this Statisticalrights differencesthe detected No the characteristic that documentand/or of rights into combined-site of fall analysis?use would contribute to pest potential Yes the This Step 3 may distribution,and owner rights No Not adverse; the subjectTest mean outside variation of be references?the violate detected difference in copy of Yes the measured Step 4 and may documentpublication, characteristic does not It Test meanexploitation outside variation No contribute to a this anyfor the crop? (“non-familiar”) biological or ecological

permissionYes concern for the crop in Step 5 prohibited No terms of pest potential Adversethe in terms of pest potential? commercial be Yes Step 6 Furthermore,any *See text for interpretation of differences detected Consequently,without Hazard identification & risk assessment on difference in the individual-site analysis

Note: A “no” answer at any step indicates that the characteristic does not contribute to a biological or ecological concern for the crop in terms of pest/weediness potential and further steps are not considered.

Figure VII-1. Flow chart for interpretation of detected differences

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VII.A.3. Evaluation of Phenotypic and Agronomic Characteristics and Ecological Interactions

VII.A.3.a. Evaluation of Seed Dormancy and Germination

Seed dormancy (e.g., hard seed) is an important characteristic that is often associated with plants that are considered as weeds (Anderson, 1996; Lingenfelter and Hartwig, 2003). Dormancy mechanisms, including hard seed, vary with species and tend to involve complex processes. For corn, the number of hard seed is negligible or nonexistent. Standardized germination assays are routinely used to measure the germination characteristics of corn seed. The Association of Official Seed Analysts and (AOSA) recommends a temperature range of 20-30°C as optimaland for germinationregime. of corn (AOSA, 2002). AG

The seed materials for the evaluation of MON 89034 were produced in 2004 at three Bayer property protectionpublishing production locations (Dayton, IA; Jerseyville,of IL; and Monmouth,contents IL) that are representative of the environmental variability found in corn producingits regions. Seed parties.data therefore materials included MON 89034, the conventional control cornand/or (H1325023)or which has a affiliates. genetic background similar to propertyMON 89034, intellectual andthird four reference commercialmay corn hybrids from each production location. its as the of and is regulatory owner. The dormancy and germination characteristicas evaluated,document temperature regimes and the any such its reproductiondocument evaluation description are presentedowner in Table VII-2this. The testsof were conducted in temperature-controlled growth chambersunder using ofthe rolledthis towel test method. Four replicates of MON and/or89034,rights thethe control, and the four references were tested in seven document to fall of rights growth chambers, each maintainedof in the darkuse under one of the following temperature the regimes This(Table VII-2): (1) constantmay distribution, targetand temperatur e of approximately 5, 10, 20, or rights owner 30°C; and (2) alternatingsubject target temperatures of approximately 10/20, 10/30, or 20/30°C. In these alternating temperature regimes, the lower temperature was maintained for 16 h be the violate and the higher temperaturecopy for 8 h. Countsof for characteristics were made four and seven and days aftermay planting. document A publication,statistical comp arison between the MON 89034 and the control was conductedIt using a SASexploitation (Statistical Analysis System) program (SAS Release 9.1, this 2002-2003). Statisticalany significance was set at p≤0.05. A summary of the results is provided below, while the permissiondetails of the materials, methods and results from individual prohibited production sites are provided in Appendix E. the commercial be A total of 29 comparisons were made between MON 89034 and the conventional control Furthermore,any seedConsequently, germinationwithout parameters across three seed production sites (Table VII-3). No statistically significant differences were detected between MON 89034 and the control for any seed characteristic measured in the AOSA temperature regime (20/30°C) or the additional temperature regimes (5, 10, 20, 30, 10/20, 10/30°C).

The observation of no viable hard seed in any temperature regime indicates that the introduction of the lepidopteran-protection trait and the expression of the Cry1A.105 and Cry2Ab2 proteins did not alter dormancy and germination of the seed of MON 89034 compared to the conventional corn seed. Thus, it is concluded that there was no change

Monsanto Company 06-CR-166U Page 116 of 262 CBI Deleted Version in the pest potential of MON 89034 compared to conventional corn based on the germination and dormancy characteristics assessed.

VII.A.3.b. Evaluation of Pollen Morphology and Viability

The purpose of this evaluation was to assess whether the introduction of the lepidopteran- protection trait and the expression of the Cry1A.105 and Cry2Ab2 proteins altered the pollen characteristics of MON 89034 compared to the conventional corn. The test material was corn pollen collected from MON 89034 plants. The control material was corn pollen collected from the conventional corn plants (H1325023) which has a genetic background similar to MON 89034. The reference materials were pollen collected from plants of commercially available conventional corn hybrids that were used to demonstrateand and regime. a range of morphology and viability characteristicsAG for corn pollen.

MON 89034, the conventional control (H1325023), and three references (DKC61-42, RX740, and Burrus 576) were grown in aBayer rando mizedproperty complete blockpublishing design with three protectioncontents replications in Carlyle, IL, under similarof agronomic conditions. Pollen collected from its five plants per plot were fixed and stained with 1:5 parties.diluteddata Alexander staintherefore (Alexander, and/oror 1980) and evaluated for viability. Whenaffiliates. exposed to the staining solution, viable pollen grains stained red (due to theproperty the presence intellectual ofthird vital cytoplasmic content),may while dead its as pollen grains stained light theblue of. Ten representativeand viable pollen grains collected from is regulatory owner. one of the five plants per plot were measured afor pollen diameter,document which were observed at 200X magnification and wereany alsuchso evaluated for general morphology.its Data analysis reproductiondocument of compared the mean of MON 89034owner to the mean of thisthe control corn for average pollen under of diameter and arcsine transformedrightsthe percent viable pollen.this No statistically significant documentand/or rights differences were detectedto at ofp ≤ 0.05fall betweenuse MON of 89034 and the control for pollen diameter or viability (Table VII-4). No statistically thedifferences between MON 89034 This may distribution,and and the control were observedrights in pollen generalowner morphology. subject These resultsbe demonstrated that the introducthe tionviolate of the lepidopteran-protection trait and copy of the expression of the Cry1A.105 and Cry2Aand b2 proteins did not alter the overall may documentpublication, morphologyIt or viability of MexploitationON 89034 pollen compared to the conventional corn. The lack of differences between the pollen collected from MON 89034 compared to the this any conventional control for the assessed characteristics demonstrate that the observed values permission were within the range of responsesprohibited familiar to what is expected for corn. the commercial be Furthermore,any Consequently,without

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Table VII-2. Seed dormancy and germination parameters evaluated

Germination Temperature regime Evaluation description characteristic (°C) Normally Germinated 20/30 Seedlings that exhibited normal developmental characteristics and possessed both a root and a shoot.

Abnormally 20/30 Germinated, but with and and regime. Germinated AG insufficient root or shoot development, may have possessed a hollow Bayer propertycoleoptile, protection orpublishing exhibited of contents mechanical damage. its parties.data therefore and/oror Total Germinated 5, 10, 20,affiliates. 30, 10/20, Seedlings that had property intellectualthird may 10/30its germinated. as the of and is regulatory owner. Dead 5, 10, 20, 30, 10/20,a Seedsdocument that had visibly any 10/30,such 20/30 deterioratedits and had reproductiondocument of owner this become soft to the touch. under of rights the this documentand/or rights Viable Hard to of5, 10,fall 20, 30, 10/20,use of Seeds that did not imbibe 10/30, 20/30 thewater and remained hard to This may distribution,and rights owner the touch. subject Viable Firmbe Swollen 5, 10, 20, 30,the 10/20, violate Seeds that had visibly copy of 10/30, 20/30and swollen (imbibed water) may documentpublication, and were firm to the touch It exploitation but lacked any evidence of this any growth. permission Note: Dormancy and germinationprohibited tests were conducted in temperature-controlled growth the chambers usingcommercial the rolled be towel test method to measure dormancy and germination characteristics. Four replicates of MON 89034, the control, and the four references were Furthermore,tested in anyseven growth chambers, each maintained in the dark under one of the following temperatureConsequently, withoutregimes: (1) constant target temperature of approximately 5, 10, 20, or 30°C; and (2) alternating target temperatures of approximately 10/20, 10/30, or 20/30°C. In these alternating temperature regimes, the lower temperature was maintained for 16 h and the higher temperature for 8 h. Counts for characteristics were made four and seven days after planting.

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Table VII-3. Germination of MON 89034 and a conventional control corn

AOSA Mean (%) Starting Temp. Viable 1 Seed Normally Abnormally Regime 2 Viable Hard Dead Firm Type Germinated Germinated (°C) Swollen Test 94.2 0.1 0.0 5.8 0.0 20/30 Control 95.3 0.2 0.0 4.5 0.0 Ref. Range 78.0 – 100.0 0.0 – 4.0 0.0 – 0.0 0.0 – 22.0 0.0 – 1.0

Additional Mean (%) Starting Temp. and Seed regime.Viable Regime1 and Type Germinated Viable Hard Dead Firm (°C) AG Swollen Test 0.0 0.0 5.7 94.3 5 Control 0.0 0.0 6.0 94.0 Bayer property protectionpublishing Ref. Range 0.0 – 0.0 of 0.0 – 0.0 0.0 – 19.0 contents 81.0 – 100.0 Test 96.0 0.0 4.0 its 0.0 10 Control 94.3 0.0 parties.data 5.7 therefore 0.0 and/or Ref. Range 82.0 – 100.0 0.0 – 0.0 0.0 – or17.0 0.0 – 1.0 affiliates. Test property95.9 intellectualthird0.0 4.1 may 0.0 20 Control 95.2its 0.0 4.8 0.0 as Ref. Range the 85.0 of – 100.0 and 0.0 – 0.0 0.0 – 15.0 0.0 – 0.0 is regulatory owner. Test 95.6 a 0.0 document4.4 0.0 any such its 30 Control 95.5 reproduction 0.0 document 4.5 0.0 Ref. Range 80.0 – 100.0owner 0.0 – 0.0this 0.0of – 20.0 0.0 – 0.0 Test 95.9 under 0.0of 4.1 0.0 rightsthe this 10/20 documentControland/or 96.3 0.0 of rights 3.8 0.0 Ref. Range to 81.0of – 100.0fall use 0.0 – 0.0 0.0 – 20.0 0.0 – 0.0 Test 95.3 0.0 the 4.7 0.0 This may distribution,and 10/30 Control rights 96.8 owner 0.0 3.3 0.0 Ref.subject Range 81.0 – 100.0 0.0 – 0.0 0.0 – 19.0 0.0 – 0.0 the 1 be violate In alternating temperaturecopy regimes the lower temperaturof e was maintained for 16 h and the higher temperature for 8 h. Counts for characteristics were made four andand seven days after planting. 2 Test = mayMON 89034;document Controlpublication, = conventional control corn (H1325023); Ref. Range = Minimum and It exploitation maximum values thisamong four replications of four references at each of three production sites (except H4242304 produced at anyIA and HEXP1184 produced at JR with three replications). permission The data in this table are the combined-siteprohibited results of the seeds from the three production sites. The results for seeds from each individualthe production site are included in Appendix E. commercial be Furthermore,any Consequently,without

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Table VII-4. Diameter and viability of pollen collected from MON 89034 and conventional control corn

Mean (SE)1 Reference Range3 Characteristic MON 89034 Control2 Minimum Maximum

Diameter (µm) 91.8 (1.29) 90.4 (0.18) 85.2 95.5

Viability (%) 96.8 (0.42) 96.4 (1.14) 90.5 99.0

1 SE = standard error. 2 and The control corn was H1325023 which has a genetic background similarand to MONregime. 89034. 3 References were three commercial corn hybrids:AG DKC61-42, RX740, and Burrus 576. Minimum and maximum mean diameter values of nine reference plants (three different reference corn × three replications). Each replication was a mean of ten viable pollen grains per plant. Minimum and maximum observed viabilitBayery valuesproperty were based onpublishing 45 reference plants protectioncontents (three reference corn × three replications ×of five plants per replication). its parties.data therefore and/oror

affiliates. VII.A.3.c. Evaluation of Phenotypicproperty aintellectualnd Agronomicthird Characteristicsmay under Field its Conditions the as of and owner. is regulatory a documentits Phenotypic and agronomic anycharacteristics such encompassing plant growth, development and reproductiondocument of yield characteristics were assessedowner under field conditions.this The purpose of these under of evaluations was to identifyrights anythe unintended changes tothis the phenotypic and agronomic documentand/or rights characteristics of MONto 89034 of relativefall to convuseentional of corn. In each study, reference corn hybrids were included to provide a range of valuesthe for the assessed characteristics This may distribution,and that are common to corn.rights In addition, certainowner growth, reproduction, and pre-harvest seed loss characteristicssubject (suc h as lodging and ear drop) can be used for an assessment of any the altered weedinessbe potential for MON 89034. Theviolate characteri stics assessed are well known copy of to experts familiar with corn breeding and agronomicand performance. may documentpublication, It exploitation Phenotypic andthis agronomic any data were collected from 18 field locations over two consecutive years: nine locations in 2004 and nine locations in 2005. Table VII-5 permission provides a list of the test site locations,prohibited soil description, cropping history, and planting information. These locationsthe provided a range of environmental and agronomic commercial be conditions representative of major U.S. corn-growing regions where commercial productionFurthermore, ofany MON 89034 would be expected. Plots were established at each of the field sitesConsequently, in a randomizedwithout complete block design with three replications. Each plot consisted of two to six rows of corn spaced approximately 30 inches apart and approximately 17.5- 20 ft in length.

The following 14 phenotypic and agronomic characteristics were evaluated during the 2004 and 2005 field trials: seedling vigor, early stand count, days to 50% pollen shed, days to 50% silking, stay green, ear height, plant height, dropped ears, stalk lodging, root lodging, final stand count, grain moisture, test weight, and yield. The evaluations and

Monsanto Company 06-CR-166U Page 120 of 262 CBI Deleted Version timing of plant assessments are already described in Table VII-1. The phenotypic and agronomic data were analyzed using SAS. Means were calculated across-sites (referred to as combined-site or across-site analysis, in which data were pooled from all test sites and analyzed statistically) for each characteristic, and MON 89034 was compared to the control. Differences were considered significant at the 5% level (p ≤ 0.05). The following is a summary of the results from the two years of phenotypic and agronomic evaluations. It is noted that only the combined-site data are presented in this section, while the results from individual sites are provided in Appendix F.

In the 2004 field trials, comparative assessments of phenotypic and agronomic characteristics were conducted on MON 89034 and a conventional control (H1325023). In addition, 23 commercially available corn hybrids were included asregime. referencesand to provide a range of values common to the crop. The 2004 fieldand trials included nine test AG sites or locations.

For the combined-site analyses, no significanBayert differences property were protection detectedpublishing between MON 89034 and the control corn for seedling ofvigor, early stand count, final standcontents count, days to its 50% pollen shed, days to 50% silking, stay green, earparties. height,data dropped ears,therefore root lodged and/or plants, grain moisture, test weight and yield (Table VII-6). Twoor differences were affiliates. detected between MON 89034property and the control,intellectualthird i.e., plant height wasmay lower for MON its 89034 compared to control (84.1 vs.as 85.4 inches), and MON 89034 had fewer stalk the of and lodged plants than the coisntrol (0.8 vs. 2.4) (Tableregulatory VII-6). owner. a document any such its reproductiondocument Although plant height for MON 89034owner was reducedthis compared ofto the control, the mean under of value observed for MONrights 89034the falls within the rangethis of values observed for the documentand/or rights commercial reference cornto hybridsof fall (Table VII-6use ). of Furthermore, the magnitude of the difference in plant height is small (approximately 1.5%),the and the difference was not detectedThis in 2005 trials (see belomayw). distribution, Therefore,and the slightly decreased plant height is rights owner unlikely to contributesubject to increased weediness potential. Likewise, the mean value for stalk lodged plants observed for MON 89034the fallsviolate within the range of values observed for be copy the commercial corn hybrids. Furthermorof e, fewer stalk lodged plants would be and agronomicallymay desirable,documentpublication, the difference was not detected in 2005 trials (see below), and It exploitation fewer stalk lodgedthis plants would not contribute to increased weediness potential. Therefore, the differencesany detected in plant height and in the number of stalk lodged permission plants during the 2004 field trialsprohibited are unlikely to be biologically meaningful in terms of increased weediness potential. the be commercial InFurthermore, the 2005 anyfield trials, two separate field studies were conducted with Study-1 involving fourConsequently, test sites andwithout Study-2 involving five test sites for a total of nine test sites. In the two studies, MON 89034 hybrids were bred to adapt to different environmental conditions (northern regions vs the Midwest). Accordingly, two different control hybrids were employed in the two studies, and each had a genetic background similar to the MON 89034 hyrbids, respectively.

In Study-1, the control corn was conventional corn hybrid DKC51-43. The background germplasm for MON 89034 hybrid and DKC51-43 hybrid used in this study was adapted

Monsanto Company 06-CR-166U Page 121 of 262 CBI Deleted Version for northern corn growing regions in the U.S. In addition, 12 commercially available corn hybrids were included as references to provide a range of values common to the crop. A total of 14 different phenotypic and agronomic characteristics were evaluated. In the combined-site analysis, no differences were detected between MON 89034 and the conventional control for any of the assessed phenotypic and agronomic characteristics (Table VII-7).

In Study-2, the control corn was conventional corn hybrid H1325023. In addition, 14 commercially available corn hybrids were included as references to provide a range of values common to the crop. A total of 14 different phenotypic and agronomic characteristics were evaluated. In the combined-site analysis, no differences were detected between MON 89034 and the control for any of the assessed phenotypic and regime.and agronomic characteristics (Table VII-8). and AG

The results of both 2004 and 2005 field trials demonstrate that the observed values of phenotypic and agronomic characteristicsBayer for MON property 89034 wereprotection withinpublishing the range of responses familiar to what is expectedof for corn. This supports a conclusioncontents that the its measured phenotypic and agronomic characteristics wereparties. notdata altered for MONtherefore 89034 as a result of the introduction of the lepidopteran-protection trait and/orand thor e expression of the affiliates. Cry1A.105 and Cry2Ab2 proteins,property and thereintellectual is thirdno altered pest potentialmay for MON 89034 its compared to the conventional corn. as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VII-5. Test site locations, planting information, soil description, and cropping history and and regime. AG 2004 field trials

Study Planting Planting Planting Plot Bayer Soil seriesproperty description; publishing2003 2002 protectioncontents Site1 Date Rate2 depth size Repsof3 organic matter (%); and pH crop crop its (mm/dd/yy) (seed/plot) (in) (ft) parties.data therefore and/oror affiliates. IA 05/29/04 35 1.5 15 x 20property 3 intellectual Siltythird clay loam; 3.9%; 6.8 may Soybean Soybean its IL-1 06/02/04 35 1.3 15 x 20 3as Silt loam; 2.0%; 6.3 Soybean Sorghum the of and IL-2 05/24/04 35 2.0 is 15 x 20 3 Siltyregulatory clay loam; 4.3%; 7.0 owner. Soybean Corn a document IN-DA 06/21/04 40 1.0 5 anyx 17.5 such 3 Crosby silt loam; 2.7%; 5.8its Corn Soybean reproductiondocument MN-GE 05/16/04 40 1.5 5 x 20 owner 3 Hamel loam;this 5.4%; 7.9of Soybean Corn MO-BE 05/17/04 40 1.0 5 x 20 3 Putnam silt loam; 1.8%; 6.9 Soybean Soybean under of this MO-CL 05/20/04 40 1.0 5rights x 20 the 3 Putnam silt loam; 1.0%; 6.1 Soybean Corn documentand/or of rights NE 05/21/04 35 1.5 to 15 x 20of fall 3 Siltuse loam; 3.0%; 6.8 Soybean Soybean OH 06/03/04 35 1.5 15 x 20 3 Loam; 2.2%;the 6.8 Non-crop Soybean This may distribution,and rights owner 1 Study sites: IA = Jefferson County, IA; IL-1subject = Jersey County, IL; IL-2 = Warren County, IL; IN-DA = Hendricks County, IN; MN-GE = be nd the violate Freeborn County, MN; MO-BE = Shelby County,copy MO; MO-CL = 2 siteof at Shelby County, MO; NE = York County, NE; OH = Fayette County, OH. and 2 document All plots were thinned to a uniform mayplant density afterpublication, seedlingexploitation vigor and early stand count data were collected at each site. 3 It Reps = replications. this any permission prohibited Table VII-5 continues on the next page. the commercial be Furthermore,any Consequently,without

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Table VII-5 (continued). Test site locations, planting information, soil description, and cropping history

and and regime. 2005 field trials, Study-1AG

Study Planting Plot size Rows/ Row Planting rate2 Planting Soil series description; plot spacing depthBayer (in) property publishing 2004 2003 Site1 Date (ft) (seed/row) organic matterprotection (%); and pH (#) of contents crop crop (mm/dd/yy) (in) its parties.data therefore MI 05/12/05 5 x 20 2 30 40 1.5 Nester loam;and/or 2.2%;or 6.5 Cabbage N/A3 affiliates. MN 05/17/05 5 x 20 2 30 40 property intellectual 1.5 third Linder sandy loam; may2.0%; 7.2 Corn Soybean its NY 05/20/05 5 x 20 2 30 40 as 1.5 Collamer silt loam; 4.0%; 6.0 Soybean Soybean WI 05/21/05 5 x 20 2 30 the 40 of 1.0and McHenry silt loam; 1-3%; 5.6-7.3 Soybean Corn is regulatory owner. a document any such its 1 reproductiondocument Study sites: MI = Ottawa County, MI; MN = Freeborn County, MN;owner NY = Wayne County,this NY; WIof = Walworth County, WI. 2 All plots at each site were thinned to a uniform plant density after seedling vigor and early stand count data were collected. under of this 3 N/A = Not available. rightsthe documentand/or of rights to of fall use the Table VII-5 continues on the next page. This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VII-5 (continued). Test site locations, planting information, soil description, and cropping history and and regime. AG 2005 field trials, Study-2

Bayer property publishing Planting protection Study Planting date Plot Rows/plot Planting rate of Soil series description; organic contents2004 2003 1 2,3 4 depth its site (mm/dd/yy) size (ft) (#) (seed/row) matter (%);parties. anddata pH thereforecrop crop (in) and/oror affiliates.Taintor silty clay loam; 3.9%;may IA 05/17/05 15 x 20 6 40 property 1.75 intellectualthird Sorghum Soybean its 6.8 as the of andSable silty clay loam; 4.3%; IL-N 05/17/05 15 x 20 6 is 40 2.0 regulatory owner.Soybean Corn a7.0 document any such its IL-S 06/17/05 5 x 17.5 2 40 2.5 Cisnereproduction silt loam;document 2.1%; 7.0 Corn Soybean owner this of MO 05/20/05 15 x 20 6 40 1.0under Putnamof silt loam; 1.8%; 6.9 Soybean Corn rightsthe this NE 05/16/05 15 x 20 6document and/or 40 2.0 Hastings silt loam;rights 3.0%; 6.8 Corn Soybean to of fall use of 1 Study sites: IA = Jefferson County, IA; IL-N -= Warren County, IL; IL-S = Clinton County,the IL; MO = Shelby County, MO; NE = York County, NE This may distribution,and rights owner 2 The IL-S site was originally planted on Maysubject 20, 2005 with six-row plots. Dry conditions at planting, however, resulted in poor seedling emergence across all plots. The study areabe was destroyed and a new studythe area wasviolate established on June 17, 2005 with two-row plots. 3 All rows were spaced 30 inches apart. copy of 4 All plots at each site were thinned to a uniform plant density after seedlingand vigor and early stand count data were collected. may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VII-6. Phenotypic and agronomic comparison of MON 89034 to the control in the combined-site analysis for 2004 field trials and and regime. AG References Phenotypic and agronomic Range1 99% Tolerance interval2 MON 89034 Control characteristic (units) Min. Max. Lower limit Upper limit Bayer property protectionpublishing Seedling vigor 6.9 6.9 of 4.3 9.0 1.7 contents 9.0 Early stand count (#/plot) 68.3 67.8 58.3 78.7 49.5 its 89.4 parties.data therefore Days to 50% pollen shed 61.9 61.5 55.3 74.7 and/or44.8or 81.6 affiliates. property intellectual52.0third 74.7 40.8 may 83.1 Days to 50% silking 60.8 60.4 its as Stay green 6.0 the 5.8 of and4.7 7.7 3.1 8.4 is regulatory owner. a document Ear height (in) 39.9 any40.5 such 34.6 54.6 27.2its 59.9 reproductiondocument of Plant height (in) 84.1* 85.4 owner 76.8 this106.6 67.0 120.4 under of this Dropped ears (#/plot) 0.1 and/orrights 0.2 the 0.0 1.0 0.0 1.2 document to of fall of rights Stalk lodged plants (#/plot) 0.8* 2.4 0.0use 6.0 0.0 6.7 the Root lodged plants (#/plot) This 1.0 0.5may distribution,and0.0 27.0 0.0 28.0 rights owner Final stand count (#/plot) 60.5subject 60.5 53.3 66.3 50.9 73.2 be the violate Grain moisture (%) 23.4 copy 22.9 of 12.6 41.7 0.0 47.7 and Test weight (lb/bu) may 52.6 documentpublication, 53.3 42.8 57.7 39.4 67.6 It exploitation 92.8 290.8 17.9 373.5 Yield (bu/ac) 192.9this any 191.3 1 permission Reference range: minimum and maximum mean values amongprohibited the 23 reference corn. 2 99% tolerance interval with 95% confidence. the be * Indicates a statistically significant differencecommercial between the test and control at p ≤ 0.05. Furthermore,any Consequently,without

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Table VII-7. Phenotypic and agronomic comparison of MON 89034 to the control in combined-site analysis for Study-1 of 2005 trials and and regime. AG References MON 89034 Control Range1 99% Tolerance Interval2 Phenotypic and agronomic Mean Mean Min. Max. Lower limt Upper limit characteristic (units) Bayer property protectionpublishing Seedling vigor 6.7 6.6 of 5.0 8.0 contents 1.8 9.0 its Early stand count (#/plot) 77.1 77.4 66.7parties. data 82.3 therefore 55.4 95.8 Days to 50% pollen shed 68.0 67.6 64.3 and/or 71.0 or 56.7 79.2 affiliates. Days to 50% silking 66.9 property 66.3 intellectual third 62.0 71.0 may 52.8 80.5 Stay green 6.3 its6.2 3.7 9.0 0.0 9.0 the as Ear height (in) 41.9 of 42.1 and 38.0 59.9 owner. 13.9 77.8 is regulatory Plant height (in) 91.6 89.8 a 82.0 document112.3 43.8 142.2 any such its Dropped ears (#/plot) 0.0 0.1 reproduction 0.0 document 0.3of 0.0 0.4 Stalk lodged plants (#/plot) 0.1 0.3owner 0.0this 2.3 0.0 3.4 under of Root lodged plants (#/plot) 0.0 rightsthe 0.0 0.0 this 0.0 nv nv documentand/or rights Final stand count (#/plot) 61.3 to of 61.8fall use 59.0of 64.0 53.5 69.7 Grain moisture (%) 18.9 18.6 17.3the 22.2 12.7 25.1 This may distribution,and Test weight (lb/bu) 55.9 rights 56.1 owner 54.0 57.0 52.0 59.4 Yield (bu/ac) subject205.5 195.1 171.0 220.0 124.4 258.5 be the violate 1 Reference range: minimum and maximumcopy valu es among the 12 ofindividual reference means. 2 99% tolerance interval with 95% confidence.document and may publication,exploitation No statistically significant differencesIt were detected between MON 89034 and the control at p ≤ 0.05. this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VII-8. Phenotypic and agronomic comparison of MON 89034 to the control in the combined-site analysis for Study-2 of 2005 trials and and regime. AG References MON 89034 Control Range1 99% Tolerance Interval2 Phenotypic and agronomic Mean Mean Bayer Min. property Max. protection publishing Lower limit Upper limit of contents characteristic (units) its parties.data therefore Seedling vigor 6.4 6.3 5.7 8.0and/or or 3.7 9.0 Early stand count (#/plot) 69.4 72.3 affiliates. 67.0 78.7 63.1 88.3 property intellectualthird may Days to 50% pollen shed 60.2 59.6its 52.3 67.0 44.3 79.7 as Days to 50% silking 60.9 the 60.2of and 52.3 69.0 42.5 82.3 is regulatory owner. Stay green 5.0 5.2 a 3.0 document 7.0 0.0 9.0 any such its Ear height (in) 38.0 39.4 32.2reproduction 50.3document of 23.6 59.8 Plant height (in) 80.3 82.0 owner 77.1 this 99.1 60.3 120.1 under of Dropped ears (#/plot) 1.4 rightsthe 0.9 0.0 this 2.0 0.0 3.0 documentand/or rights Stalk lodged plants (#/plot) 9.6 to of 5.4fall use 0.0 of 49.0 0.0 66.6 Root lodged plants (#/plot) 0.6 0.6 0.0 the 7.3 0.0 12.2 This may distribution,and Final stand count (#/plot) 58.9 rights 59.8 owner 52.7 63.0 48.8 71.2 Grain moisture (%) subject 19.6 19.4 16.3 25.8 7.0 33.5 be the violate Test weight (lb/bu) 56.0copy 55.7 of 49.1 63.7 40.5 71.8 Yield (bu/ac) 126.8document 125.7 and 31.7 203.5 0.0 374.8 may publication,exploitation 1 Reference range = Minimum and Itmaximum values among the 14 individual reference means. this 2 99% tolerance interval with 95% confidence. any

No statistically significant differences were detected betweenpermission MON 89034 and the control at p≤0.05. prohibited the commercial be Furthermore,any Consequently,without

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VII.A.3.d. Evaluation of Ecological Interactions

In the two years of field trials for evaluation of phenotypic and agronomic characteristics of MON 89034, observational data on the presence of and differential response to biotic (insects, diseases) and abiotic (drought, wind, nutrient deficiency etc.) stressors were also collected to examine the ecological interactions of MON 89034 compared with those of the conventional control corn. The observed stressors were “natural” (i.e., no artificial infestation or interference was used). Therefore, the same stressors were not necessarily observed at each field site.

Ecological interactions were assessed qualitatively. Observation of plant interactions with insect pests and diseases, and plant responses to abiotic stressors were collectedand and regime. from each of the 18 field locations in 2004 and 2005.AG The purpose of these evaluations was to assess whether plant-insect or plant-disease interactions, or plant response to abiotic stressors of MON 89034 were altered compared to the conventional control corn. For the plant-insect interactions, plant-diseasBayere interactions, property and plant responsespublishing to abiotic protectioncontents stressors, the reported values representof th e range of ratings observed across the three its replications at each site. MON 89034 and the controlparties. datawere consideredtherefore qualitatively and/oror different in response to a stressor if theaffiliates. ratings between MON 89034 and the control corn did not overlap across all threeproperty replications intellectual for thirdthat particular stressor may(e.g. “none” rating its as vs “slight-moderate” rating).the ofThe ratingsand observed among the commercial reference is regulatory owner. hybrids provide qualitative assessment dataa common documentto the crop for each stressor assessed. any such its reproductiondocument of owner this under of In the 2004 field trials, rightsacrossthe the nine field locations,this 11 insect and other animal documentand/or rights categories (species or group),to of 12 diseasefall categoriesuse of(species or group), and eight abiotic stressor categories were evaluated (Table VII-9). Forthe the plant-insect or plant-other This may distribution,and animal interactions, norights qualitative differencesowner were observed between MON 89034 and the control amongsubject the 37 * plant-insect or plant-other animal interactions evaluated (Table VII-9). It shouldbe be noted that in these fitheeld trials,violate efficacy testing of MON 89034 was copy of not the objective. The entire plot area atand each location would have been treated with insecticidesmay if needed,document aspublication, indicated by the insect pressure in the control and reference It exploitation plots observed. Thus, it is not unexpected when no difference was observed for the plant this any response to lepidopteran insects like corn borer between MON 89034 and the control permission corn. For plant-disease interaction,prohibited no qualitative differences were observed between MON 89034 and the controlthe be for 52 out of the 54 interactions assessed (Table VII-9). At the IA and IN-DAcommercial locations a slight incidence of ear rot was observed in the control and noFurthermore, ear rot wasany observed in MON 89034. This difference was not observed at the other 7 locationsConsequently, wherewithout ear rot data were recorded.

For the plant-abiotic stressor interactions, no qualitative differences were observed between MON 89034 and the control for 35 out of the 38 interactions assessed (Table VII-9). At the seedling stage at MO-BE, excess moisture stress was not observed in

* The number of interactions is on a per stressor/test location/plant growth stage basis. For example, aphids were assessed at the IA and MO-BE sites, both during the reproductive stage. This was counted as two interactions.

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MON 89034, while a slight incidence of excess moisture stress was observed in the control. Excess moisture stress was assessed at various growth stages at four sites, but differences between MON 89034 and the control were only observed at MO-BE. This qualitative difference was likely due to the lack of uniformity of moisture (i.e., standing water) across plots at MO-BE. Wind damage was assessed at various growth stages at six sites, but differences between MON 89034 and the control were only observed at IL-2 and MO-BE. At IL-2, during harvest, wind damage was not observed in MON 89034, while slight wind damage was observed in the control. At the reproductive stage at MO- BE, slight wind damage was observed in MON 89034, while no wind damage was observed in the control. This qualitative difference was not considered biologically meaningful since there was no consistent trend observed between MON 89034 and the control regarding to the response to wind damage across sites where differences were regime.and observed. Furthermore, no differences were observed betweenand MON 89034 and the AG control at four additional sites where wind damage was assessed (Table VII-9).

In Study-1 of the 2005 field trials, a totalBayer of 6 insectproperty categories protection (speciespublishing or group), 8 disease categories (species or group), andof 7 abiotic stressors were evaluatedcontents (Table VII- its 10). Within these categories, 16 plant-insect interactions,parties.data 24 plant-diseasetherefore interactions, 16 plant responses to abiotic stressors were evaluated. For theand/or plant-disor ease interactions affiliates. or plant-abiotic stressor interactions,property no intellectualqualitativethird differences were mayobserved in MON its 89034 plant responses compared to theas control (Table VII-10). For the 16 plant-insect the of and interactions evaluated, fisour qualitative differencesregulatory were observed betweenowner. MON 89034 a document and the control for corn borerany andsuch corn earworm damage (Table VII-10its ). Specifically, reproductiondocument corn borer and corn earworm damagesowner were not observedthis on MONof 89034 while slight damage was observed in the control atunder MI an d WI.of This was expected because of the rightsthe this introduced lepidopteran-protectiondocumentand/or trait for MON 89034. Ifrights there was lepidopteran insect to of fall use of pressure (such as corn borer or corn earworm) and theif insecticide application was not conductedThis in a timely fashion,may one distribution,wouldand expect to see the difference between MON rights owner 89034 (resistantsubject to lepidopteran insects) and the control corn (susceptible to lepidopteran insects). the violate be copy of and In Study-2may of 2005 documentfieldpublication, trials, a total of 9 insect and other animal categories (species or It exploitation group), 7 diseasethis categories (species or group), and 5 abiotic stressors were evaluated (Table VII-11). Withinany these categories, 21 plant-insect and other animal interactions, permission 30 plant-disease interactions andprohibited 19 plant responses to abiotic stressors were evaluated and, no qualitative differences were observed in the ecological interactions of MON the be 89034 comparedcommercial to those of the control (Table VII-11). Furthermore,any In summary,Consequently, basedwithout on 255 comparative observations recorded over 2 years, no consistent differences were observed across sites between MON 89034 and the control in their susceptibility or tolerance to the ecological stressors assessed during 2004 and 2005 field trials. These results support the conclusion that compared to the conventional corn, the ecological interactions between MON 89034 and insects, diseases, and abiotic stressors were not altered except for the introduced lepidopteran-protection trait.

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Table VII-9. Ecological stressor incidence of MON 89034, the control, and references in 2004 field trials

Seedling Vegetative Reproductive Harvest 4 and Stressors Sites Test Contr. Ref. Test Contr. Ref. Test andContr. Ref.regime. Test Contr. Ref. Insects AG Aphids IA — — — — — — NO NO NO-MO — — — MO-BE — — — — — — SL NO-SL NO-SL — — — 1 Armyworm IL-1 — — — — — Bayer— propertyNO protectionNO publishingNO — — — IL-2 NO NO NO — — of — — — —contents — — — MO-CL — — — NO-SL NO NO-SL — — its — NO NO NO 2 parties.data therefore Corn borer IL-1 — — — — — — — and/or— or — NO NO NO-SL IL-2 NO NO NO NO NO NO — — — NO-SL SL NO-SL affiliates. may MN-GE NO NO NO NOproperty NO-SL intellectual NOthird NO NO-SL NO-SL NO-SL NO-SL NO-SL its MO-BE — — — NO-SL NOas NO-SL — — — — — — the of and NE — — — is NO NO NOregulatory — — owner.— NO NO-MO NO-MO a document OH — — — anyNO suchNO NO NO NOits NO NO NO NO reproductiondocument Corn earworm IN-DA — — — — owner— — this— —of — NO-SL SL SL Corn rootworm IN-DA SL-MO SL-MO SL-MO SL-MO SL-MO SL-MO SL SL SL-MO — — — under of this NE — — —and/or rights — the — — NO NO NO — — — 3 document rights Cutworms IA NO NO NO to —of fall— use— of — — — — — — IN-DA NO NO NO — — — the— — — — — — MO-BE NO-SL ThisNO-SL NO-SL —may distribution,— and — — — — — — — rights owner NE NO NO subjectNO — — — — — — — — — Flea beetles MO-CL NO NO-SL NO-SL — — — — — — — — — be the violate OH NO NO copyNO — —of — — — — — — — Grasshoppers IA — — — NO NO-SLand NO-SL — — — — — — may documentpublication, MO-BE — It — — —exploitation — — — — — NO NO-SL NO-SL MO-CL — — — — — — NO NO NO — — — this any Japanese beetles IL-1 — — — NO NO NO — — — — — — permission Wireworms IL-1 NO NO NO — prohibited— — — — — — — — Seedling, vegetative, reproductive, and harvest refer to different growth stages. Test = MON 89034; Contr. = Control corn; Ref. = Reference corn; NO = None; the SL = Slight; MO = Moderate; SE = Severe. Dash commercial(—) denotes no be observations taken. The severity of stressor symptoms per plot were rated using the following scale: 0 = none (no symptoms observed), 1–3 = slight (symptoms observed but not detrimental to plant growth and development), 4–6 = moderate (intermediate between slight and severe), and Furthermore,7–9 = severeany (symptoms observed and detrimental to plant growth and development). 1 Including fall armyworm. 2 Including European corn borer. 3 Including blackConsequently, cutworm. without4 Refer to Table VII-5 for test site descriptions. Table VII-9 continues on next page.

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Table VII-9 (continued). Ecological stressor incidence of MON 89034, the control, and references in 2004 field trials

Seedling Vegetative Reproductive regime.and Harvest Stressors Sites Test Contr. Ref. Test Contr. Ref. Test andContr. Ref. Test Contr. Ref. Other animals AG Rodents IL-1 NO-SL NO-MO NO-MO — — — — — — — — — Diseases Bayer property publishing Anthracnose MO-CL — — — NO NO NO — protection— —contents — — — 5 of Ear rot IA — — — — — — — — its — NO SL NO-MO IL-1 — — — — — — parties.— data — —therefore NO-SL SL SL-MO and/oror IL-2 — — — — affiliates.— — — — — NO NO-SL NO-SL property intellectualthird may IN-DA — — — — its — — — — — NO SL SL as MN-GE — — — the— of — and— — — — NO NO NO is regulatory owner. MO-BE — — — — — a— —document — — SL NO-SL NO-SL any such its MO-CL — — — — — — reproduction— document — — NO-SL SL NO-SL owner this of NE — — — — — — — — — NO NO NO under of this OH — — — rights— the — — — — — NO NO NO-SL documentand/or of rights Eyespot MO-CL — — — to —of fall— use— NO-SL NO-SL NO-SL — — — Fusarium MN-GE NO NO NO — — — the— — — — — — This may distribution,and Gray leaf spot IL-2 — — — rightsNO NO ownerNO SL SL SL — — — IN-DA — — subject— — — — — — — SL SL SL MO-BE — — be — NO-SL NO-SLthe NO-SLviolate NO-SL NO-SL NO-SL — — — copy of MO-CL — — — — — — — — — SL SL SL document and Leaf blight IA — may— — publication,SL NO-SL SL — — — — — — It exploitation IN-DA — — — — — — SL SL SL — — — this any MN-GE — — — — — — — — — NO-SL NO-SL NO-SL permission NE — — — — prohibited— — NO NO NO — — — OH — — — the — — — — — — NO NO NO commercial be 5 Ear rot data collected on 5 plants/plot at all sites. Table VII-9 continues on next page. Furthermore,any Consequently,without

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Table VII-9 (continued). Ecological stressor incidence of MON 89034, the control, and references in 2004 field trials

Seedling Vegetative Reproductive regime.and Harvest Stressors Sites Test Contr. Ref. Test Contr. Ref. Test andContr. Ref. Test Contr. Ref. Diseases AG Pythium IA NO NO NO — — — — — — — — — IL-2 NO NO NO — — — — — — — — — Bayer property protectionpublishing IN-DA NO NO-SL NO-SL — — of — — — —contents — — — MO-BE NO-SL SL NO-SL — — — — — its — — — — parties.data therefore Root rot IL-1 NO NO NO — — — — and/or— or — — — — Rust IA — — — — affiliates.— — NO NO NO-SL SL-SE SL-SE SL-MO property intellectualthird may IL-1 — — — SL its SL SL — — — — — — as IL-2 — — — the— of — and— SL SL SL — — — is regulatory owner. IN-DA — — — SL SL aSL-MO —document — — — — — any such its MN-GE — — — NO NO NO-SLreproduction SL document SL SL SL SL SL owner this of MO-BE — — — — — — — — — SL-MO MO SL-MO under of this MO-CL — — —and/or rights — the — — — — — SL-MO SL-MO SL-MO document fall of rights NE — — — to NOof NO useNO — — — — — — OH — — — NO NO-SL NO-SL theNO NO-SL NO-SL — — — This may distribution,and Seedling blight MO-CL NO NO NO rights— — owner— — — — — — — subject OH NO NO NO — — — — — — — — — be the violate copy of Table VII-9 continues on next page. and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VII-9 (continued). Ecological stressor incidence of MON 89034, the control, and references in 2004 field trials

Seedling Vegetative Reproductive regime.and Harvest Stressors Sites Test Contr. Ref. Test Contr. Ref. Test andContr. Ref. Test Contr. Ref. Diseases AG Stalk rot6 IA — — — — — — — — — NO-SL NO-SL NO-SL IL-1 — — — — — — — — — NO-SL NO-SL NO-SL Bayer property protectionpublishing IL-2 — — — — — of — — — —contents NO-SL SL-MO SL-MO IN-DA — — — — — — — — its — NO NO NO parties.data therefore MN-GE — — — — — — — and/or— or — NO-SL NO-SL NO-SL MO-BE — — — — affiliates.— — — — — NO-SL SL NO-SL property intellectualthird may MO-CL — — — — its — — — — — SL SL-MO SL as NE — — — the— of — and— — — — NO NO NO is regulatory owner. a document OH — — — any— such— — — — its — SL SL SL reproductiondocument Stewart’s wilt IL-1 — — — — owner— — thisNO NOof NO — — — NE NO NO NO — — — — — — — — — under of this Abiotic rights the documentand/or of rights Cold weather MO-CL NO NO NO to —of fall— use— — — — — — — Compaction OH NO NO NO NO NO-SL NO-SL the— — — — — — Drought-Heat IA — This— — —may distribution,— and — NO NO NO — — — rights owner IL-1 — — subject— NO NO NO NO NO NO — — — IL-2 NO NObe NO NO NO the NOviolate — — — — — — IN-DA — — copy— — —of — NO NO NO — — — MO-CL — — — — — and— NO NO NO — — — may documentpublication, OH — It — — —exploitation — — NO NO NO — — — Frost IN-DA — — this —any — — — — — — MO MO MO Hail IL-1 NO NO NO — — — — — — — — — permission NE NO NO NO NO prohibitedNO NO NO NO NO NO NO NO 6 the Stalk rot data collected on 5 plants/plot at all sites.commercial The ranges beof the mean plot ratings are presented in this table. At IL-1, IL-2, and NE, stalk rot data were collected both on a per plot basis and on 5 plants/plot. For these sites, the mean ratings from the 5 plants/plot were combined with the overall plot ratings and the ranges are presented in thisFurthermore, table. any Table VII-9 continues on next page. Consequently,without

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Table VII-9 (continued). Ecological stressor incidence of MON 89034, the control, and references in 2004 field trials

Seedling Vegetative Reproductive regime.and Harvest Stressors Sites Test Contr. Ref. Test Contr. Ref. Test andContr. Ref. Test Contr. Ref. Abiotic AG Nutrient MN-GE NO NO NO — — — SL SL SL NO-SL NO-SL NO-SL Excess moisture IL-2 NO NO NO — — — — — — — — — Bayer property protectionpublishing IN-DA — — — SL NO-SLof NO-SL — — —contents — — — MN-GE NO NO NO NO-SL NO NO-SL — — — — — — its MO-BE NO SL NO-SL NO NO NO-SL parties.NO data NO NO-SLtherefore — — — and/or Wind IA SL SL SL NO NO NO-SL — — or — NO NO NO affiliates. IL-1 NO NO NO — property— intellectual—third — — may— NO NO NO its IL-2 — — — — —as — — — — NO SL SL-MO the of and MO-BE — — — is — — —regulatory SL NO owner.NO-SL NO-SL NO NO-SL a document MO-CL — — — anyNO suchNO NO — — its — NO NO NO reproductiondocument OH — — — — owner— — this— —of — NO NO NO under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VII-10. Ecological stressor incidence of MON 89034, the control, and references in Study-1 of 2005 trials

Seedling Vegetative Reproductive regime.and Harvest Stressors Sites3 and Test Contr. Ref. Test Contr. Ref. AGTest Contr. Ref. Test Contr. Ref. Insects Corn borer1 MI — — — NO SL NO-SL NO SL SL — — — Bayer MN — — — NO NO NO NOproperty NO protectionNOpublishing NO NO NO of contents NY — — — — — — — — — NO NO NO its WI — — — NO SL NO-SL —parties. data — — therefore NO NO-SL NO-SL and/or Corn or affiliates. earworm MI — — — — property— intellectual— third — — —may NO SL SL its Corn as rootworm NY NO NO NO —the of — and— NO NO NO — — — is regulatory owner. WI NO NO NO — — —a — document— — — — — any such its 2 reproductiondocument Cutworm MI NO NO NO — —owner — —this — of — — — — MN NO NO NO — — — of— — — — — — rights under this Grasshopper WI — — — and/or— the— — NO NO NO — — — document to of fall of rights Stalk borer NY — — — NO NO NOuse — — — — — — the Seedling, vegetative, reproductive, and harvestThis refer to different growthmay states.distribution, and Test = MON 89034; Contr. = Control; Ref. = References; NO = none; SL = rights owner slight; MO = moderate; SE = severe. Dashes (—)subject indicate no observations taken. The severity of stressor symptoms per plot were rated using the following scale: 0 = none (no symptoms observed), 1–3 = slight (symptoms observed but not detrimental to plant growth and development), 4–6 = moderate (intermediate be the violate between slight and severe), and 7–9 = severe (symptomscopy observed and detrimentalof to plant growth and development). 1 Includes corn borer and European corn borer. and 2 document Includes cutworm and black cutworm. may publication,exploitation 3 It Refer to Table VII-5 for test site descriptions. this any Table VII-10 continues on next page. permission prohibited the commercial be Furthermore,any Consequently,without

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Table VII-10 (continued). Ecological stressor incidence of MON 89034, the control, and references in Study-1 of 2005 trials

Seedling Vegetative Reproductive regime.and Harvest Stressors Sites and Test Contr. Ref. Test Contr. Ref. AG Test Contr. Ref. Test Contr. Ref. Diseases Ear rot4 MI — — — — — — — — — NO-SL NO-SL NO-SL Bayer property protectionpublishing MN — — — — — of — — — — contentsNO-SL NO-SL NO-SL NY — — — — — — — — —its NO NO NO parties.data therefore WI — — — — — — — —and/or or — NO-SL NO-SL NO-SL Eyespot WI — — — — — affiliates.— NO NO NO — — — property intellectualthird may Northern leaf its blight NY — — — NO NO as NO NO NO NO NO NO NO the of and Pythium MI NO NO NO is — — — regulatory— — —owner. — — — a document NY NO NO NO —any such— — — — its— — — — reproductiondocument of Rust5 MI — — — NO NOowner NO NOthis NO NO — — — under of MN — — — NOrights the NO NO SL this SL NO-SL SL SL SL documentand/or rights WI NO NO NO to — of —fall — use —of — — — — — Seedling the blight MN NO ThisNO NO — may —distribution, and— — — — — — — rights owner 4 Ear rot data collected from 5 plants/plot at each site.subject The ranges of the individual plant ratings at each site are presented in this table. 5 Includes rust and leaf rust. the violate be copy of and may documentpublication, Table VII-10 continues on next page. It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VII-10 (continued). Ecological stressor incidence of MON 89034, the control, and references in Study-1 of 2005 trials

Seedling Vegetative Reproductive regime.and Harvest Stressors Sites and Test Contr. Ref. Test Contr. Ref. AGTest Contr. Ref. Test Contr. Ref. Diseases Smut MI — — — — — — — — — NO NO NO-SL WI — — — NO NO BayerNO —property — protection —publishing NO-SL NO NO-SL of contents Stalk rot6 MI — — — — — — — — — NO-SL NO-SL NO-SL its MN — — — — — — —parties. data — — thereforeNO-SL NO-MO NO-MO and/oror NY — — — — — — — — — NO NO NO affiliates. WI — — — — property— intellectual— third — — —may NO-MO NO-SL NO-SL its Abiotic as the of and Cold stress NY NO NO NO is — — — regulatory— — —owner. — — — a document Drought7 MI — — — NOany suchNO NO NO NO itsNO — — — reproductiondocument of NY — — — — —owner — NOthis NO NO NO NO NO under of WI NO NO NO NOrights the NO NO — this — — NO-SL NO-SL NO-SL documentand/or rights Frost MI — — — to — of —fall — use —of — — NO NO NO Heat stress NY — — — NO NO NO — the — — — — — Herbicide This may distribution,and rights owner injury MI NO-SL NO NO-SLsubject — — — — — — — — — MN NO NO be NO-SL — — the— violate— — — — — — copy of Nutrient 8 document and deficiency MN — may — — publication, NOexploitation NO NO SL SL SL SL SL SL Wind WI — It — — — — — NO NO NO — — — this any 6 Data collected from 5 plants/plot at each site. The ranges of the individual plant ratings at each site are presented in this table. 7 permission Includes drought and drought/heat. prohibited 8 Includes nutrient deficiency and nitrogen deficiency. the commercial be Furthermore,any Consequently,without

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Table VII-11. Ecological stressor incidence of MON 89034, the control, and references in Study-2 of 2005 trials

Seedling Vegetative Reproductive regime.and Harvest 3 and Stressors Sites Test Contr Ref Test Contr Ref AG Test Contr Ref Test Contr Ref Insects Aphids IL-S — — — — — — MO MO MO-SE NO-SL NO NO-SL MO — — — — — Bayer— propertySL protectionSL publishing SL — — — Black of contents cutworm IA NO-SL SL NO-SL — — — — — its — — — — parties.data therefore IL-N NO NO NO — — — — and/or— or — — — — MO NO-SL NO-SL NO-SL — —affiliates. — — — — — — — property intellectualthird may NE NO NO NO — its — — — — — — — — as 1 the of and Corn borer IA — — — is — — —regulatory — — owner.— SL SL-MO SL-SE a document IL-N — — — anySL suchSL SL SL SL its NO-SL SL SL-MO NO-MO reproductiondocument MO — — — NO-SL NO-SLowner NO-SL this— of— — — — — NE — — — NO NOunder NO of — — — — — — rightsthe this Corn documentand/or of rights earworm MO — — — to —of fall— use— — — — SL-MO MO SL-MO Corn the 2 This may distribution,and rootworm IA — — — rights— — owner— NO-SL NO-SL NO-SL — — — NE — — subject— — — — NO NO NO-SL NO NO NO the violate Grasshopper IL-S — — be copy— — — — NO-MO NO-SL NO-MO — — — Japanese of beetle IL-S MO SL-MO SL-MOdocument — — and — — — — — — — may publication,exploitation Leaf hopper IA — It — — SL SL NO-SL — — — — — — this any Seedling, vegetative, reproductive, and harvest refer to different growth stages. Contr = Control; Ref = References; NO = none; SL = slight; MO = moderate; SE permission = severe. Dashes (—) indicate no observations taken on stressor. Theprohibited severity of stressor symptoms per plot were rated using the following scale: 0 = none (no symptoms observed), 1–3 = slight (symptoms observed butthe not detrimental to plant growth and development), 4–6 = moderate (intermediate between slight and severe), and 7–9 = severe (symptoms observed andcommercial detrimental to beplant growth and development). 1 Including European corn borer. 2 Including corn rootworm adults.Furthermore, any 3 Refer to Table VII-5 for test site descriptions.Consequently, without Table VII-11 continues on next page.

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Table VII-11 (continued). Ecological stressor incidence of MON 89034, the control, and references in Study-2 of 2005 trials Seedling Vegetative Reproductive Harvest and Stressors Sites Test Contr Ref Test Contr Ref Test andContr Refregime. Test Contr Ref Diseases AG Anthracnose IA — — — NO NO-SL NO-SL SL SL SL SL-MO SL SL-MO IL-S — — — — — — — — — SE MO-SE MO-SE Bayer property protectionpublishing MO — — — SL SLof SL — — contents— — — — Blight4 IA NO-SL NO-SL NO-SL its parties.data therefore IL-N NO NO NO — — — — and/or— or — — — — affiliates. IL-S NO NO NO —property — intellectual—third SL SL-MO maySL-MO — — — its NE — — — — —as — NO NO NO — — — 5 the of and Ear/kernel rot IA — — — is — — —regulatory — — owner. — NO NO-SL NO-SL a document IL-N — — — any— such — — — — its — NO-SL NO-MO NO-MO reproductiondocument IL-S — — — — owner — — this — of — — NO-SE NO-MO NO-SE under of MO — — — rights— the — — this — — — NO-SE SL-SE SL-SE NE — —document and/or— — — — — rights — — NO NO NO to of fall use of Fusarium MO NO-SL NO-SL NO — — — the— — — — — — NE NO ThisNO NO NOmay distribution, NO and NO — — — — — — rights owner Gray leaf spot IA — — subject — — — — — — — MO MO MO-SE IL-N — — — — — the violate — NO NO NO SL SL SL be copy MO — — — — —of — SL SL SL SL-MO MO SL-MO document and NE — may — — publication,exploitation— — — — — — SL SL SL-MO Rust IL-N —It — — SL SL SL — — — — — — this any Stalk rot5 IA — — — — — — — — — NO-SL NO-SE NO-SL permission IL-N — — — — prohibited — — — — — NO-MO NO-SE NO-MO IL-S — — — the — — — — — — SL-SE SL-SE SL-SE commercial be MO — — — — — — — — — NO-SL NO-SL NO-MO NE Furthermore, — any— — — — — — — — NO-SL NO-SL NO-SL 4 Including seedling blight, pythium,Consequently, southern leafwithout blight, and northern corn leaf blight. 5 Data collected on 5 plants/plot at each site. The ranges of the individual plant ratings are presented in this table. Table VII-11 continues on next page.

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Table VII-11 (continued). Ecological stressor incidence of MON 89034, the control, and references in Study-2 of 2005 trials

Seedling Vegetative Reproductive regime. and Harvest Stressors Sites and Test Contr Ref Test Contr RefAG Test Contr Ref Test Contr Ref Abiotic Drought6 IA — — — NO NO NO SL SL SL-NO MO MO MO-SE IL-N NO NO NO MO MO BayerMO property MO MOpublishing MO — — — protectioncontents IL-S MO-SE MO-SE MO-SE — —of — SE MO-SE SE — — — its MO — — — MO-SE MO-SE MO-SE parties. SEdata MO-SE MO-SEtherefore MO-SE MO-SE MO-SE and/or NE — — — — — — NO NOor NO NO NO NO affiliates. Nutrient property intellectualthird may its deficiency IL-N — — — — as— — — — — NO NO NO Soil the of and is regulatory owner. compaction NE SL SL SL SL SL a SL document — — — — — — any such its Wet soil MO SL SL NO-MO — — —reproduction —document — — — — — owner this of Wind IA SL SL SL — under— —of — — — — — — rightsthe this IL-S — — documentand/or— — — — of —rights — — MO-SE SL-SE MO-SE Other to of fall use Deer the This may distribution,and damage NE — — — rights — — owner— — — — NO NO-SL NO-SE subject the 6 be violate Including drought/heat. copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 141 of 262 CBI Deleted Version

VII.B. Compositional Assessment of MON 89034

Compositional analyses were conducted to assess whether the nutrient, anti-nutrient and secondary metabolite levels in the grain and forage tissues derived from MON 89034 are comparable to those in the conventional control corn which has a genetic background similar to MON 89034. Additional conventional corn hybrids (references) currently in the marketplace were also included in the analysis to establish a range of natural variability for each analyte, where the range of variability is defined by a 99% tolerance interval for that particular analyte. Results of the comparisons indicate that MON 89034 is compositionally and nutritionally equivalent to conventional corn hybrids currently in commerce. regime.and and AG In 2004, grain and forage tissues of MON 89034 and conventional control corn were harvested from plants grown in three replicated plots at each of five field sites. The field sites were located in regions of the U.S. Bayerthat are conduciveproperty toprotection the growthpublishing of corn, and representative of commercial corn production.of The five test sites were: Sitecontents 1-Jefferson its County, IA; Site 2-Jersey County, IL; Site 3-Warrenparties. County,data IL; Site 4-Yorktherefore County, NE; and Site 5-Fayette County, OH. Fifteen conventionaland/or cornor hybrids were also affiliates. included as references by growingproperty three differeintellectualthirdnt hybrids at each of fivemay sites for a total its of 15 references. These reference cornas hybrids were included to provide data for the the of and development of a 99% toleranceis interval for eachregulatory component analyzed.owner. This tolerance a document interval is expected to contain,any withsuch 95% confidence, 99% of the itsvalues obtained from reproductiondocument the population of commercial corns.owner It is importantthis to estaboflish the 99% tolerance interval from representative conventionalunder corn hybridsof for each of the analytes, because rightsthe this such data illustratedocument and/orthe compositional variability that naturallyrights occurs in commercially to of fall use of grown varieties. By comparison to the 99% tolerancethe interval, any statistically significantThis difference betweenmay MON distribution, 89034and and the control may be put into perspective, rights owner and can be assessedsubject for biological relevance in the context of the natural variability in corn. the violate be copy of and The compositionalmay documentanalysespublication, were conducted on a total of 77 components - nine in forage It exploitation and 68 in grain.this Components were selected based on recommendations from the OECD (OECD, 2002). Compositionalany analyses of the forage samples included proximates permission (protein, fat, ash, and moisture),prohibited acid de tergent fiber (ADF), neutral detergent fiber (NDF), minerals (calcium and phosphorus), and carbohydrates by calculation. the be Compositional analysescommercial of the grain samples included proximates (protein, fat, ash, and moisture),Furthermore, ADF,any NDF, total dietary fiber (TDF), amino acids, fatty acids (C8-C22), vitaminsConsequently, (B1, Bwithout2, B 6, E, niacin, and folic acid), anti-nutrients (phytic acid and raffinose), secondary metabolites (furfural, ferulic acid, and p-coumaric acid), minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc), and carbohydrates by calculation. Methods for analysis were based on internationally- recognized procedures and published literature.

Statistical analyses of the compositional data were conducted using a mixed model analysis of variance with data from each of the five sites, and a combination of all five

Monsanto Company 06-CR-166U Page 142 of 262 CBI Deleted Version field sites. Each individual analyte for MON 89034 was compared to that of the conventional control, for the combination of all five sites (i.e., the combined-site) and for each of the five sites. The statistical significance was defined at the level of p < 0.05. Of the 77 components analyzed, 16 components had greater than 50% of the analytical values that were below the limit of quantitation (LOD), and were not included in the statistical analyses.

Statistical analyses of the remaining 61 components were conducted for comparison of MON 89034 with control corn. The overall data set was examined for evidence of biologically relevant changes. Based on this evaluation and the results of statistical analyses, analytes for which the levels were not statistically different were deemed to be present at equivalent levels between MON 89034 and the control. Analyses using data regime.and from the combination of all five sites (combined-site) indicatedand that there were no AG statistical differences for 58 of the 61 components. Details of the combined-site analysis results are provided in Appendix G. Analyses using data from the five single sites indicated that there were no statistically Bayersignificant propertydifferences protection in 261publishing out of the 305 comparisons made between MON 89034of and the control. contents its parties.data therefore Table VII-12 shows all the statistical differences in bothand/or theor combined-site and affiliates. individual site (total of 5 sites)property analyses. intellectual Forthird the combined-site analyses,may statistical its differences between MON 89034 andas control corn were observed for three analytes, the of and which included phosphorusis in forage, and 18:0regulatory stearic and 20:0 arachidicowner. acids in grain. a document The differences observed anyare generallysuch small (3.4 – 19.2%) consideringits the natural reproductiondocument variability, and the mean levels andowner ranges of thesethis an alytes ofof MON 89034 are well within the 99% tolerance intervals forunder commercial of corn. The levels of phosphorus in rightsthe this forage, and 18:0document stearicand/or and 20:0 arachidic acids in grain, rightswere also within the ranges in to of fall use of the International Life Sciences Institute (ILSI) Crop Compositionthe Database (ILSI-CCD, 2006), asThis well within publishedmay literature distribution,and ranges. Therefore, it is concluded that MON rights owner 89034 and the subjectcontrol corn are compositionally equivalent based on analyses of the combined-site data. The reported ILSI andthe published violate literature ranges for the analytical be copy components present in corn are summarizedof in Appendix G. and may documentpublication, It exploitation The reproducibilitythis and trends at individual sites were also examined, and comparisons made to conventionalany corn hybrids using the 99% tolerance intervals. Of the 44 permission statistical differences observed prohibitedin the indivi dual site analyses, 33 were only observed at one site (out of the total of 5 sites). There were no consistent trends and the mean and the be ranges of the analytescommercial in MON 89034 were well within the 99% tolerance interval for conventionalFurthermore,any corn. The mean levels and ranges for calcium and methionine in grain were slightlyConsequently, outsidewithout the 99% tolerance interval but within the ILSI database. It is concluded that these differences are not biologically significant. Of the remaining 11 differences observed at more than one site, there were no analytes that were consistently and statistically different across five or four sites. In addition, there were no analytes that showed statistically significant differences in three sites that had not been previously observed to be different in the combined-site analysis. Statistically significant differences were observed in as many as two sites for three analytes (carbohydrates, copper, iron), which were not previously found to be different in the combined-site

Monsanto Company 06-CR-166U Page 143 of 262 CBI Deleted Version analysis. For carbohydrates and iron, the observed differences from the control were small and lower at one site and higher at the other site. As there is no evidence of any reproducibility across sites, it is concluded that the statistical differences are not biologically relevant for carbohydrates and iron. For copper, the differences for MON 89034 were higher than the control at both sites. The observed differences are small in magnitude, and the mean levels and ranges of MON 89034 are well within the 99% tolerance interval. Therefore, it is concluded that the statistical differences for copper are not biologically relevant.

Based on the data and information presented above, it was concluded that corn grain and forage derived from MON 89034 are compositionally and nutritionally equivalent to those of conventional corn. The few statistical differences between MON 89034 and regime.and control corn likely reflect the natural variability of the componentsand since the mean levels AG of analytes for MON 89034 are well within the 99% tolerance intervals for conventional corn, and/or within the ranges in the ILSI database (ILSI-CCD, 2006) and the scientific literature. Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VII-12. Summary of differences (p<0.05) for the comparison of corn component levels for MON 89034 vs the control and commercial references

Mean Difference (Test minus Control) MON 89034 Control % of Significance MON 89034 and Commercial Analytical Component (units)1 and regime. Mean Mean ControlAG (p-Value) (Range) Tolerance Int.2 Statistical Differences Observed in Combined-site Analyses Mineral Forage Phosphorus (% DW) 0.25 0.21 Bayer 19.24 property 0.01 protection (0.22publishing - 0.32) [0.071,0.32] of contents its Fatty Acid parties.data therefore Grain 18:0 Stearic (% Total FA) 1.89 1.82 3.97 0.002 and/oror (1.79 - 2.03) [0.86,2.98] affiliates. Grain 20:0 Arachidic (% Total FA) 0.39 property0.38 intellectual 3.43third <0.001 (0.36may - 0.42) [0.23,0.54] its as Statistical Differences in More Than One Site the of and is regulatory owner. Proximate a document its Site IA Grain Carbohydrates (% DW) 83.38 any 84.52such -1.34reproduction 0.008 (83.29 - 83.55) [81.08,88.80] this documentof Site OH Grain Carbohydrates (% DW) 84.26 83.8owner 0.55 0.009 (83.99 - 84.59) [81.08,88.80] under of rightsthe this documentand/or rights Mineral to of fall use of Site IL-1 Grain Copper (mg/kg DW) 1.76 1.36 29.35 the 0.023 (1.51 - 2.21) [0,4.20] Site NE Grain Copper (mg/kg DW) This 2.15 may1.67 distribution, and 28.66 0.023 (1.92 - 2.38) [0,4.20] rights owner Site IL-1 Grain Iron (mg/kg DW) subject20.86 19.48 7.11 0.048 (19.23 - 21.79) [8.88,34.51] Site OH Grain Iron (mg/kg DW) be 21.37 25.74 the -17violate 0.006 (20.59 - 21.76) [8.88,34.51] copy of Fatty Acid and may documentpublication, Site IL-1 Grain 18:0 Stearic (% Total FA)It 1.96 exploitation1.82 7.94 <0.001 (1.89 - 2.02) [0.86,2.98] Site IL-2 Grain 18:0 Stearic (% Total FA) this 1.98any 1.82 9.05 <0.001 (1.93 - 2.03) [0.86,2.98] Site IL-1 Grain 20:0 Arachidic (% Total FA) 0.41 0.39 5.23 0.007 (0.40 - 0.42) [0.23,0.54] permission Site IL-2 Grain 20:0 Arachidic (% Total FA) 0.39 prohibited 0.37 6.83 0.021 (0.38 - 0.40) [0.23,0.54] Site OH Grain 20:0 Arachidic (% Total FA) 0.38 the 0.37 3.12 0.035 (0.38 - 0.39) [0.23,0.54] commercial be 1DW = dry weight; FW = fresh weight; FA = fatty acid; Combined-site = analyses of the combined data from each of the five replicated field trials. 2With 95% confidence, interval containsFurthermore, 99% of theany values expressed in the population of commercial lines. Negative limits were set to zero. Consequently,without Table VII-12 continues on the next page.

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Table VII-12 (continued). Summary of differences (p<0.05) for the comparison of corn component levels for MON 89034 vs the control and commercial references

Mean Difference (Test minus Control) MON 89034 Control % of Significance MON 89034 and Commercial Analytical Component (units)1 and regime. Mean Mean ControlAG (p-Value) (Range) Tolerance Int.2 Statistical Differences Observed in One Site Only Amino Acid Site IA Grain Alanine (% DW) 0.88 0.81 Bayer 7.83 property 0.03 protection (0.87publishing - 0.88) [0.48,1.08] Site IA Grain Arginine (% DW) 0.51 0.46 of 10.83 0.005 (0.50 contents- 0.52) [0.33,0.56] its Site IA Grain Aspartic acid (% DW) 0.77 0.71 8.66 parties. 0.003data (0.77 - 0.78)therefore [0.43,0.90] Site IA Grain Cystine (% DW) 0.25 0.23 7.54 0.014 and/oror (0.24 - 0.26) [0.18,0.27] affiliates. Site IA Grain Glutamic acid (% DW) 2.27 property2.09 intellectual 8.66third 0.011 (2.26may - 2.28) [1.25,2.75] Site IA Grain Glycine (% DW) 0.41 0.its38 6.94 0.02 (0.40 - 0.41) [0.28,0.46] as Site IA Grain Histidine (% DW) 0.34 the of0. 32 and 7.16 0.022 (0.34 - 0.34) [0.22,0.38] is regulatory owner. Site IA Grain Leucine (% DW) 1.49 1.37 8.96a document 0.032 its (1.48 - 1.51) [0.77,1.92] Site IA Grain Lysine (% DW) 0.35 any such0.32 6.66 reproduction 0.028 (0.33 - 0.36) [0.20,0.40] this documentof Site IA Grain Methionine (% DW) 0.25 0.23owner 11.2 0.003 (0.25 - 0.27) [0.14,0.25] under of Site IA Grain Phenylalanine (% DW) 0.58 rightsthe 0.53 9.45 this 0.028 (0.57 - 0.59) [0.32,0.73] documentand/or rights Site IA Grain Proline (% DW) 1.05 to of 0.98fall 7.29use of 0.028 (1.04 - 1.05) [0.68,1.21] Site IA Grain Serine (% DW) 0.6 0.56 8.28 the 0.004 (0.60 - 0.61) [0.34,0.71] This may distribution,and Site IA Grain Threonine (% DW) 0.37 rights 0.34 8.45owner 0.004 (0.37 - 0.37) [0.24,0.41] Site IA Grain Tyrosine (% DW) subject0.43 0.36 17.5 0.006 (0.42 - 0.43) [0.17,0.52] the violate be copy Fatty Acid of Site IA Grain 18:3 Linolenic (% Total FA) 1.21document 1.34 and -9.4 0.009 (1.20 - 1.23) [0.63,1.77] may publication,exploitation Site IL-1 Grain 16:1 Palmitoleic (% TotalIt FA ) 0.13 0.14 -6.87 0.012 (0.12 - 0.13) [0,0.28] this Site IL-2 Grain 18:1 Oleic (% Total FA) 24.any75 23.82 3.93 0.003 (24.14 - 25.25) [7.51,46.46] Site IL-2 Grain 18:2 Linoleic (% Total FA) 61.87 permission63.17 -2.07 0.001 (61.19 - 62.42) [39.41,76.74] Site NE Grain 20:1 Eicosenoic (% Total FA) 0.28 prohibited0.29 -1.5 0.03 (0.28 - 0.28) [0.15,0.39] the be 1DW = dry weight; FW = fresh weight; FA = fatty acid; Combined-sitecommercial = analyses of the combined data from each of the five replicated field trials. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero. Furthermore, any Consequently,without Table VII-12 continues on the next page.

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Table VII-12 (continued). Summary of differences (p<0.05) for the comparison of corn component levels for MON 89034 vs the control and commercial references

Mean Difference (Test minus Control) MON 89034 Control % of Significance MON 89034 and Commercial Analytical Component (units)1 and regime. Mean Mean ControlAG (p-Value) (Range) Tolerance Int.2 Mineral Site IA Grain Calcium (% DW) 0.0064 0.0058 10.96 0.012 (0.0062 - 0.0066) [0.0016,0.0059] Site IA Grain Manganese (mg/kg DW) 8.34 6.99 Bayer 19.32 property 0.017 protection (7.62publishing - 9.32) [3.17,9.99] Site IA Forage Calcium (% DW) 0.24 0.26 of -8.77 0.033 (0.24 contents- 0.24) [0.016,0.38] its Site NE Forage Phosphorus (% DW) 0.25 0.17 46.95 parties. 0.036data (0.23 - 0.28)therefore [0.071,0.32] and/oror affiliates. Proximate property intellectual third may Site IA Grain Protein (% DW) 11.89 10.85its 9.59 0.005 (11.73 - 11.98) [7.54,13.13] as Site IL-1 Forage Moisture (% FW) 69.03 the of66.53 and3.76 0.031 (68.50 - 69.40) [57.62,86.45] is regulatory owner. Site NE Forage Ash (% DW) 3.2 4.39 -27.12a document 0.021 its (2.93 - 3.38) [1.93,6.31] Site NE Forage Carbohydrates (% DW) 88.16 any 84.98such 3.74 reproduction0.004 (86.86 - 88.84) [83.05,90.74] this documentof owner under of Fiber rightsthe this documentand/or rights Site NE Grain Neutral Detergent Fiber (% DW) 10.52 to of 9.05fall 16.27use of 0.028 (10.43 - 10.69) [5.93,13.63] Site OH Forage Acid Detergent Fiber (% DW) 31.31 23.58 32.78 the0.012 (26.92 - 34.93) [16.76,43.76] Site OH Forage Neutral Detergent Fiber (% This may distribution,and 43.21 rights 37.87 14.11owner 0.027 (40.07 - 46.82) [25.94,55.67] DW) subject

be the violate Vitamin copy of Site IL-2 Grain Folic Acid (mg/kg DW) 0.37 0.32 and 13.81 <0.001 (0.35 - 0.38) [0.012,0.69] may documentpublication, It exploitation Secondary Metabolite this any Site OH Grain p-Coumaric Acid (µg/g DW) 218.38 185.63 17.64 0.032 (187.79 - 253.04) [0,378.57] permission 1DW = dry weight; FW = fresh weight; FA = fatty acid; Combined-site = analysesprohibited of the combined data from each of the five replicated field trials. 2With 95% confidence, interval contains 99% of the values expressedthe in the population of commercial lines. Negative limits were set to zero. commercial be Furthermore,any Consequently,without

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VII.C. Conclusions of Phenotypic, Agronomic, Ecological Interactions and Compositional Assessment of MON 89034

An extensive set of information and robust data were used to assess whether the introduction of the lepidopteran-protection trait and the expression of the Cry1A.105 and Cry2Ab2 proteins altered the plant pest potential of MON 89034 compared to the control. The assessment was based on thorough phenotypic, agronomic, ecological interactions and compositional characterization and comparison of MON 89034 to control and conventional reference corn hybrids. Data were collected for five seed dormancy/germination parameters, two pollen characteristics, 14 phenotypic and agronomic characteristics during plant growth and development, more than 70 regime.and observations for each of the plant-insect, plant disease and plant-abiotic stressor AG interactions, and 305 comparisons in compositional analyses.

Results from the phenotypic and agronomicBayer assessments property indicate protection thatpublishing MON 89034 does not possess characteristics that would conferof a plant pest risk or result incontents meaningfully its altered ecological impact compared to conventional corn.parties. Datadata on ecologicaltherefore interactions also indicate that MON 89034 does not confer any increased susceptibilityand/oror or tolerance to affiliates. specific disease, abiotic stressors,property or insectsintellectual thirdexcept for the introducedmay lepidopteranits protection trait. Data from compositionas an alyses support the conclusion of compositional the of and equivalence between MONis 89034 and conventionalregulatory corn in nutritional,owner. anti-nutritional, a document and secondary metabolite levelsany insuch grain and forage. Taken together,its these data support a reproductiondocument conclusion that MON 89034 is notowner likely to pose increasedthis plantof pest risk or result in meaningfully altered ecological impact undercompared toof conventional corn. rightsthe this documentand/or rights to of fall use of VII.D. References the This may distribution,and rights owner Alexander, M.P.subject 1980. A versatile stain for pollen fungi, yeast and bacteria. Stain be the violate Technology.copy 55:13-18. of Anderson, W.P. 1996. Weed Science andPrinci ples and Applications, 3rd ed. West may documentpublication, It Publishing Company,exploitation St. Paul, MN. pp 27-37. this AOSA. 2002. Rulesany for Testing Seeds. Association of Official Seed Analysts. Lincoln, permission NE. 166pp. prohibited the ILSI-CCD. 2006.commercial International be Life Science Institute Crop Composition Database. Version 3.0. Available at http://www.cropcomposition.org/ Furthermore,any Lingenfelter,Consequently, D.D.without and N.L. Hartwig. 2003. Introduction to Weeds and Herbicides. Agricultural Research and Cooperative Extension CAT UC175. The Pennsylvania State University, University Park, PA. OECD. 2002. Consensus document on compositional considerations for new varieties of maize (Zea mays): key food and feed nutrients, anti-nutrients and secondary plant metabolites. Organisation for Economic Co-operation and Development. Environmental Health and Safety Publications, Paris, France. ENV/JM/MONO(2002)25.

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VIII. Environmental Assessment and Impact on Agronomic Practices

This section provides an environmental assessment of MON 89034 and considers the impact of the introduced Cry1A.105 and Cry2Ab2 proteins on non-target organisms (NTOs) and endangered species, the potential for gene flow, and the weediness potential of MON 89034. The section also provides a brief summary of the current production and the agronomic practices for corn in the U.S., and an assessment of the impact from the introduction of MON 89034 on the agronomic practices and insect resistance management (IRM).

The assessment of the impact of MON 89034 and the introduced proteins on NTOs and regime.and endangered species concludes that risk to these organisms fromand the use of MON 89034 is AG negligible. This risk assessment took into consideration several components, including familiarity with the mode of action of Cry proteins; the activity spectra of the Cry1A.105 and Cry2Ab2 proteins; the expression levelsBayer of theproperty two proteins protection inpublishing MON 89034; the environmental fate of the proteins; theof lack of synergistic or antagonisticcontents interaction its between the two proteins; and feeding tests with eachparties. of thedata two proteins ortherefore MON 89034 corn materials to representative non-target organisms. and/oror affiliates. property intellectualthird may its The evaluation of weediness potentialas and gene flow concludes that MON 89034 is no the of and more likely to become a isweed than conventionalregulatory corn, and MON 89034owner. is expected to be a document similar to conventional cornany regarding such the potential for gene flow. itsPollen-mediated gene reproductiondocument flow would be expected to occur onlyowner within cultivatedthis corn in theof U.S. The probability for horizontal gene flow is exceedinglyunder small. Evenof if it were to occur, the consequences rightsthe this would be negligibledocumentand/or since the genes introduced into MONrights 89034 are of bacterial origin to of fall use of and the two Cry proteins produced have no meaningfulthe toxicity to humans and other NTOs underThis the conditions of mayuse. distribution,and rights owner subject An assessment of current corn agronomicthe prviolateactices indicates that the introduction of be copy MON 89034 will not impact the currentof U.S. corn cultivation practices and the and managementmay of weeds,document publication,diseases and insects except for the control of lepidopteran insect It exploitation pests. In addition,this MON 89034 will provide an effective IRM tool due to the production of both the Cry1A.105any and Cry2Ab2 proteins in a single plant. permission prohibited VIII.A. Environmental Assessment of the Cry1A.105 and Cry2Ab2 Proteins the be commercial VIII.A.1.Furthermore, Modeany of Action of Cry1A.105 and Cry2Ab2 Proteins TheConsequently, history of withoutsafe use of Bt microbial pesticides and Bt crops and the well understood mode of action of Bt Cry proteins are important considerations in the environmental safety assessment of these proteins. Bt microbial pesticides have been used for several decades. Bt corn producing Cry1Ab or Cry1F (YieldGard®, Herculex® I) and Bt cottons

® YieldGard and Bollgard are trademarks of Monsanto Company. Herculex and WideStrike are trademarks of Dow AgroSciences LLC.

Monsanto Company 06-CR-166U Page 149 of 262 CBI Deleted Version producing Cry1Ac, Cry1F, and Cry2Ab2 (Bollgard®, Bollgard® II, WideStrike®) have been in commerce for up to a decade.

As discussed in Section VI, MON 89034 produces two Cry proteins – Cry1A.105 and Cry2Ab2. Cry1A.105 is a full-length Cry1 protein (protoxin) with a molecular weight of 133 kDa. It is a chimeric protein which consists of domains I and II from Cry1Ab or Cry1Ac, domain III from Cry1F, and the C-terminal domain from Cry1Ac. The Cry2Ab2 protein produced in MON 89034 is also derived from Bt and shares 99.8% sequence identity with the wild-type Cry2Ab2 differing by one single amino acid.

The general mode of action of Bt Cry proteins is well understood. The bacterially- produced crystal proteins are first solubilized in the insect midgut, followed by activation regime.and of the protoxins (full-length proteins) to active toxins (proteolyticand resistant cores) by AG midgut proteases. A similar process occurs when Cry proteins are expressed in plants. The activated proteins then bind to midgut membrane receptors in susceptible insects, insert into the apical membrane and formBayer pores. Formationproperty ofprotection the porespublishing causes loss of osmotic regulation, and eventually leadsof to cell lysis, which is thought tocontents be responsible its for insect death (Gill et al., 1992; Schnepf et al., 1998;parties. Zhuangdata and Gill, 2003).therefore and/oror affiliates. Cry1 protoxins (such as Cry1Ab,property Cry1Ac, intellectual and Cry1F)third are 130 to 140 kDamay in size, and are its activated by proteases to active coresas of 65 to 70 kDa. The crystal solubilization is the of and facilitated by an alkalineis pH. The typical midgutregulatory pH is between 9-11owner. in lepidopteran a document larvae. During the solubilizationany such and activation of Cry1 proteins, anits N-terminal peptide reproductiondocument of 25-30 amino acids and approximatelyowner half of thethis sequences fromof the C-terminus are cleaved (Choma et al., 1990; Gill et al.,under 1992; Schnepfof et al., 1998; Bravo et al., 2002; rightsthe this Zhuang and Gill,document 2003).and/or The role of the C-terminal domainrights is believed to be in the to of fall use of formation of crystalline inclusion bodies within the Btthe bacterium and is not required for insecticidalThis activity (Park et al.,may 2000; distribution, Deand Maagd et al., 2001). The 25-30 amino acid rights owner residues at the N-terminussubject play a role in promoting crystallization of the protoxin in the bacterium, but do not contribute to toxicitythe toviolate insects (Choma et al., 1990; Gill et al., be copy 1992; Schnepf et al., 1998). In fact, it ofwas shown with Cry1Ac that proteolytic removal and of the N-terminalmay peptidedocumentpublication, is essential before the protein becomes fully active (Bravo et It exploitation al., 2002). Cry2,this Cry3, Cry10, Cry11 and Cry20 proteins appear to be naturally truncated, and they anyare present in crystals as 65 to 86 kDa proteins. Although naturally permission truncated, these 65 to 86 kDa proteinsprohibited also require proteolytic activation, resulting in 60 to 65 kDa active cores (Lee and Gill, 1997; Zhuang and Gill, 2003). the be commercial TheFurthermore, 3-dimensionalany structures of three members of the Cry protein family, which may wellConsequently, prove to bewithout representative of all Cry proteins, reveal the presence of three structural domains (Li et al., 1991; Grochulski et al., 1995; Morse et al., 2001). Domain I, consisting of seven α-helices, is involved in membrane insertion and pore formation. Domain II, consisting of three β-sheets in a Greek key conformation, is involved in specific receptor recognition and binding. Domain III, which consists of two β-sheets in a jellyroll conformation, has been suggested to maintain the structural integrity of the protein molecule (Li et al., 1991) and also to contribute to specificity (De Maagd et al., 2000; 2001). All three domains are included in the N-terminal portion of the protoxins

Monsanto Company 06-CR-166U Page 150 of 262 CBI Deleted Version during the formation of active toxins in insect guts. Since domains II and III can both contribute to the specificity, the difference in these domains among different Cry proteins would account for the diversity of insecticidal activities. These domains may dictate whether and how binding occurs between the Cry proteins and the insect midgut. Only those insects with specific receptors are affected by Cry proteins and no toxicity is observed in species that lack these receptors (Crickmore et al., 1998; De Maagd et al., 2001). Monsanto’s research data indicates that there are important differences between the Cry1A.105 and Cry2Ab2 proteins regarding their binding to the lepidopteran midgut. The data demonstrate that Cry1A.105 and Cry2Ab2 proteins bind to some unique components on European corn borer brush border membrane. They also share many common binding sites. By screening a limited number of glycosylated bovine serum albumins, it was shown that galactosamine is recognized by Cry1A.105 only, while regime.and Cry2Ab2 demonstrated a high affinity for N-acetylglucosamineand and galactosamine. AG These differences are advantageous to insect resistant management for MON 89034 because they result in a low probability of insect cross-resistance between the two proteins (see Section VIII.F.). Bayer property protectionpublishing of contents its VIII.A.2. Activities against Target Insects parties.data therefore and/oror affiliates. property intellectualthird may VIII.A.2.a. Laboratory Tests on itsActivi ties against Target Insect Pests as the of and is regulatory owner. Lepidopteran pest larvae feed on leaves, stalks,a silk and earsdocument of corn and can significantly any such its reproductiondocument affect yield. Furthermore, feedingowner can also affect thisthe overallof qua lity of the crop by providing an opportunity for infection underby fungi whichof produce mycotoxins. The major rightsthe this lepidopteran documentinsect and/orpests include corn borers, suchof as rightsEuropean corn borer (ECB, Ostrinia nubilalis), southwesternto of cornfall borer (SWCB,use Diatraea grandiosella), sugarcane the borer (SCB,This Diatraea saccharalismay), Africandistribution,and stalk borer (Busseola fusca), and Asian corn borer (ACB, Ostrinia funicalisrights ). ECB is the ownerprimary pest in the U.S. In addition, corn subject leaves, silks and ears are the primary food source for fall armyworm (FAW, Spodoptera be the violate frugiperda), corn copyearworm (CEW, Helicoverpaof zea), and black cutworm (BCW, Agrotis ipsilon) and feeding damage from these pestsand ne gatively affects growth and development may documentpublication, of theIt plants. exploitation this any Studies were conducted topermission evaluate the spectra of the insecticidal activities of the Cry1A.105 and Cry2Ab2 proteinsprohibited with a variety of agronomically-important insects from the three major taxa.commercial Insect species be tested included four representative lepidopterans: ECB, CEW, FAW, and BCW; two representative coleopterans: boll weevil (Anthonomus grandisFurthermore, grandisany ) and southern corn rootworm (Diabrotica undecimpunctata howardi); without and Consequently,two representative hemipterans: western tarnished plant bug (Lygus hesperus) and green peach aphid (Myzus persicae). The insects were exposed for either 5 or 7 days to a range of concentrations of Cry1A.105 or Cry2Ab2 protein up to 80 or 100 µg of protein per ml of insect diet using either diet-incorporation or diet-overlay bioassay procedures. The results showed that Cry1A.105 and Cry2Ab2 proteins had activities against all four representative lepidopteran insects. There was no indication of activity of these two proteins against the two coleopteran or two hemipteran species evaluated. The results

Monsanto Company 06-CR-166U Page 151 of 262 CBI Deleted Version from these assays confirm that, under expected agricultural use, these proteins have the targeted range of insecticidal activity against lepidotperan insect pests.

VIII.A.2.b. Field Efficacy Trials

The field efficacy of MON 89034 against major lepidopteran pests of corn was evaluated at multiple locations in the U.S. and Puerto Rico during 2004 and 2005. The 2004 field trials were conducted at 10 locations in Illinois, Tennessee, Mississippi, Alabama, Arkansas, Oregon, and Puerto Rico. The 2005 field trials were conducted at 18 locations in Wisconsin, Illinois, Indiana, Missouri, Arkansas, Tennessee, Mississippi, Alabama, Louisiana, Nebraska, and Colorado. MON 89034 was tested along with a conventional control corn and one current Bt corn (Yieldgard® corn borer, i.e., MON 810)and as a and regime. reference. The experimental design for all locationsAG was a randomized complete block with four replications. Experimental units were either one-, two- or four-row plots, ranging from 5.3 to 6.1 m in length depending upon location. Each row was thinned to 35 plants, and row spacing was 0.76 m. TheBayer entire trialproperty areas were surroundedpublishing by at least protectioncontents two conventional corn border rows and ofbuffered by a complete range of hybrid plants to its further eliminate border effects. parties.data therefore and/oror affiliates. Seven insect pests were tested propertyduring the intellectualtwo yearsthird of field trials, includingmay ECB SWCB, its as FAW, CEW, SCB, BCWthe and ofwestern beanand cutworm (WBCW, Striacosta albicosta). is regulatory owner. The infestations were either artificially introduceda to thedocument plants at appropriate growth stages (using neonate larvaeany for such ECB, SWCB, FAW, CEW andits BCW; and eggs for reproductiondocument of WBCW) or relied on heavy naturalowner insect pressurethis (for SCB). Insect damage was under of assessed on 10-24 plants perrights plotthe (damage rating systemsthis varied by insect species). Mean documentand/or rights separation tests were performedto of at αfall = 0.05 leveluse to assessof treatment differences. the This may distribution,and The results demonstraterights that the high level ofowner efficacy of MON 89034 against ECB and SWCB was equivalentsubject to the commercial reference Bt corn, MON 810. MON 89034 provided outstandingbe control of FAW, whichthe wasviolate superior to that of MON 810. MON copy of 89034 was also superior to MON 810 forand the control of CEW. SCB control by MON 89034 wasmay comparabledocument publication,to that of MON 810. MON 89034 appeared to be efficacious It exploitation against BCW and have potential for the control of WBCW. However, since the pressure this any was light and the number of locations limited for these two pests, additional testing is permission needed to confirm this activity.prohibited The performance of MON 89034 to reduce ear damage was most evident underthe combined be infestations of both WBCW and CEW. In conclusion, the broader spectrumcommercial of activ ity of MON 89034 compared to MON 810 provides greater insuranceFurthermore, againstany yield loss from lepidopteran pests, and further enhances agronomic qualities,Consequently, ease ofwithout harvest, and grain quality.

VIII.A.3. Impact on Non-target Organisms

Evaluation of the potential risks to NTOs is an important component of an overall environmental risk assessment of a biotechnology-derived crop. Assessment of the potential risks to NTOs associated with the introduction of a biotechnology-derived crop producing insecticidal proteins is based on the characteristics of the crop and introduced

Monsanto Company 06-CR-166U Page 152 of 262 CBI Deleted Version proteins. Since risk is a function of hazard and exposure, it is critical to determine the potential hazards and exposure scenarios that are most likely and that require evaluation through experimental studies. Selection of the test organisms and test material are important decisions that are based on the characteristics of the trait and the product. In the U.S., regulatory guidelines for NTO risk assessment of insect-protected crops were developed by the EPA. The testing is conducted according to a tier-based system (EPA, 1996; 2001a-d; 2004a-b). Additionally, the EPA has convened several Scientific Advisory Panel (SAP) meetings to make recommendations and provide guidance for NTO testing and risk assessment for agricultural products produced by methods of biotechnology (EPA, 2001a-b; 2002; 2004a-b).

For the Cry proteins tested in laboratory assays to date, potentially significant adverse regime.and effects have been observed for only a very few NTO species thatand are closely related to the AG target species (Mendelsohn et al., 2003; Romeis et al., 2006). However, field studies conducted over the past decade by industry and the academic community on registered insect-protected crops that produce a varietyBayer of Cry proteinsproperty have protection demonstratedpublishing that these crops have no adverse effects on the diveof rsity and abundance of non-targetcontents arthropod its communities including predators, parasitoids and otherparties. ecologicallydata importanttherefore non-target arthropods (Bitzer et al., 2005; Bhatti et al., 2005; Daly and Buntin,and/oror 2005; Dively, 2005; affiliates. Head et al., 2005; Lopez, et al.,property 2005; Pilcherintellectual thirdet al., 2005; Torres andmay Ruberson, 2005; its Whitehouse et al., 2005; Naranjo et al.,as 2005; Naranjo, 2005a-b; Lozzia et al., 1998; Orr the of and and Landis, 1997; Pilcheris et al., 1997). Additionally,regulatory Cry1 and Cry2 owner.proteins have been a document shown to pose low risk to populationsany such of non-target lepidopterans (Hellmichits et al., 2001; reproductiondocument Mattila et al., 2005; Pleasants et al.,owner 2001). For example,this theof impact of exposure to pollen containing Cry1 and Cry2 proteinsunder on populationsof of lepidopteran species has been rightsthe this evaluated in adocument numberand/or of empirical studies and several riskrights assessments (Mattila et al., to of fall use of 2005; Mendelsohn et al., 2003; Wolt et al., 2005), andthe the risk has been shown to be negligible.This may distribution,and rights owner subject The NTO risk assessment of Cry1A.105 andthe Cry2Ab2violate proteins was a multi-step process be copy taking into consideration hazard identificationof and characterization, exposure assessment, and and riskmay characterization. documentpublication, The hazard identification and characterization included testing It exploitation the spectra of insecticthis idal activities of the Cry1A.105 and Cry2Ab2 proteins, efficacy of MON 89034 againstany target corn insect pests, and the potential for interaction between the permission Cry1A.105 and Cry2Ab2 proteins.prohibited These studies demonstrated that the Cry proteins produced in MON 89034 perform in the expected manner based on the extensive the be knowledge and commercialexperience on Cry proteins. The insecticidal activity spectra of the Cry1A.105Furthermore, anyand Cry2Ab2 proteins were found to be typical for the Cry1 and Cry2 classes of BtConsequently, proteins.without The activity was most evident against insects within the order Lepidoptera. An assessment of the potential for interaction between Cry1A.105 and Cry2Ab2 proteins demonstrated that there was no interaction between the two proteins. The Cry1A.105 and Cry2Ab2 proteins were tested alone and in combination against two sensitive lepidopteran species – ECB and CEW. Two species were tested to demonstrate reproducibility thereby strengthening the validity of the study. The insects were exposed to purified Cry1A.105 and Cry2Ab2 proteins in diet-incorporation bioassays. The results demonstrated that these proteins only showed additive effect in insecticidal activity, and

Monsanto Company 06-CR-166U Page 153 of 262 CBI Deleted Version no synergistic or antagonistic effects were observed. The result is consistent with the findings in an earlier study that demonstrated no interaction between the Cry1Ac and Cry2Ab2 proteins (Greenplate et al., 2003). The lack of interaction between Cry1A.105 and Cry2Ab2 allows for each protein to be tested and evaluated independently.

The exposure assessment of the NTO risk assessment is comprised of three components: estimation of the expression levels of the Cry1A.105 and Cry2Ab2 proteins in tissues from MON 89034; a conservative calculation of margins of exposure based on the maximum amount of corn tissue that might be exposed to NTOs; and assessment of the environmental fate of the Cry1A.105 and Cry2Ab2 proteins in soil. Expression values from several tissue types were used to calculate the doses used in the NTO toxicity tests, while the results from soil degradation studies were used to characterize the potential regime.and exposure to soil organisms and the likelihood that each proteiand n could persist and AG accumulate in agricultural soils.

Dosing in the NTO tests was based on theBayer estimated property maximum protection expectedpublishing environmental concentrations (MEEC) of the Cry1A.105of and/or Cry2Ab2 protein presentcontents in the tissue(s) its likely to be ingested by the representative NTO. A parties.targeteddata margin of exposuretherefore (MOE) of at least 10-times greater than the MEEC was used in theand/or tests.or The maximum affiliates. expression levels for the Cry1A.105property (8.8 µintellectualg/g fwt)third and Cry2Ab2 (0.47may µg/g fwt) proteins its in pollen were used to determine the doseas levels for honeybee, ladybird beetle, a parasitic the of and wasp, and minute pirate isbug, for which pollen reregulatorypresents the major routeowner. of exposure. a document any such its reproductiondocument The principal route of exposure forowner soil-d welling organismsthis suchof as Collembola and earthworms was assumed to be from decomunderposing of plant tissue containing the Cry1A.105 rightsthe this and Cry2Ab2document proteins.and/or The use of a soil MEEC to calculaterights MOEs is appropriate to of fall use of because these organisms feed on detritus that is madethe up of soil and decaying plant and other material.This Therefore, themay maximum distribution,and amount of plant tissue entering the soil rights owner environment andsubject the maximum concentration of the Cry1A.105 and Cry2Ab2 proteins in the plant tissue were considered in detethermining violate the MEEC for the Cry1A.105 and be copy Cry2Ab2 proteins in the top 15 cm of ofsoil. The EPA model assumes that corn plants in and the fieldmay are tilled intodocument thepublication, top 6 inches (15 cm) of soil, and the plants uniformly express It exploitation the introduced thisproteins at the maximum concentration. However, because expression of Cry proteins varies anyin a tissue-specific and temporal manner this hazard assessment was permission generated based on the maximumprohibited in planta expression of the Cry1A.105 (240 µg Cry1A.105/g dwt) andthe the be Cry2Ab2 (210 µg Cry2Ab2/g dwt) proteins at a late developmental commercialstage (pre-tasseling) of corn. The protein levels in late plant developmentalFurthermore,any stage represent a probable exposure scenario. These data as well as additionalConsequently, parameterwithout estimates 1 were used to calculate the soil MEECs for Collembola and earthworm.

1 The soil MEEC for Collembola and earthworm was calculated using the following parameter assumptions: 25,000 corn plants/acre; a corn plant dry weight is 650 g/plant; a bulk density of soil of 1500 kg/cubic meter; a soil depth is 0.15 m (about 6 inches) and a soil volume in a one-hectare 0.15 m layer is 1500 cubic meters. The Cry1A.105 and Cry2Ab2 maximum expression values were taken for leaves at the pre-tasseling stage and were 240 and 210 µg/g dwt, respectively.

Monsanto Company 06-CR-166U Page 154 of 262 CBI Deleted Version

The August 2002 EPA SAP reports (EPA, 2002) recommended that nontarget testing be focused on species exposed to the crop being registered (i.e., beneficial insects and avian species). The exposure of fish to Cry proteins in corn is negligible in agricultural settings. According to EPA "There is no evidence of sensitivity of aquatic (including endangered) species to Cry proteins. Toxicity studies with aquatic organisms show very limited hazard for fish or invertebrates exposed to either corn pollen or bacterially- expressed Cry protein" (EPA, 2001d). The requirements for a freshwater fish were waived for a number of Bt corn products based on a lack of any substantial exposure of fish to Bt proteins produced in corn (EPA, 2003). Therefore, a static renewal freshwater fish toxicity study was not performed. The exposure of aquatic invertebrates to corn pollen, if it occurs at all, would be of a very short duration and at extremely low levels that are not expected to produce any adverse effects. Nevertheless, an acute aquatic regime.and invertebrate species test was performed with Daphnia magnaand using pollen from MON AG 89034. No treatment mortality or other adverse effects were observed at the concentration of 100 mg pollen/l over a 48-hour exposure period. Under a well accepted toxicity classification system for freshwatBayerer ecotoxicology, property ifprotection no adversepublishing effects are observed at a concentration of 100 mg/l,of the tested material is classifiedcontents as practically its non-toxic (EPA, 1985). Therefore, no hazard is anticipatedparties.data to aquatic invertebratestherefore from exposure to the registered uses of MON 89034. and/oror affiliates. property intellectualthird may its Based on the results from the productas characterization and exposure assessment, an the of and evaluation of the potentialis toxicity to selected NTOsregulatory (hazard assessment)owner. was conducted. a document The detailed hazard assessmentany includedsuch toxicity testing against oneits mammalian species reproductiondocument (mice), two avian species (broiler ownerchicken and bobwhitethis quail),of one aquatic species (Daphnia), two species of soil decomposersunder (Collembolaof and earthworm), and four rightsthe this beneficial insectdocument speciesand/or [honeybee (Apis mellifera); rightsminute pirate bugs (Orius to of fall use of insidiosus), ladybird beetle (Coleomegilla maculata);the and parasitic wasp (Ichneumon promissoriusThis )]. The test materialsmay distribution,wereand selected for each study based on the species rights owner being evaluatedsubject and whether more ecologically relevant exposures (plant tissues) could be achieved without compromising the perforthe manceviolate of the study. In many cases, pure be copy proteins were used because ingestion ofof the material could be ensured using artificial and diets containingmay highdocument levelspublication, of either Cry1A.105 or Cry2Ab2 protein. When tissues were It exploitation used (e.g. Collembola),this the expression levels for both Cry1A.105 and Cry2Ab2 proteins were estimated to calculateany the doses. The NOECs (no observed effect concentrations) of permission Cry1A.105 and Cry2Ab2 proteinsprohibited determined for each of the tested NTOs are summarized in Table VIII-1. the be commercial MOEsFurthermore, for theany non-target arthropods were also calculated based on the ratio of the NOECs to theConsequently, MEECs.without The result showed that the MOEs were ≥14 fold of the potential maximum exposure level for these NTOs (Table VIII-2). MOEs that exceed 10 are considered as indicative of minimal risk by many regulatory authorities. Therefore, as with other Cry proteins, Cry1A.105 and Cry2Ab2 are not likely to produce adverse effects at field exposure levels on tested representative terrestrial beneficial invertebrate species. This conclusion is in agreement with prior published literature which reported no adverse effects on non-target organisms from insect-protected crops that produce Cry1

Monsanto Company 06-CR-166U Page 155 of 262 CBI Deleted Version or Cry2 proteins (Mendelsohn et al., 2003; Daly and Buntin, 2005; Dively, 2005; Naranjo et al., 2005, Pilcher et al., 2005).

Table VIII-1. No observed effect concentrations (NOECs) of Cry1A.105 and Cry2Ab2 proteins for non-target organisms

Test organism NOECs for Cry1A.105 NOECs for Cry2Ab2

Collembola 80 µg/g 70 µg/g Earthworm 178 mg/kg dry soil 330 mg/kg dry soil 1 and Honeybee larvae 1100 µg/ml as a single dose 100and µg/ml as a regime.single dose Honeybee adult 550 µg/ml AG 68 µg/ml Minute pirate bugs 120 µg/g 100 µg/g Ladybird beetle 240 µg/g 120 µg/g Bayer property protectionpublishing Parasitic wasp 240 µg/mlof 100 µg/mlcontents Mouse 2072 mg/kg 2198its mg/kg parties.data therefore Quail 50% corn grain from MONand/or 89034or in diet affiliates. Broiler property50% to 55%intellectual cornthird grain from MON 89034may in diet its Daphnia as100 mg/l pollen from MON 89034 the of and 1 The NOEC for the honeybeeis larval assay is based on theregulatory concentration of the dosingowner. solution. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 156 of 262 CBI Deleted Version

Table VIII-2. Estimated margins of exposure (MOEs) to non-target arthropods for the Cry1A.105 and Cry2Ab2 proteins

Test organism Cry1A.105 Cry2Ab2and and regime. Source MEEC1 NOEC MOEAG2 Source MEEC NOEC MOE Collembola Leaf 4.3 mg/kg dry ≥80 µg/g ≥19 Leaf 3.7 mg/kg dry ≥70 µg/g ≥19 soil 3 soil3 Bayer property protectionpublishing Earthworm E. coli 4.3 mg/kg dry ≥178 mg/kg of ≥41 Bt 3.7 mg/kg contentsdry ≥330 mg/kg ≥89 soil dry soil soilits dry soil parties.data therefore Honeybee larvae E. coli 8.8 µg/g fw ≥1100 µg/ml as ≥125 Bt and/or 0.47or µg/g fw ≥100 µg/ml ≥213 (pollen level) affiliates.4 (pollen level) a singleproperty dose intellectualthird may as a single its dose4 as Honeybee adult E. coli 8.8 µg/g fw ≥the550 ofµg/ml and≥ 63 Bt 0.47 µg/g fw ≥68 µg/ml ≥145 is regulatory owner. (pollen level) a document(pollen level) any such its reproductiondocument Minute pirate bugs E. coli 8.8 µg/g fw 120 µg/g owner 14 thisE. coli of0.47 µg/g fw ≥100 µg/g ≥213 (pollen level) under of (pollen level) rightsthe this Ladybird beetle E. coli 8.8 µg/gdocument fwand/or ≥ 240 µg/g ≥27 E. colirights 0.47 µg/g fw ≥120 µg/g ≥255 to of fall use of (pollen level) (pollen level) the Parasitic wasp E. coli This8.8 µg/g fw ≥240may µg/ml distribution, and ≥27 E. coli 0.47 µg/g fw ≥100 µg/ml ≥213 rights owner (pollen subjectlevel) (pollen level) 1 the violate Expression levels determined from MONbe 89034.copy 2 of Margins of Exposure (MOE) were calculated based on the ratio of theand NOEC to MEEC. The MOE was determined based on the may documentpublication, expression level of the Cry1A.105It and Cry2Ab2 proteinsexploitation in the tissue from MON 89034 deemed most relevant to the NTO exposure. this any 3 The MEEC for Collembola and earthworm was calculated using the following parameter assumptions: 25,000 corn plants/acre; corn permission plant dry weight is 650 g /plant; the bulk density of soil isprohibited 1500 kg/cubic meter; soil depth is 0.15 m (about 6 inches); soil volume in a one-hectare 0.15 m layer is 1500 cubic meters.the The Cry1A.105 and Cry2Ab2 expression values were taken for leaves at the pre- commercial be tasseling stage and were 240 and 210 µg/g dwt, respectively. 4 The NOEC for the honeybeeFurthermore, larval assayany is based on the concentration of the dosing solution. Consequently,without

Monsanto Company 06-CR-166U Page 157 of 262 CBI Deleted Version

VIII.A.4. Impact on Endangered Species

As discussed in the above sections, Cry proteins are known to have biological activity exclusively toward insect species. Extensive literature references support the observation that Cry proteins have a high degree of specificity and will not pose a significant hazard to non-insect animals (Federici, 2002; Romeis et al., 2006). This observation has been confirmed through testing with a standard battery of terrestrial and aquatic non-target organisms, including mammals, birds, water fleas and earthworms and beneficial insects, for Bt crop registrations (Mendelsohn et al., 2003). These data establish that the Cry proteins pose negligible risk to non-insect animals and the vast majority of non-target and insects. Based on the demonstrated low hazard of Cry proteinsand to non-insectregime. animals, no adverse effects are expected for endangered mammals,AG birds, non-insect aquatic animals, and non-insect soil organisms.

Bayer property protectionpublishing Monsanto has conducted extensive studiesof testing the Cry1A.105 and Cry2Ab2contents proteins for activity against a range of both target and non-target insect speciesits (refer to Sections parties.data therefore VIII.A.2 and VIII.A.3). Experiments were also conducted and/orto examineor any potential interaction between the two proteins,affiliates. and the results concluded the activities of property intellectualthird may Cry1A.105 and Cry2Ab2 on representativeits lepidopteran species are additive. There are as no synergistic or antagonisticthe interactionsof and between the two proteins. The results also is regulatory owner. show that both the Cry1A.105 and Cry2Ab2a prot eins aredocument highly specificits in insecticidal activity against lepidopteranany insects such and have reproductionlittle activity against non-lepidopteran this documentof insects. These data indicate that theowner only potential risk to endangered species resides under of with endangered butterfliesrights and themoths in the order Lepidopterathis . documentand/or rights to of fall use of Risk assessments of potential effects of Bt crops onthe the federally listed threatened or This may distribution,and endangered lepidopteransrights began with an examinationowner of an overlay map showing the county level distributionsubject of endangered lepidopteran species as listed by the U.S. Fish be the violate and Wildlife Service,copy relative to corn productionof counties in the U.S. The result showed that most endangered lepidopteran speciesand do not occur in agricultural counties where may documentpublication, corn isIt grown. Of the 11 endangeredexploitation lepidop teran species that do occur in counties that also produce corn,this anany analysis of the endangered species critical habitat showed that only the habitat of the Karner blue butterfly (Lycaeides melissa samuelis) has the potential to permission occur in proximity to corn fields.prohibited A recent academic analysis of Karner blue butterfly the phenology and corncommercial pollen shed be showed that there are only two Wisconsin counties in the Midwestern corn belt where there is a potential for temporal overlap between corn pollen shedFurthermore, and theany presence of Karner blue larvae. At present there is no evidence that Karner blueConsequently, larvae arewithout exposed to corn pollen in these two locations (Peterson et al., 2006). Based on its feeding habits and phenology, Karner blue larvae will have a low likelihood of exposure to corn pollen from MON 89034.

Although the likelihood of exposure of Karner blue larvae to corn pollen from MON 89034 is low, a risk analysis based on conservative hazard using the most sensitive species approach and exposure estimates was used to determine the margin of safety. Since Karner blue larvae use wild lupine plant (Lupinus perennis) exclusively as a host

Monsanto Company 06-CR-166U Page 158 of 262 CBI Deleted Version plant, the only route of exposure of Cry1A.105 and Cry2Ab2 proteins to Karner blue larvae is through deposition of pollen from MON 89034 onto lupine plants. The interactions among Karner blue, its lupine host, and corn pollen are very similar to those among the monarch butterfly, milkweed plant, and corn pollen. Therefore, the approaches used for evaluating potential effects of corn pollen on Monarch butterfly (Pleasants et al., 2001; Stanley-Horn et al., 2001, Sears et al., 2001; Dively et al., 2004) were used for assessing the effects of MON 89034 pollen on Karner blue. The exposure level of Karner blue to MON 89034 pollen was estimated based on the potential pollen density on the lupine plants and the combined maximum expression level of Cry1A.105 and Cry2Ab2 proteins in the pollen. Since toxicity testing of Karner blue larvae directly is not possible due to its endangered status, the LC50 value of Cry1A.105 and Cry2Ab2 proteins for larvae of a most sensitive lepidopteran insect – European corn borer (ECB) – regime.and was used in the risk analysis. The results of this risk analysisand showed that there was a AG minimum 12-fold margin of safety for Karner blue larvae in the event that they would be exposed to the highest possible pollen concentration from MON 89034. The margin of safety was calculated as the quotient of theBayer LC50 valueproperty for ECB protectiondividedpublishing by the maximum exposure level of the two proteins. Therefore,of it can be concluded that pollencontents from MON its 89034 is not likely to adversely affect Karner blue larvae.parties. data therefore and/oror affiliates. VIII.A.5. Environmental Fateproperty of Cry1A.105 intellectual thirdand Cry2Ab2 Proteinsmay and Impact on its Soil-dwelling Organisms as the of and is regulatory owner. a documentits Soil organisms may be exposedany tosuch the Cry1A.105reproduction and Cry2Ab2 proteins by contact with this documentof roots, the above-ground plant residuesowner after falling or tilling into the soil, or by pollen under of deposited on the soil. Micrrightsobialthe exposure may occur bythis feed ing on living or dead corn documentand/or rights biomass or by ingestionto or absorptionof fall of the Cryuse proteinsof after their release into the soil. Several soil factors (e.g., pH and clay content) can influenthe ce the degradation rate of Cry This may distribution,and proteins. Published studiesrights s uggest that Cry proteinsowner may bind to the clay components of soil and becomesubject more resistant to degradation by soil microorganisms (Tapp and Stotzky, be the violate 1998). However,copy soil pH near or aboveof neutra lity substantially increases the degradation rate of Cry proteins. Under most corn productionand conditions, corn would be grown on may documentpublication, soils Itthat are near neutral pHexploitation (Wright et al 2004), i.e., under conditions favorable to degradation of thisCry anyproteins. Indeed, published field monitoring studies have shown no persistence or accumulation of Cry proteins in fields where corn expressing Cry1Ab permission protein (Dubelman et al., 2005)prohibited or Cry3Bb1 protein (Ahmad et al., 2005), and cotton the expressing Cry1Accommercial protein be(Head et al., 2002) were grown continuously for several years. Furthermore,any A laboratoryConsequently, aerobicwithout soil degradation study showed that the Cry1A.105 and Cry2Ab2 proteins dissipated rapidly in soils from several sources (MO, IL and ND) that contained significant proportions of clay (up to 41%), organic matter (up to 4.5%) and a range of soil pH (5.9-8.0). Purified Cry1A.105 and Cry2Ab2 proteins were added to each soil in a large excess relative to the maximum (worst-case) concentration possible under corn growing conditions. The large excess was used to improve the accuracy of the analytical quantifications, and to ensure that the DT50 and DT90 (time to 50% and 90% dissipation of the proteins) could be calculated from the measured values. This excess also provided

Monsanto Company 06-CR-166U Page 159 of 262 CBI Deleted Version an increased margin of environmental safety, including possible exudation of the Cry proteins through the roots into soil. A sensitive soil insect bioassay showed that the DT50 ranged 2 to 7 days, and the DT90 ranged 7 to 19 days for the Cry1A.105 protein. The DT50 ranged 0.5 to 3 days and the DT90 ranged 3 to 13 days for the Cry2Ab2 protein.

Published studies on the effect of Cry proteins on soil-dwelling organisms show little or no impact on the soil microflora from the use of biotechnology-derived plants producing Bt proteins. For example, a season-long field study conducted with the Cry3A protein expressed in biotechnology-enhanced potato, showed no adverse effects towards soil- dwelling microorganisms (Donegan et al., 1996). In a study conducted in Kansas during the 2000 and 2001 growing seasons, the numbers of soil mites, Collembola and nematodes observed in plots planted with Cry3Bb1-producing corn were similar to those regime.and observed in plots planted with conventional corn (Al-Deeb et al.,and 2003). Other published AG reports showed that Cry proteins had no microbiocidal or microbiostatic activity in vitro against selected bacteria, fungi, and algae (Koskella and Stotzky, 2002), and had no apparent effect on earthworms, nematodes,Bayer protozoa, property bacteria protection and publishingfungi (Saxena and Stotzky, 2001). of contents its parties.data therefore These results suggest that there will be negligible risk to soil-dwellingand/oror organisms from affiliates. their exposure to MON 89034 propertywhich expr intellectualesses Cry1A.105third and Cry2Ab2may proteins. its as the of and VIII.B. Weediness Potentialis of MON 89034 regulatory owner. a document any such its reproductiondocument In the United States, corn is not listedowner as a weed in thethis major weedof references (Crockett, 1977; Holm et al., 1979; Muenscher, 1980),under nor isof it present on the lists of noxious weed rightsthe this species distributeddocument byand/or the federal government (7 CFR Partrights 360). Furthermore, corn has to of fall use of been grown throughout the world without any report thethat it is a serious weed. Modern corn cannotThis survive as a weedmay because distribution, ofand past selection in the evolution of corn. During rights owner domestication ofsubject corn, traits often associated with weediness such as seed dormancy, a dispersal mechanism, or the ability to formthe reproducing violate populations outside of cultivation be copy have not been selected. For example, theof corn ear is enclosed with husks. Consequently, and seed dispersalmay of individualdocumentpublication, kernels is limited. Even if individual kernels of corn were It exploitation distributed withinthis a field or along transportation routes from the fields to storage or processing facilities,any sustainable volunteer corn populations are not found growing in permission fence rows, ditches, and road prohibitedsides. Corn is poorly suited to survive without human assistance and is not capable of surviving as a weed (Baker, 1965; Keeler, 1989; Galinat, the be 1988). Althoughcommercial corn seed can overwinter into a crop rotation with soybeans, mechanicalFurthermore, anyand chemical measures can be used to control volunteers. Consequently,without In comparative studies conducted between MON 89034 and a conventional control, dormancy and germination, growth and development, and reproductive characteristics were evaluated for changes that would impact plant pest potential, and in particular, plant weediness potential (see Section VII). The results of these studies support a conclusion that the introduction of the insect protection trait did not unexpectedly alter the assessed characteristics compared to the control. Thus, the results support a conclusion of no increased weediness potential of MON 89034 compared to the conventional corn.

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Furthermore, extensive postharvest monitoring of field trial plots planted with MON 89034 under USDA-APHIS notifications (see Appendix H for a complete list of USDA notifications approved for MON 89034) did not reveal any differences in survivability or persistence relative to other corn. These data suggest that MON 89034 is no more likely to become a weed than conventional corn.

VIII.C. Gene Flow

Gene flow (often used synonymously with the term “outcrossing” or “cross pollination”) is a natural biological process that occurs in most crop species including corn. Pollen- mediated gene flow is a term used to describe the movement of plant genes from one plant to another via pollen. The rate of pollen-mediated gene flow depends on biotic and regime.and abiotic factors such as plant biology, pollen biology/volume, plantand phenology, overlap of AG flowering times, proximity of the pollen source and sink, ambient conditions such as temperature and humidity, and field architecture. Bayer property protectionpublishing VIII.C.1. Vertical Gene Flow – Transferof of Genetic Information amongcontents Species with its which Corn can Interbreed parties.data therefore and/oror affiliates. property intellectualthird may Corn morphology fosters cross-pollination;its therefore, high levels of pollen-mediated gene as flow can occur in this species.the of Researchers and also recognize that 1) the amount of gene is regulatory owner. flow that occurs can be high because of open-pollination;a document 2) the percentits gene flow will vary by population, hybridany or inbred;such 3) the levelreproduction of gene flow decreases with greater this documentof distance between the source and recipientowner plants ; 4) environmental factors affect the level under of of gene flow; 5) corn pollenrights is viablethe for a short periodthis of time under field conditions; 6) documentand/or rights corn produces ample pollento of over fallan extendeduse periodof of time; and, 7) there are no purposeful insect pollinators of corn but that pollinatingthe insects, especially bees, are This may distribution,and occasional visitors to therights tassels but rarely visitowner silks of corn. subject be the violate For gene flow to copyoccur by normal sexualof transmission, certain conditions must exist: 1) the two parents must be sexually compatibandle; 2) there must be overlapping phenology; may documentpublication, and 3)It a suitable factor (sucexploitationh as wind or insects) must be present and capable of transferring pollenthis betwany een the two parents.

permission Several studies have been conductedprohibited on the extent of pollen-mediated gene flow in corn. the As expected, resultscommercial were befound to vary depending on the experimental design, environmental conditions and detection method. In general, percent gene flow was found toFurthermore, diminish anywith increasing distance from the source field. It was previously reported that Consequently,corn cross-pollinationwithout rate fell below 1% at distances >200 m (Jemison and Vayda, 2000; Luna et al., 2001). A more recent research showed that corn cross pollination rate decreased below 0.9% level beyond 15 m (Bannert, 2006)

As discussed in Section II.G, corn and annual teosinte (Zea mays subsp. mexicana Schrad.) are genetically compatible, wind-pollinated and, in areas of Mexico and Guatemala, freely hybridize when in close proximity to each other. Corn easily crosses with teosinte; however, teosinte is not present in the U.S. other than as an occasional

Monsanto Company 06-CR-166U Page 161 of 262 CBI Deleted Version botanical garden specimen. These specimens would only flower at the same time as corn if they were subject to artificial day length shortening for several weeks at a time (Wilkes, 1967). Differences in factors such as flowering time, geographical separation and development factors make natural crosses in the U.S. highly unlikely.

In contrast with corn and teosinte, which easily hybridize, it is only with extreme difficulty and special techniques that corn and Tripsacum hybridize. Furthermore, the offspring of the cross show varying levels of sterility (Galinat, 1988; Mangelsdorf, 1974; Russell and Hallauer, 1980). (see Section II.G.). No cases of gene flow between corn and its wild relatives are known to occur naturally in the U.S.

VIII.C.2. Horizontal Gene Flow – Transfer of Genetic Information toregime. Speciesand with which Corn Cannot Interbreed and AG

Monsanto is aware of no reports of the transfer of genetic material from corn to other Bayer property protectionpublishing species with which corn cannot sexuallyof interbreed. The probability for contentshorizontal gene flow to occur is judged to be exceedingly small. Even if it wereits to occur, the parties.data therefore consequences would be negligible since the genes introducedand/or intoor MON 89034 are of bacterial origin and the two Cry proteinsaffiliates. produced have no meaningful toxicity to property intellectualthird may humans and other NTOs under theits conditions of use. as the of and is regulatory owner. VIII.D. Production and Current Agronomica Practices documentfor U.S. Fieldits Corn any such reproduction this documentof This section provides a review of U.S.owner agr onomic practices in corn and the anticipated under of environmental consequencesrights fromthe th e commercializationthis of MON 89034. documentand/or rights to of fall use of VIII.D.1. Production the This may distribution,and rights owner subject Corn is the largestbe U.S. crop in terms of acreagthe violatee planted and net crop value. In the past 10 years (1996-2005),copy total annual cornof acrerage planted varied from approximately 76 to 82 million acres, totaldocument annual production rangedand from 9 to 11.8 billion bushels, and total may publication,exploitation annualIt value fluctuated from 17 to 25 billion dollars depending on the production output this and commodity pricesany ( Table VIII-3). permission prohibited Corn is planted in almost every state in the continental U.S. For convenience the states the can be grouped commercialinto the following be regions (number of states): Midwest (12), Northeast (8), Mid-Atlantic (5), Southeast (9), Great Plains (5), Northwest (4) and Southwest (5). Furthermore,any However,Consequently, the majoritywithout of the corn (86%) is grown in the Midwest region (Table VIII-4). Yields vary considerably from region to region because of the diversity in rainfall/irrigation, climatic conditions and soil productivity.

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Table VIII-3. Field corn production in the U.S. (1996 – 2005)

Acres Acres Yield Production Price Value Year planted harvested (x 1000) (x 1000) (bu/acre) (x 1000 bu) ($/bu) ($ billion) 1996 79,229 72,644 127.1 9,232,557 1997 79,537 72,671 126.7 9,206,832 1998 80,165 72,589 134.4 9,758,685 1999 77,386 70,487 133.8 9,430,612 2000 79,551 72,440 136.9 9,915,051 and and regime. 2001 75,702 68,768 138.2 AG 9,502,580 2002 78,894 69,330 129.3 8,966,787 2003 78,603 70,944 142.2 10,089,222 Bayer property protectionpublishing 2004 80,929 73,631 of 160.4 11,807,086 contents its 2005 81,759 75,107 147.9 11,112,072parties.data therefore and/oror Source: USDA-NASS, 2006c affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VIII-4. Field corn production by regions and states in the U.S. (2005)

Acres % to national Acres planted Yield Production State harvested total (x 1000) (x1000) (bu/acre) (x1000 bu) production Midwest region Illinois 12,100 11,950 143 1,708,850 15.38 Indiana 5,900 5,770 154 888,580 8.00 Iowa 12,800 12,500 173 2,162,500 19.46 Kentucky 1,250 1,180 132 155,760 1.40 Michigan 2,250 2,020 143 288,860 2.60 and Minnesota 7,300 6,850 174 and1,191,900 regime. 10.73 AG Missouri 3,100 2,970 111 329,670 2.97 Nebraska 8,500 8,250 154 1,270,500 11.43 North Dakota 1,410 1,200Bayer property 129 protection 154,800publishing 1.39 of contents Ohio 3,450 3,250 143 464,750its 4.18 parties.data therefore South Dakota 4,450 3,950 119 and/or 470,050or 4.23 Wisconsin 3,800 affiliates.2,900 148 429,200 3.86 property intellectualthird may Regional total 66,310 its 62,790 9,515,420 85.63 as the of and is regulatory owner. a document Northest region any such its reproductiondocument New York 990 owner 460 this 124 of 57,040 0.51 under of Pennsylvania 1,350rights the 960 122this 117,120 1.05 documentand/or rights Connecticut to 28of fall use of Maine 26 the This may distribution,and Massachusetts rights20 owner subject New Hampshire 15 be the violate Rhode Island copy 2 of and Vermontmay documentpublication,95 It exploitation Regional total this 2,526 1,420 174,160 1.57 any permission Mid-Atlantic prohibited region the be Delaware commercial160 154 143 22,022 0.20 Furthermore,Maryland any 470 400 135 54,000 0.49 NewConsequently, Jersey without 80 62 122 7,564 0.07 Virginia 490 360 118 42,480 0.38 West Virginia 45 28 109 3,052 0.03 Regional total 1,245 1,004 129,118 1.16 Source: USDA-NASS, 2006c Table VIII-4 continues on the next page.

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Table VIII-4 (continued). Field corn production by regions and states in the U.S. (2005)

Acres % to national Acres planted Yield Production State harvested total (x 1000) (x1000) (bu/acre) (x1000 bu) production Southeast region Alabama 220 200 119 23,800 0.21 Arkansas 240 230 131 30,130 0.27 Florida 65 28 94 2,632 0.02 Georgia 270 230 129 29,670 0.27 and Louisiana 340 330 136 and44,880 regime. 0.40 Mississippi 380 365 AG 129 47,085 0.42 North Carolina 750 700 120 84,000 0.76 South Carolina 300 285Bayer property 116 protection 33,060publishing 0.30 of contents Tennessee 650 595 130 77,350 0.70 its Regional total 3,215 2,963 parties.data 372,607 therefore 3.35 and/oror affiliates. Great plains property intellectualthird may its region the as of and owner. Colorado is1,100 950 regulatory148 140,600 1.27 a documentits Kansas 3,650any such 3,450 reproduction135 465,750 4.19 this documentof Oklahoma 290 owner 250 115 28,750 0.26 under of this Texas and/or2,050rights the 1,850 114 210,900 1.90 document to fall of rights Wyoming 80of 49 use 140 6,860 0.06 the RegionalThis total 7,170may distribution, 6,549and 852,860 7.68 rights owner subject Northwest be the violate region copy of Idaho document235 and60 170 10,200 0.09 may publication,exploitation MontanaIt 65 17 148 2,516 0.02 this Oregon any 53 25 160 4,000 0.04 permission Washington 150 prohibited 80 205 16,400 0.15 Regional total the 503be 182 33,116 0.30 commercial Furthermore,Southwest any regionConsequently, without Arizona 50 22 195 4,290 0.04 California 540 110 172 18,920 0.17 Nevada 5 0.00 New Mexico 140 55 175 9,625 0.09 Utah 55 12 163 1,956 0.02 Regional total 790 199 34,791 0.31

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VIII.D.2. Corn Cultivation and Management

The key considerations for corn production include soil quality, tillage practices, hybrid selection, moisture, nutrients, and the management of insects, weeds and diseases. A summary of the corn cultivation and management practices is provided below.

VIII.D.2.a. Soil Quality and Tillage Practices

Corn is grown in a variety of soils in the U.S. ranging from the sandhills of Nebraska and Colorado to the clays of delta regions, from strongly acidic to strongly alkaline soils, and and from shallow soils on residual material to deep soils in loess,and till or alluvium.regime. Within these ranges, however, there is a corresponding variationAG in the crop’s productivity. Ideal soils are those that have intermediate textures of loam to silt loam in the surface horizon and somewhat higher clay content as silt loam to silty clay loam in the subsoil. This Bayer property protectionpublishing combination, complemented with good structurof al properties, allows good storagecontents of water and nutrients and a degree of permeability favorable to water intake itsand air exchange. parties.data therefore Corn growth is optimal on soils with pH levels ranging betweenand/oror 6.0 and 7.0 and affiliates. moderate-to-high fertility (Jones,property 2003). intellectualthird may its as There are three main tillagethe practices of employeand d in corn production: conventional tillage, is regulatory owner. reduced tillage, and conservation tillage. Conventia onal tillagedocument practices leave <15% crop any such its reproductiondocument residue cover after planting and involveowner the use of athis moldboa rdof plow or other intensive tillage procedure. Reduced tillage practicesunder leaveof between 15-30% crop residue cover rightsthe this after plantingdocument and and/orexclude the use of a moldboardof plowrights or other intensive tillage procedure. Conservationto tillageof is falla system thatuse covers 30% or more of the soil surface the with cropThis residue after plantingmay to distribution,reduceand soil erosion by water, and consists of three subtypes: no-till, ridge-tillrights and mulch-till. owner These subtypes differ in the timing of subject cultivation of the seedbed and type of equipment used. In recent years, there has been a be the violate trend toward the copyincreased use of conservationof tillage practices. In 1996, the total acres planted to corn were tilled as follows: conservationand tillage (38%), reduced tillage (32%), may documentpublication, and conventionalIt tillage (30%)exploitation (USDA-ERS, 2002). this any VIII.D.2.b. Corn Hybrids permission prohibited the In the U.S. hundredscommercial of corn be hybrids are marketed by seed companies. Their selection is primarily based on yield potential, climatic environment, and disease/pest resistance for thatFurthermore, locale. any One key determinant is the selection of seed with the appropriate maturity without groupConsequently, for the local region (see Section II.E). Corn hybrids with relative maturities of 100 to 115 days are typically grown in the U.S. corn belt. To maximize yield, planting of corn on 30-in rows with a density of between 28,000 to 32,000 kernels per acre is typically recommended for the corn belt (Aldrich et al., 1986; Iowa State University, 2002).

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VIII.D.2.c. Moisture and Nutritional Requirements

In the five major corn belt states, corn uses more moisture for evapotranspiration than is provided by rainfall, especially during the critical months of July and August (Shaw, 1988). The additional moisture requirement can be supplemented by irrigation, which is practiced on 10-11 million acres or approximately 15% of the total corn acreage (USDA- ERS, 2002). Irrigation practices also facilitate the application of agricultural chemicals and fertilizers.

Corn, like all higher plants, requires at least 13 elements from the soil for growth and development (Olson and Sander, 1988). The 13 elements include the primary elementsand and regime. (nitrogen, phosphorus, and potassium), secondaryAG elements (calcium, magnesium, sulphur), and micronutrient elements (iron, manganese, zinc, copper, boron, molybdenum and chlorine). By far the most important are the primary elements, which are depleted in the soil as the corn plant develops. WhereasBayer nitrogen property and phos phatepublishing uptake continues protectioncontents until maturity, potassium absorption is oflargely completed by the silking stage. This is its why fertilization of corn fields is essential to ensureparties. productiondata and profitability.therefore In and/oror 2005, nitrogen was applied to 96% ofaffiliates. the plante d corn acreage at an average usage rate of 138 pounds of nitrogen per acreproperty per crop year.intellectual thirdPhosphate was applied mayto 81% of the corn its as acreage at an average rate theof 58 of pounds perand acre per crop year. Potassium, applied at 84 is regulatory owner. pounds per acre per crop year, was applied to a65% of the documentacreage planted to corn (USDA- NASS, 2006b). any such its reproductiondocument of owner this under of VIII.D.2.d. Management rightsof Diseasesthe and Insects this documentand/or rights to of fall use of Management of diseases and insects during corn growththe and development is essential for This may distribution,and protecting the yield of rightsthe harvested grain. Estimatesowner for annual yield losses because of diseases have rangedsubject from 7 to 17% (Shurtleff, 1980). Incidence of disease infestation is be the violate highly variable andcopy depends on manyof factors such as location, climate, and other environmental factors. Most corn hybrids andon the market today have acceptable levels of may documentpublication, resistanceIt to common diseasesexploitation. The diseases found to occur in corn grown in the U.S. are summarizedthis in any Table VIII-5. In addition, several nematode species have been known to cause diseases in corn (Dicke and Guthrie, 1988). The use of fungicides in permission corn is limited because the incidenceprohibited and severity of most diseases tends to be low and the quite variable. commercial The fungicide be s currently used on corn plants in the U.S. include azoxystrobin, chlorothalonil, and propiconazole (USDA-NASS, 2006b). Furthermore,any TheConsequently, corn crop iswithout subject to attack by a complex of insects from the time it is planted until it is used as food and feed. The economically important insect pests in North America include wireworms, the black cutworm, European corn borer, Southwestern corn borer, the corn rootworms, grasshoppers, fall armyworm, and corn earworm. Table VIII-6 lists the insect pests in corn grown in the U.S. Approximately 27 active ingredients are registered for use in corn for the control of insect pests. In its annual survey of agricultural chemical usage, USDA determined that 23% of the corn acreage was treated with insecticides in 2005 (USDA-NASS, 2006b). Tefluthrin, cyfluthrin, and

Monsanto Company 06-CR-166U Page 167 of 262 CBI Deleted Version tebupirimphos were the most widely applied insecticides, at 7, 7, and 6%, respectively, to the acres planted to corn. Chlorpyrifos was only applied to 2% of the acres, but total quantity applied is more than 3 times greater than next highest insecticide at 2.0 million pounds.

The introduction of biotechnology-derived Bt corn has offered growers an alternative and effective solution for the control of major insect pests in corn. The Bt corn products which have been commercialized in the U.S. include YieldGard® Corn Borer corn (producing Cry1Ab), YieldGard® Rootworm corn (Cry3Bb1), Herculex® I (Cry1F), Herculex® RW (Cry34Ab1 and Cry35Ab1), and Bt11 (Cry1Ab). These Bt corn products provide control of a spectrum of lepidopteran and coleopteran insect pests. In 2006, approximately 40% of the total corn acreage in the U.S. was planted with hybrids regime.and possessing insect protection traits (USDA-NASS, 2006a). and AG

VIII.D.2.e. Weed Management Bayer property publishing protectioncontents Weeds cause significant losses and requireof careful management by the growers because its they interfere with corn plants by competing for availableparties.data resources includingtherefore water, and/oror nutrients and light. Economicallyaffiliates. damaging weeds in corn include annuals and perennials, grasses, broadleaf propertyand sedge intellectual species.third Some weeds can maytolerate cold, wet its as conditions better than corn,the and ofcan get a headand start prior to planting. Fields infested with is regulatory owner. perennial weeds present special problems afo r corn growers.document Like annual weeds, perennials can reproduce byany seeds, such but they also regrow and spreadits vegetatively. This reproductiondocument of means that their rhizomes, thickenedowner roots or tubers thispropogate new shoots, usually soon under of after corn is planted. Unlessrights effectivelythe controlled, perennialthis weeds can quickly gain a documentand/or rights season-long advantage overto theof cornfall crop. use of the This may distribution,and Corn yield loss is generallyrights proportional to thownere amount of weeds present. While the ratio is not always one-to-one,subject some studies suggest that for every pound of weed dry matter, there is a reductionbe of approximately one thepound violate of corn plant dry matter (Gianessi et al., copy of 2002). Competition for light, nutrients, andand moisture resources by the crop and weeds can leadmay to proportional documentpublication, reductions in yield (Knake et al., 1990). Numerous studies have It exploitation shown that weed control early in the growing season is necessary to reduce yield losses in this any corn. Weed species such as giant foxtail, barnyardgrass and pigweed can reduce corn permission yields by up to 13, 35 and 50%prohibited respectively (Bosnic and Swanton, 1997; Fausay et al., 1997; Knake and Slife,the 1965). be In a study of mixed weed populations competing with corn, corn yieldscommercial were reduced by up to 20% when the weed plants reached a height of eightFurthermore, inchesany (Carey and Kells, 1995). Consequently,without Corn is typically planted in wide rows (30 inches) and has an upright leaf orientation. As a result, corn is not successful in competing with weeds early in the growing season. Corn is also usually planted early when soil temperature and weather conditions favor weed over corn growth. A survey of Extension Service weed scientists solicited estimates of the percent of corn acreage infested with individual weed species by state or region, as well as the the potential impact on corn yields if the species were left uncontrolled. In this survey, 12 annual broadleaf, nine annual grass, and seven perennial

Monsanto Company 06-CR-166U Page 168 of 262 CBI Deleted Version species were identified as troublesome weeds (Table VIII-7) (Gianessi et al., 2002). Estimates of yield loss ranged from a low of 15% due to wirestem muhly and sandburs to a high of 48% from burcucumber.

Pigweed is the most widespread weed species, infesting corn fields throughout the U.S. despite the fact that it is readily controlled by most corn herbicides. This is because it is a prolific seed producer and those seeds remain viable in the soil for years. Some weed species are problems at a regional level, e.g., hemp dogbane is a problem in Missouri and Illinois, wirestem muhly is a problem in Pennsylvania, and woolly cupgrass is a problem in Wisconsin and Iowa.

Until the early 1950s, tillage and cultivation practices were primarily used for weed regime.and control in corn, but since then they have been largely replaced byand the use of herbicides. AG Herbicide use in corn became widespread by the end of the 1970s. In 2005, herbicides were applied to 97% of the corn planted acreage (USDA-NASS 2006b). Atrazine continues to be the most widely applied heBayerrbicide withproperty 66% of protectionthe plantedpublishing acreage being treated. It was applied at an averageof rate of 1.133 pounds per acre.contents Glyphosate its isopropylamine salt was applied to 31% of planted parties.acres,data up from 19% intherefore 2003, at an average rate of 0.963 pounds per acre. In terms of area applied,and/or that orwas followed closely affiliates. by S-metolachlor and acetochlor,property at 23% ofintellectual the thirdplanted corn acreage treated.may Glyphosate its (Roundup) is the most effective herbicideas in corn. Roundup Ready corn developed by the of and Monsanto makes the applicationis of glyphosateregulatory easy for control of a owner.broad spectrum of a document weeds. any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VIII-5. Diseases of corn

Common Name Causative Agent [transmittal agent] Seed rots and seedling blights Fusarium moniliform, Pythium spp. Foliar Diseases Bacterial leaf blight and stalk rot Pseudomonas avenae Bacterial stripe Pseudomonas andropogonis Stewart’s wilt Erwinia stewartii Chocolate spot Pseudomonas coronafaciens Goss’s wilt Clavibacter michiganense Holcus spot Pseudomonas syringe Anthracnose Colletotrichum graminicola and Eyespot Kabatiella zeae and regime. Gray leaf spot CercosporaAG zeae-maydis Northern leaf spot Bipolaris zeicola Northern corn leaf blight Exserohilum turcicum Physoderma brown spot PhysodermaBayer maydisproperty publishing protectioncontents Southern corn leaf blight ofBipolaris maydis its Yellow leaf blight Phyllosticta maydisparties.data therefore and/or Common rust Puccinia sorghi or affiliates. Southern corn rust property Pucciniaintellectualthird polysora may its Common corn smut as Ustilago maydis the of and Systemic Diseases is regulatory owner. a document Head smut any such Sphacelotheca reiliana its reproductiondocument Crazy top ownerSclerophthora this macrospora of Sorghum downy mildew Peronosclerospora sorghi under of this Maize dwarf mosaic virus rightsthe [aphids] documentand/or of rights Maize chlorotic dwarf virusto of fall [leafhoppers]use Corn lethal necrosis [chrysomelid beetles]the This may distribution,and Maize white line mosaicrights virus [not identified]owner Corn stunt subject [leafhoppers] Maize bushybe stunt [leafhoppers]the violate copy of Stalk and root rots and Gibberellamay stalk rot documentpublication, Gibberella zeae It exploitation Diplodia stalk rotthis Stenocarpella maydis Anthracnose stalk rotany Colletotrichum graminicola Charcoal rot permission Macrophomina phaseolina prohibited Fusarium stalk rot Fusarium moniliforme the Pythium stalk rotcommercial be Pythium aphanidermatum Bacterial stalk rot diseases Erwinia chrysanthemi Furthermore,Root rots any Pythium spp. without Ear Consequently,rots and storage molds Fusarium ear rot Fusarium moniliforme Gibberella ear rot Gibberella zeae Diplodia ear rot Diplodia maydis Aspergillus ear and kernel rot Aspergillus flavus Storage molds Penicillium spp., Aspergillus spp. Source: Smith and White, 1988

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Table VIII-6. Insect pests of corn

Common Name Latin name Soil Insects Northern corn rootworm Diabrotica barberi Western corn rootworm Diabrotica virgifera virgifera Southern corn rootworm Diabrotica undecimpunctata Black cutworm Agrotis ipsilon Wireworms A. mancus, Horistonotus uhlerii, Melanotus cribulosus, others Billbugs Sphenophorus spp. White grubs Phyllophaga spp. and Corn root aphid Anuraphis maidiradicisand regime. Seedcorn maggot Delia platuraAG Grape colaspis Colaspis brunnea Seed corn beetle Stenolophus lecontei Bayer property publishing protectioncontents Insects attacking the leaf, stalk, and ear of its Corn earworm Helicoverpa parties.zea data therefore and/or European corn borer Ostrinia nubilalis or affiliates. Corn leaf aphid property Rhopalosiphumintellectualthird maidis may its Fall armyworm as Spodoptera frugiperda the of and Stalk borers is Diatraearegulatory spp. owner. a document Armyworm any such Pseudaletia unipuncta its reproductiondocument Lesser stalk borer ownerElasmopalpus this lignosellus of Chinch bug Blissus leucopterus leucopterus under of this Grasshoppers rightsthe Melanoplus differentialis documentand/or of rights Corn flea beetle to of fall Chaetocnemause pulicaria Japanese beetle Popillia japonicathe This may distribution,and rights owner Other insects subject Thrips be Anaphothripsthe violate spp., Frankliniella spp. copy of Leafhoppers Trigonotylusand brevipes, others Westernmay bean cutwormdocument publication, Striacosta albicosta It exploitation Corn blotch leafthis miner Agromyza parvicornis Spider mites any Oligonychus spp., Tetranychus spp. Pink scavenger caterpillar permission Pyroderces rileyi prohibited Garden symphlan Scuttigerella immaculata the Hop-vine borer commercial be Hydraecia immanis Sod webworms Subfamily Cramdinae Furthermore,Leaf rollers any without StinkConsequently, bugs

Insect disease vectors Several

Sources: Dicke and Guthrie, 1988; and University of Missouri, 1998.

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Table VIII-7. Troublesome weeds in U.S. corn field

Acreage Infested Potential Yield Loss Weed Species Area Infested 1 (%) (%) Annuals Broadleaves Burcucumber PA/OH/TN/SE 5-10 48 Cocklebur MW/NP/SE 20-60 33 Jimsonweed MW/CO 5-20 17 Kochia NP/NW 10-70 33 Lambsquarters MW/SE/NE/CA 15-80 33 Morningglory MW/SE/SP 20-75 33 and Nightshade MW/NP/CA 25-50 and regime.26 Pigweeds/Waterhemp US AG 30-90 36 Ragweed, Common MW/SE/NE 20-70 30 Ragweed, Giant MW/NP 10-45 28 Smartweeds MW/SD/NE/SE Bayer property30-70 publishing22 protectioncontents Velvetleaf MW/NE/NPof 25-70 28 its parties. data therefore and/or Grasses or affiliates. Barnyardgrass SP/NW/CAproperty intellectualthird 80-90 may 23 its Bermudagrass MD/SE/UT/CAas 10-20 47 the of and Crabgrass spp. is MW/SE/NE regulatory20-80 owner. 29 a document Cupgrass, Woolly any IA/WIsuch 15-20 its 29 reproductiondocument Foxtail spp. MW/NE/NPowner this50-90 of 31 Millet, Wild-Proso UT/WY/CO/ID 15-40 31 under of this Panicum, Fall rightsMW/SE/NE/NPthe 15-80 30 documentand/or of rights Sandburs to NP/UT/WYof fall use 5-30 15 Shattercane MW/SP the5-40 33 This may distribution,and rights owner Perennials subject Bindweed, Fieldbe ND/SW/CA the violate 40-80 18 copy of Dogbane, Hemp IL/MO and 2-20 21 Johnsongrassmay documentpublication,MW/SE/SW/CA 20-60 45 It exploitation Muhly, Wirestemthis PA 2 15 Nutsedge, Yellow any MW/SE/NE/NP/CA 10-70 21 Quackgrass permissionMW/NE/UT 10-70 27 prohibited Thistle, Canada NE/MW/NP/CO 5-25 26 the be Source: Gianessi et commercialal., 2002. 1 Furthermore,Regions any States MW:Consequently, Midwest without US:United States CA: California OH: Ohio NE: Northeast CO: Colorado PA: Pennsylvania NP: Northern Plains ID: Idaho SD: South Dakota NW: Northwest IA: Iowa TN: Tennessee SE: Southeast MD: Maryland UT: Utah SW: Southwest MO: Missouri WI: Wisconsin SP: Southern Plains ND: North Dakota WY: Wyoming

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VIII.D.2.f. Volunteer Management

Volunteer corn commonly occurs in rotational crops in the season following corn cultivation regardless of whether or not the corn was conventional or biotechnology- derived. Volunteers in rotational crops do not occur with corn grown for silage (approximately 9% of the US corn acres) since corn harvested for silage does not produce grain. In the warmer climates of the Southeast and Southwest, the occurrence of volunteer corn is rare because any corn grain remaining after harvest is likely to germinate in the fall and the resulting plants can usually be controlled by tillage or by freezing temperatures in the winter. In the Northern corn-growing regions, volunteer corn does not always occur in the rotational crop because of seed decomposition andover the and regime. winter, efficient harvest procedures, and tillage priorAG to planting rotational crops.

The first step to manage volunteer corn in rotational crops is to minimize or reduce the potential for volunteers. The followingBayer practices propertyshould be implementedpublishing to reduce protectioncontents volunteer corn in rotational crops: 1) adjustof harvest equipment to minimize the amount of its corn grain lost in the field, 2) plant corn hybrids thatparties. reducedata the extent oftherefore ear drop, 3) and/oror choose corn hybrids with superior stalkaffiliates. strength and reduced lodging, and 4) practice no- till production to significantly propertyreduce the intellectualpotentialthird for volunteer growthmay in the rotational its as crop. If volunteer corn thedoes ofoccur in subsand equent crops, preplant tillage or in-crop is regulatory owner. cultivation is very effective in managing it. Selectivea herbicidesdocument labeled for the control of volunteer corn in the particularany suchrotational crop are available. Assureits II® (quizalofop), reproductiondocument of Fusilade® DX (fluazifop), Fusion® owner(fluazifop + fenoxaprop),this Poast® (sethoxydim), and under of Select® 2EC (clethodim) providerightsthe effective postemergencethis control of volunteer corn in documentand/or rights labeled crops. These productsto of are labeledfall for use in eightof field crops, including soybeans, cotton, sugar beet and alfalfa, and eleven vegetable cropsthe id entified as rotational crops for This may distribution,and corn. rights owner subject be the violate VIII.E. Impact ofcopy the Introduction ofof MON 89034 on Agronomic Practices and may documentpublication, No negativeIt impact is expectedexploitation from the introduction of MON 89034 on current cultivation andthis management any practices for corn. MON 89034 has been shown to be no different from conventional corn in its phenotypic, ecological, and compositional permission characteristics (refer to Sectionprohibited VII). Thus, MON 89034 is expected to be similar in its agronomic characteristics,the and have the same levels of resistance to insects and diseases commercial be as current commercial corn, except for the introduced trait of enhanced protection from feedingFurthermore, damageany caused by lepidoperan pests. Biotechnology-derived Bt corn has been cultivatedConsequently, and consumedwithout in the US since 1997 and has proven to have a positive impact on agricultural practices. These products have provided simple, inexpensive and highly effective means for controlling lepidopteran and coleopteran pests. They are

® Assure II is a trademark of E.I. DuPont de Nemours, Inc. ® Fusilade and Fusion are trademarks of Syngenta Group Company. ® Poast is a trademark of BASF Corporation. ® Select is a trademark of Valent U.S.A. Corporation.

Monsanto Company 06-CR-166U Page 173 of 262 CBI Deleted Version environmentally benign so that they preserve beneficial insects, decrease cultivation input, and require less chemical pesticide applications. MON 89034, with two Cry proteins in a single plant, will provide an even more effective tool for corn farmers to control lepidopteran pests compared with Bt corn which only produces one Cry protein.

VIII.F. Insect Resistance Management

A critical component for the long-term use of biotechnology-derived Bt crops containing insecticidal proteins is to implement Insect Resistance Management (IRM) programs to prevent or delay the onset of resistance in the target insect species. Research by industry as well as academic scientists over the past decade has improved understanding and gained broad agreement for the major elements of IRM plans for Bt crops. The core regime.and element of an IRM plan is the use of a refuge to ensure anand adequate population of AG susceptible insects of the target species is available to mate with any resistant insects that survive exposure to the Cry protein produced by the crop. This refuge may include wild host plants, other crops, or non-Bt plantingsBayer of theproperty crop in question.protectionpublishing Monsanto has developed an IRM plan for MON 89034,of which has been submitted separatelycontents to the its EPA. A summary of the plan is included as follows. parties. data therefore and/oror affiliates. The combination of the Cry1A.105property and Cry2Ab2intellectualthird insecticidal proteinsmay in a single plant its MON 89034 provides both better insectas control than current Bt corn products and a more the of and effective approach for IRM.is Mathematical modelingregulatory conducted by Monsantoowner. indicates a document that biotechnology-derivedany plants such expressing two Cry proteins willits have significantly reproductiondocument greater durability than plants producingowner either ofthis the single ofproteins if: (1) cross- resistance between the Cry proteins isunder low; and (2)of the mortality of susceptible insects rightsthe this caused by eachdocument of theand/or individual proteins is at least 90%, rightsand preferably >95%. Several to of fall use of lines of evidence indicate that the Cry1A.105 and Cry2Ab2the proteins have important differencesThis in the way in whichmay they distribution, bindand to the lepidopteran midgut. Therefore, the rights owner probability of subjectcross-resistance between these proteins is very low. Comparable biophysical studies also indicate that Cry1A.105the violate differs from Cry1Ab in the mode of be copy action with regard to binding to the insectof midgut. Therefore, Cry1A.105 and Cry2Ab2 and have distinctmay modesdocument of actionpublication, regardi ng to receptor binding. Furthermore, in vitro and in It exploitation planta studies thiswith Cry1A.105 and Cry2Ab2 demonstrate that both proteins are highly active against the primaryany lepidopteran pests of corn (ECB, SWCB, CEW, and FAW), permission particularly ECB, achieving closeprohibited to or greater than the critical 95% level of control in all cases. With these properties, MON 89034 should be durable with a significantly smaller the be structured refugecommercial than is necessary for a Bt corn product containing only a single insecticidalFurthermore, anyprotein. Based on these data and appropriate mathematical models, a 5% structuredConsequently, refugewithout in the U.S. corn belt (where current Bt corn products have a 20% refuge), and a 20% structured refuge in cotton growing regions (where current Bt corn products have a 50% refuge), is predicted to preserve the durability of MON 89034 for more than 20 years.

The specific requirements of the IRM plan for MON 89034 are described below.

1) Refuge Requirements - Planting of a 5% non-B.t. corn refuge in the U.S. corn belt and a 20% non-B.t. corn refuge in cotton growing regions.

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2) Grower Agreements - Requirement for the growers to sign a contractual agreement, which imposes an obligation on each grower to comply with the refuge requirements. 3) Grower Education – Requirement to develop, implement, and report to EPA on a program to educate growers about IRM requirements for MON 89034. 4) Compliance Assurance Program - Requirement to develop, implement, and report to EPA on a program to evaluate and promote grower compliance with IRM requirements. 5) Monitoring - Requirement to develop, implement, and report to EPA on programs to evaluate whether there are statistically significant and biologically relevant changes in target insect susceptibility to the Cry1A.105 or Cry2Ab2 proteins.and and regime. 6) Mitigation - Requirement to develop and,AG if triggered, to implement a remedial action plan in the event that any insect resistance is detected. 7) Reporting to EPA - Submit annualBayer reports onproperty sales of MONpublishing 89034 seed, IRM protectioncontents grower agreement results, complianceof assurance, and educational program. its parties.data therefore and/oror VIII.G. Conclusions of the Environmenaffiliates.tal and Agronomic Practice Assessment property intellectualthird may its as An environmental assessmentthe ofof MON 8903and 4 was conducted to assess the potential is regulatory owner. impact of the introduced Cry1A.105 and Cray2Ab2 proteinsdocument on non-target organisms, any such its endangered species and soil-dwelling organisms,reproduction the potentialdocument for gene flow, and the owner this of weediness potential of MON 89034. Based on the results of this assessment, it has been under of this concluded that the potentialrights riskthe of MON 89034 and the Cry1A.105 and Cry2Ab2 documentand/or of rights proteins to cause adverseto effectsof onfall NTOs anduse endangered species is negligible. MON 89034 is no more likely to become a weed than conventionalthe corn, and MON 89034 is This may distribution,and also expected to be similarrights to conventional coownerrn regarding to the potential for gene flow. subject Introduction of MON 89034 will not impact current corn cultivation practices and the be the violate management of weeds,copy diseases and insectsof except for the control of lepidopteran insect pests. The combination of the Cry1A.105and and Cry2Ab2 insecticidal proteins in a single may documentpublication, plant Itprovides not only a widerexploitation spectrum of pest control but also a more effective insect resistance managementthis any tool. permission VIII.H. References prohibited the commercial be Ahmad, A., G.E. Wilde, and K.Y. Zhu. 2005. Detectability of coleopteran-specific Furthermore,Cry3Bb1any protein in soil and its effect on nontarget surface and below-ground Consequently,arthropods.without Environ. Entomol. 34:385-394. Al-Deeb, M.A., G.E. Wilde, J.M. Blair, and T.C. Todd. 2003. Effect of Bt corn for corn rootworm control on nontarget soil microarthropods and nematodes. Environ. Entomol. 32:859-865. Aldrich, S.A., W.O. Scott., and R.G. Hoeft. 1986. Page 48 in Origin of Corn: Modern Corn Production. 3rd ed. A&L Publications Inc., Champaign, Illinois.

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Baker, H.G. 1965. Characteristics and modes of origin of weeds. Page 147-172 in The Genetics of Colonizing Species. Baker, H.G. and G.L. Stebbins (eds.). Academic Press, New York. Bannert, M. 2006. Simulation of transgenic pollen dispersal by use of different grain colour maize. Doctor of Sciences dissertation. Swiss Federal Institute of Technology Zurich. Diss. ETH No. 16508. Bhatti, M.A., J. Duan, G. Head, C. Jiang, M.J. McKee, T.E. Nickson, C.L. Pilcher, and C.D. Pilcher. 2005. Field evaluation of impact of corn rootworm (Coleopteran: Chrysomelidae)-protected Bt corn on ground-dwelling invertebrates. Environ. Entomol. 34:1325-1335. Bitzer, R.J., M.E. Rice, C.D. Pilcher, C.L. Pilcher, and W.-K.F. Lam. 2005. Biodiversityand and regime. and community structure of epedaphic andAG euedaphic springtails (Collembola) in transgenic rootworm Bt corn. Environ. Entomol. 34:1346-1376. Bosnic, A.C. and C.J. Swanton. 1997. Influence of barnyardgrass (Echinochloa crus- Bayer property protectionpublishing galli) time of emergence and densityof of corn (Zea mays). Weed Sci.contents 45:276-282. its Bravo, A., J. Sanchez, T. Kouskoura, and N. Crickmore.parties. data2002. N-terminaltherefore activation is an essential early step in the mechanism of action of and/orthe Bacillusor thuringiensis affiliates. Cry1Ac insecticidal toxin.property J. Biol. intellectualChem.third 277:23985-23987. may its Carey, J.B. and J.J. Kells. 1995. Timingas of total postemergence herbicide applications to the of and maximize weed controlis and corn (Zea maysregulatory) yield. Weed Technol.owner. 9:356-361. a document any such its Choma, C.T., W.K. Surewicz, P.R. Carey, M. Pozsgay,reproduction T. Raynor,document and H. Kaplan. 1990. owner this of Unusual proteolysis of the protoxin and toxin from under of Bacillus thuringiensis: structural implications.rights Eur.the J. Biochem. 189:523-527.this documentand/or rights to of fall use of Crickmore, N., D.R. Ziegler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, the andThis D.H. Dean. 1998. Revisionmay distribution, ofand the nomenclature for the Bacillus thuringiensis rights owner pesticidalsubject crystal proteins. Microbiol. Mol. Biol. Rev. 62:807-813. Crockett, L. be1977. Wildly Successful Plants:the violate North American Weeds. University of copy of Hawaii Press, Honolulu, Hawaii. and may documentpublication, Daly, ItT. and D. Buntin. 2005.exploitation Effect of Bacillus thuringiensis transgenic corn for lepidopteranthis any control on nontarget arthropods. Environ. Entomol. 34:1292-1301. permission De Maagd, R.A., A. Bravo, andprohibited N. Crickmore. 2001. How Bacillus thuringiensis has evolved specificthe toxins to colonize the insect world. Trends Genet. 17:193-199. commercial be De Maagd, R.A., M. Weemen-Hendriks, W. Stiekema, and D. Bosch. 2000. Bacillus Furthermore,thuringiensisany delta-endotoxin Cry1C domain III can function as a specificity Consequently,determinantwithout for Spodoptera exigua in different, but not all, Cry1-Cry1C hybrids. Appl. Environ. Microbiol. 66:1559-1563. Dicke, F.F. and W.D. Guthrie. 1988. The most important corn insects. In Corn and Corn Improvement, 3rd ed. Sprague, G.G. and J.W. Dudley (eds.). American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Madison, Wisconsin.

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Dively, G., R. Rose, M. Sears, R. Hellmich, D. Stanley-Horn, D. Calvin, J. Russo, and P. Anderson. 2004. Effects on monarch butterfly larvae (Lepidoptera: Danaidae) after continuous exposure to Cry1Ab expressing corn during anthesis. Environ. Entomol. 33:1116-1125. Dively, G.P. 2005. Impact of transgenic VIP3A x Cry1Ab lepidopteran-resistant field corn on nontarget arthropod community. Environ. Entomol. 34:1267-1291. Donegan, K.K., D.L. Schaller, J.K. Stone, L.M. Ganio, G. Reed, P.B. Hamm, and R.J. Seidler. 1996. Microbial populations, fungal species diversity and plant pathogen levels in field plots of potato plants expressing the Bacillus thuringiensis var. tenebrionis endotoxin. Transgenic Res. 5:25-35. Dubelman, S., B.R. Ayden, B.M. Bader, C.R. Brown, C. Jiang, and D. Vlachos.and 2005. and regime. Cry1Ab protein does not persist in soil AGafte r 3 years of sustained Bt corn use. Environ. Entomol. 34:915-921. EPA. 1985. Hazard evaluation division, standard evaluation procedure: acute toxicity for Bayer property protectionpublishing freshwater invertebrates. PB86-129269.of contents its EPA. 1996, Harmonized OPPTS Testing Guidelinesparties. 712-C-96-280data Februarytherefore 1996 as defined in 40 CFR 152.20. U.S. Environmental Protectionand/or Agency.or Washington, affiliates. DC. property intellectualthird may its EPA. 2001a. Regulation under the FIFRAas for plant-incorporated protectants (formerly the of and plant-pesticides).is Federal Register: 59: 37772regulatory – 6054737817. U.S.owner. Environmental a document Protection Agency. anyWashington, such DC. its reproductiondocument owner this of EPA. 2001b. SAP Report No. 2000-07. FIFRA Scientific Advisory Panel Meeting, under of October 18-20, 2000;rights Finalthe Report: “Sets of Scientificthis Issues Being Considered documentand/or of rights by the Environmentalto ofProtection fall Agencyuse Rega rding: Bt Plant-Pesticides Risk and Benefits Assessments.” U.S. Environmental Protectionthe Agency, Washington, DC This may distribution,and http://www.epa.gov/scipoly/sap/meerights tings/2000/october/octoberfinal.pdfowner subject EPA. 2001c.be SAP Report No. 2000-07.the FIFRA violate Scientific Advisory Panel Meeting, October 18-20,copy 2000. “Issues ofpertaining to the Bt plant pesticides Risk and Benefit Assessments”,document Section andC: Environmental Assessment. U.S. may publication,exploitation It Environmental Protection Agency, Washington, DC. this http://www.epa.gov/scipoly/sap/any meetings/2000/october/brad3_ enviroassessment.pdf.permission prohibited EPA. 2001d. Biopesticidesthe beRegi stration Action Document: Bacillus thuringiensis (Bt) Plant-incorporatedcommercial Protectants (October 15, 2001). U.S. Environmental Protection Furthermore,Agency,any Washington, DC. Consequently, http://www.epa.gov/pesticides/biowithout pesticides/pips/bt_brad.htm EPA. 2002. Corn Rootworm Plant-Incorporated Protectant Nontarget Insect and Insect Resistance Management Issues Part B: Insect Resistance Management Issues. FIFRA Scientific Advisory Panel Meeting, August 27-29, 2002, SAP Meeting Minutes No., 2002-05A. EPA. 2003. Biopesticide Rgistration Ation Dcument (BRAD). Event MON 863 Bacillus thuringiensis Cry3Bb1 corn. U.S. Environmental Protection Agency.

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EPA. 2004a. FIFRA Scientific Advisory Panel Meeting. SAP Report No. 2004-05 Product characterization, human health risk, ecological risk, and insect resistance management for Bacillus thuringiensis (Bt) cotton products. U.S. Environmental Protection Agency, Washington, DC. EPA. 2004b. Overview of the Ecological Risk Assessment Process in the Office of Pesticide Programs, U.S. Environmental Protection Agency - Endangered and Threatened Species Effects Determinations. http://epa.gov/espp/consultation/ecorisk-overview.pdf. Fausay, J.C., J.J. Kells, S.M. Swinton, and K.A. Renner. 1997. Giant foxtail interference in non-irrigated corn. Weed Sci. 45:256-260. Federici, B. 2002. Case study: Bt crops-a novel mode of insect resistance. Pages 164-200and and regime. in Genetically Modified Crops: AssessingAG Safety. Atherton, K. (Ed.), Taylor & Francis Group, London. Galinat, W.C. 1988. The origin of corn. Pages 1-31 in Corn and Corn Improvement. Bayer property protectionpublishing Sprague, G.F. and J.W. Dudleyof (eds.). American Society of Agronomy,contents Crop Science Society of America, and Soil Science Society of America,its Madison, parties.data therefore Wisconsin. and/oror affiliates. Gianessi, L.P., C.S. Silvers, S.property Sankula, intellectualand J.E.third Carpenter. 2002. Plantmay biotechnology: its current and potential impact foras improving pest management in U.S. agriculture. the of and an analysis of 40is case studies. Nationalregulatory Center for Food & Agriculturalowner. Policy, a document Washington, D.C. any such its reproductiondocument Gill, S.S., E.A. Cowles, and P.V. ownerPietrantonio. 1992.this The modeof of action of Bacillus under of thuringiensis endotoxins.rightsthe Annu. Rev. Entomol. 37:615-636.this documentand/or of rights Greenplate, J.T., J.W. Mullins,to of S.R.fall Penn, A. useDahm, B.J. Reich, J.A. Osborn, P.R. Rahn, L. Ruschke, and Z.W. Shappley. 2003. Partialthe characterization of cotton plants This may distribution,and expressing tworights toxin proteins fromowner Bacillus thuringiensis: relative toxin contribution,subject toxin interaction, and resistance management. J. Appl. Entomol. be the violate 127:340-347.copy of Grochulski, P., L. Masson,document S. Borisova, M.and Pusztai-Carey, J.L. Schwartz, R. Brousseau, may publication,exploitation It and M. Cygler. 1995. Bacillus thuringiensis Cry1A(a) insecticidal toxin-crystal this structure andany channel formation. J. Mol. Biol. 254:447-464. permission Head, G., J.B. Surber, J.A. Watson,prohibited J.W. Martin, and J.J. Duan. 2002. No detection of Cry1Ac protein thein soil be after multiple years of transgenic Bt cotton (Bollgard) use. Environ. commercialEntomol. 31:30-36. Head,Furthermore, G., anyW. Moar, M. Eubanks, B. Freeman, J. Ruberson, A. Hagerty, and S. without Consequently,Turnipseed. 2005. A multiyear, large-scale comparison of arthropod populations on commercially managed Bt and non-Bt cotton fields. Environ. Entomol. 34:1257-1266. Hellmich, R.L., B.D. Siegfried, M.K. Sears, D.E. Stanley-Horn, M.J. Daniels, H.R. Mattila, T. Spencer, K.G. Bidne, and L.C. Lewis. 2001. Monarch larvae sensitivity to Bacillus thuringiensis-purified proteins and pollen. Proc. Natl. Acad. Sci. USA 98:11925-11930.

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Holm, L., J.V. Pancho, J.P. Herberger, and D.L. Plucknett. 1979. Introduction. Pages i-vii in A Geographical Atlas of World Weeds. John Wiley and Sons, New York. Iowa State University. 2002. Agronomy 212 -- Grain and Forage Crops. http://www.agron.iastate.edu/courses/agron212/Calculations/calculations.htm Jemison, J.M. and M. Vayda. 2000. Pollen transport from genetically modified corn. Page 145 in 2000 Agronomy Abstracts. American Society of Agronomy (ASA), Madison, Wisconsin. Jones, J.B. Jr. 2003. Agronomic Handbook: Management of Crops, Soils, and Their Fertility. Page 26. CRC Press LLC, Boca Raton, Florida. Keeler, K. 1989. Can genetically engineered crops become weeds? BioTechnol. 7:1134- and 1139. and regime. Knake, E.L. and F.W. Slife. 1965. Giant foxtailAG seeded at various times in corn and soybeans. Weeds 13:331-334. Knake, E.L., J.D. Doll, J.F. Miller and O.E.Bayer Strand. 1990.property Annual Grasspublishing Weeds in Corn. protectioncontents National Corn Handbook, Michiganof State University. its parties.data therefore Koskella, J. and G. Stotzky. 2002. Larvicidal toxins from Bacillusand/or thuringiensisor subspp. kurstaki, morrisoni (strain tenebrionsisaffiliates. ), and israelensis have no microbiocidal or property intellectualthird may microbiostatic activity againstits selected bacteria, fungi, and algae in vitro. Can. J. as Microbiol. 48:262-267.the of and is regulatory owner. a document Lee, H. and S.S. Gill. 1997.any suchMolecular cloning and characterizationits of a novel reproductiondocument mosquitocidal protein geneowner from Bacillus thisthuringiensis of subsp. fukuokaensis. Appl. Environ. Microbiol. 63:4664-4670.under of rightsthe this documentand/or rights Li, J., J. Carroll, and D.J.to Ellar.of 1991.fall Crystaluse structure of of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 Å resolution. theNature 353:815-821. This may distribution,and Lopez, M., J. Prasifka, rightsD. Bruck, and L. Lewis.owner 2005. Utility of ground beetle species in subject field tests of potential nontarget effects of Bt crops. Environ. Entomol. 34:1317- be the violate 1324. copy of and Lozzia, mayG.C., C. Furlanis,documentpublication, B. Manachini, I. E. Rigamonti. 1998. Effects of Bt corn on It exploitation Rhopalosiphumthis padi L. (Rhynchota Aphidae) and on its predator Chrysoperla carnea Stephenany (Neuroptera Chrysopidae). Boll. Zool. Agr. Bachic. 30:153-164. permission Luna, S.V., J.M. Figueroa, B.M.prohibited Baltazar, R.L. Gomez, R. Townsend, and J.B. Schoper. 2001. Maize pollenthe longevity and distance isolation requirement for effective commercial be pollen control. Crop Sci. 41:1551-1557. Mangelsdorf,Furthermore,any P.C. 1974. Corn: its Origin, Evolution, and Improvement. Harvard Consequently,without University. Press. Cambridge, Massachusetts. Mattila, H.R., M.K. Sears, and J.J. Duan. 2005. Response of Danaus plexippus to pollen of two new Bt corn events via laboratory bioassay. Entomologia Experimentalis et Applicata. 116:31-41. Mendelsohn, M., J. Kough, Z. Vaituzis, and K. Matthews. 2003. Are Bt crops safe? The US EPA analysis of Bt crops finds that they do not pose significant risk to the environment or human health. Nat. Biotechnol. 21:1003-1009.

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Morse, R.J., T. Yamamoto, and R.M. Stroud. 2001. Structure of Cry2Aa suggests an unexpected receptor binding epitope. Structure 9:409–417. Muenscher, W.C. 1980. Weeds. 2nd ed. Cornell University Press, New York. Naranjo, S.E. 2005a. Long-term assessment of the effects of transgenic Bt cotton on the abundance of nontarget arthropod natural enemies. Environ. Entomol. 34:1193- 1210. Naranjo, S.E. 2005b. Long-Term Assessment of the Effects of Transgenic Bt Cotton on the Function of the Natural Enemy Community. Environ. Entomol. 34:1211-1223. Naranjo, S.E., G. Head, and G.P. Dively. 2005. Field studies assessing arthropod nontarget effects in Bt transgenic crops: introduction. Environ. Entomol. 34:1178-1180. and Olson, R.A. and D.H. Sander. 1988. Corn production. In Cornand and Corn regime.Improvement, 3rd ed. Sprague, G.G. and J.W. Dudley (eds.).AG American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Madison, Wisconsin. Bayer property publishing protectioncontents Orr, D.B. and D.A. Landis. 1997. Ovipositiof on of European corn borer (Lepidoptera: its Pyralidae) and impact of natural enemy populationsparties.data in transgenic versustherefore isogenic and/oror corn. J. Econ. Entomol. 90: 905-909. affiliates. property intellectualthird may Park, H.W., D.K. Bideshi, and B.A.its Federici. 2000. Molecular genetic manipulation of as truncated Cry1C proteinthe of synthesis andin Bacillus thuringiensis to improve stability is regulatory owner. and yield. Appl. Environ. Microbiol. 66:4449-4455.a document any such its reproductiondocument Peterson, R., S. Meyer, A. Wolf, J.owner Wolt, and P. Davis.this 2006.of Genetically engineered plants, endangered species and underrisk: a temporalof and spatial exposure assessment rightsthe this for Karnerdocument blueand/or butterfly larvae and Bt maize pollen.rights Risk Analysis 26:845-858. to of fall use of Pilcher, C.D., J.J. Obrycki, M.E. Rice, and L.C. Lewis.the 1997. Preimaginal development, This may distribution,and survival, and fieldrights abundance of insect predatorsowner on transgenic Bacillus thuringiensis corn. Environ.subject Entomol. 26:448-454. be the violate Pilcher, C.D., M.E.copy Rice, and J.J. ofObrycki. 2005. Impact of transgenic Bacillus thuringiensis corn and crop phenologyand on five nontarget arthropods. Environ. may documentpublication, It Entomol. 34:1302-1316.exploitation this Pleasants, J., R. Hellmich,any G. Dively, M. Sears, D. Stanley-Horn, H. Mattila, J. Foster, T. permission Clark and G. Jones. 2001.prohibited Corn pollen deposition on milkweeds in and near cornfields. Proc.the Natl. Acad. Sci. USA 98: 11919-11924. commercial be Romeis, J., M. Meissle, and F. Bigler. 2006. Transgenic crops expressing Bacillus Furthermore,thuringiensisany toxins and biological control. Nat. Biotechnol. 24:63-71. Consequently,without Russell, W.A. and A.R. Hallauer. 1980. Corn. Pages 299-312 in Hybridization of Crop Plants. Fehr, W.R. and H.H. Hadley (eds.). American Society of Agronomy, and Crop Science Society of America, Publishers, Madison, Wisconsin. Saxena, D. and G. Stotzky. 2001. Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil. Soil Biol. Biochem. 33:1225- 1230.

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Schnepf, E.; N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Zeigler, and D.H. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:775-806. Sears, M., R. Hellmich, D. Stanley-Horn, K. Oberhauser, J. Pleasants, H. Mattila, B. Siegfried and G. Dively. 2001. Impact of Bt corn pollen on monarch butterfly populations: a risk assessment. Proc. Natl. Acad. Sci. USA 98:11937-11942. Shaw, R. H. 1988. Climate requirement. In Corn and Corn Improvement, 3rd ed. Sprague, G.F. and J.W. Dudley (eds.). American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Madison, Wisconsin. Shurtleff, M.C. 1980. Compendium of Corn Diseases. 2nd ed. Americanand and regime. Phytopathological Society, St. Paul, Minnesota.AG Smith, D.R. and D.G. White. 1988. Diseases of corn. Pages 687-766 in Corn and Corn Improvement, 3rd ed. Sprague, G.G. and J.W. Dudley (eds.). American Society of Bayer property protectionpublishing Agronomy, Crop Science Societyof of America, and Soil Sciencecontents Society of America, Madison, Wisconsin. its parties.data therefore Stanley-Horn, D., G. Dively, R. Hellmich, H. Mattila, M. Sears,and/or R.or Rose, L. Jesse, J. affiliates. Losey, J. Obrycki andproperty L. Lewis. intellectual 2001.third Assessing the impactmay of Cry1Ab- its expressing corn pollen on monarchas butterfly larvae in field studies. Proc. Natl. the of and Acad. Sci. USA 98:11931-11936.is regulatory owner. a document Tapp, H. and G. Stotzky.any 1998. such Persistence of the insecticidalits toxin from Bacillus reproductiondocument thuringiensis subsp. kurstakiowner in soil. Soil Biol.this Biochem. 30:471-476.of under of Torres, J.B. and J.R. Rubersonrights. 2005.the Canopy- and ground-dwellingthis predatory arthropods in documentand/or of rights commercial Bt toand non-Btof fall cotton fieluseds: patterns and mechanisms. Environ. Entomol. 34:1242-1256. the This may distribution,and rights owner University of Missouri.subject 1998. Corn Insect Pests: A Diagnostic Guide. O’Day, M., A. Becker,be A. Keaster, L. Kabrick,the K. violateSteffey (eds.). University of Missouri- Columbia,copy Columbia, Missouri. of and USDA-ERS.may 2002.document Soil,publication, nutrient, and water management systems used in U.S.. Corn It exploitation Production Agriculture Information Bulletin. No. 774. Electronic report from the this any Economic Research Service, United States Department of Agriculture. permission http://www.ers.usda.govprohibited. the USDA-NAAS. commercial2006a. USDA-NASS. be 2006. National Agricultural Statistics Service, United States Department of Agriculture. Acreage Report, Cr Pr2-5 (6-06) a. June Furthermore,30, 2006.any Consequently,without USDA-NASS. 2006b. Agricultural chemical usage 2005 field crops summary. United States Department of Agriculture, National Agricultural Statistics Service. Ag Ch1 (06). USDA-NASS. 2006c. Online database (www.nass.usda.gov) at the United States Department of Agriculture, National Agricultural Statistics Service (USDA- NASS). Accessed on June 26, 2006.

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Whitehouse, M., L. Wilson, and G. Fitt. 2005. A comparison of arthropod communities in transgenic Bt and conventional cotton in Australia. Environ. Entomol. 34:1224- 1241. Wilkes, H.G. 1967. Introduction. Pages 1-2. Discussion and Summary. Pages 121-127 in Teosinte: the Closest Relative of Maize. Bussey Institution, Harvard University, Cambridge, Massachusetts. Wolt, J.D., C.A. Conlan, and K. Majima. 2005. An ecological risk assessment of Cry1F maize pollen impact to pale grass blue butterfly. Environ. Biosafety Res. 4:243- 251. Wright, D., J. Marois, J. Rich, and R. Sprenkel. 2004. Field corn production guide. Document SS-AGR-85, Department of Agronomy, Florida Cooperative Extensionand and regime. Service, Institute of Food and AgriculturAG al Sciences, University of Florida. Available online at http://edis.ifas.ufl.edu/AG202. Accessed September 19, 2006. Zhuang, M. and S.S. Gill, S. S. 2003. Mode of action of Bacillus thuringiensis toxins. Bayer property protectionpublishing Pages 213-236 in Chemistry ofof Crop Protection, Progress andcontents Prospects in Science and Regulation. Voss, G. and Ramos, G. (ed), Wiley-VCH,its Weinheim. parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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IX. Adverse Consequences of Introduction

Monsanto knows of no study results or observations associated with MON 89034 that would be anticipated to result in adverse environmental consequences from its introduction. MON 89034 provides protection from feeding damage caused by lepidopteran insect pests. As demonstrated by field results and laboratory tests, the only phenotypic difference between MON 89034 and conventional corn is the presence of the Cry1A.105 and Cry2Ab2 proteins.

The data and information presented in this petition demonstrate that MON 89034 is unlikely to pose an increased plant pest potential or to have an adverse environmental regime.and consequence compared to conventional corn. This conclusionand is reached based on AG multiple lines of evidence developed from a detailed characterization of the product compared to conventional corn, followed by risk assessment on detected differences. The characterization studies included molecularBayer and proteinproperty analyses, protection whichpublishing confirmed the insertion of a single functional copy of ofcry1A.105 and cry2Ab2 expressioncontents cassettes at a its single locus within the corn genome and that the twoparties. proteinsdata were expressedtherefore in tissues at levels that are efficacious for the control of target insect pests. and/orExtensiveor characterization affiliates. of the plant phenotype includingproperty compositional intellectualthird analysis of key nutrientmay and antinutrients its also indicated that MON 89034, withas the exception of intended modification, was the of and unchanged compared tois conventional corn. regulatoryAllergenicity assessmentowner. concluded that a document Cry1A.105 and Cry2Ab2 proteinsany such are unlikely to be allergens for humans.its Toxicity tests reproductiondocument including an acute mouse oral gavageowner and other selectedthis non-targetof organisms with either equivalent proteins produced by recombinantunder strainsof of E. coli or MON 89034 tissues rightsthe this showed no signsdocument of adverseand/or effects at high doses. An endangeredrights species risk assessment to of fall use of also concluded that MON 89034 is unlikely to have adversethe effects on these organisms, includingThis endangered lepidopteranmay insects.distribution,and Therefore, the risks for humans, animals, and rights owner other non-targetsubject organisms from MON 89034 are negligible under the conditions of use. the violate be copy The introduction of MON 89034 is expectedof to increase economic, environmental and and health benefitsmay duedocument to publication,the protection of corn yields, decrease of chemical insecticide It exploitation usage, reductionthis of mycotoxin levels in corn grain, and increase of Bt corn product durability. The introductionany of MON 89034 will not impact cultivation practices and the permission management of weeds, diseasesprohibited and insects except for the control of lepidopteran insect pests. The combination of the Cry1A.105 and Cry2Ab2 insecticidal proteins in a single the be plant provides notcommercial only a wider spectrum of pest control but also a more effective insect resistanceFurthermore, managementany tool. Consequently,without

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Appendix A. Materials and Methods for Molecular Characterization and Results of PCR Analysis on MON 89034 Insert

Molecular analysis was performed using genomic DNA isolated from MON 89034 in order to characterize the integrated DNA. MON 89034 genomic DNA was analyzed by Southern blot analysis for the number of integration sites, the number of copies of the integrated DNA at each locus, the integrity of the inserted gene cassettes, the presence or absence of plasmid backbone sequence and the stability of the introduced DNA across multiple generations. Additionally, PCR amplification and DNA sequencing were used to confirm the 5' and 3' insert-to-plant junctions, confirm the organization of the elements within the insert, and determine the complete DNA sequence of the integrated DNA. regime.and and AG A.1. Test Material

The test material was grain, leaf or seed fromBayer corn MONproperty 89034. protection publishing of contents its A.2. Control Material parties.data therefore and/oror affiliates. The control material was conventionalproperty cornintellectual withthird a gene tic backgroundmay similar to MON its 89034. Where applicable, a secondas conventional corn with a different genetic the of and background to MON 89034is was also included toregulatory provide an additional owner.negative control. a document any such its reproductiondocument A.3. Reference Materials owner this of under of rightsthe this PV-ZMIR245document was usedand/or as a positive hybridization controlrights for Southern blots and as a to of fall use of template control for PCR analyses. The plasmid was theisolated and its identity confirmed by restrictionThis enzyme digestion.may distribution,and rights owner subject The 1 kb DNA extension ladder, low DNAthe massviolate ladder, high DNA mass ladder, and λ be copy DNA/Hind III fragments from Invitrogenof (Carlsbad, CA) were used for size estimations and of DNAmay fragments ondocument Southernpublication, blots and agarose gels. It exploitation this A.4. Characterizationany of Test and Control Materials permission prohibited Event specific PCR assays were used to confirm the identity of test and control materials. the be The test and controlcommercial materials were considered stable during storage if they did not appearFurthermore, visiblyany degraded on ethidium bromide-stained gels and/or yielded interpretable signalsConsequently, on the Southernwithout blots.

A.5. Genomic DNA Isolation

Grain and seed samples were processed and genomic DNA was extracted using a CTAB (hexadecyltrimethylammonium bromide)-based method (Rogers and Bendich, 1985). For a single DNA extraction, chloroform was used rather than chloroform:isoamyl alcohol. For leaf tissue, genomic DNA was extracted using a Sarkosyl extraction method

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(Fulton et al, 1995). Extracted DNA solutions were stored in a 4°C refrigerator and/or a -20°C freezer. For purification of plasmid DNA, Qiagen QIA filter Plasmid Mini Kit was used.

A.6. Quantitation of Genomic DNA

Extracted DNA was quantified using Hoefer’s DyNA Quant 200 Fluorometer. Molecular size marker IX (Roche, Indianapolis, IN) was used as the calibration standard.

A.7. Restriction Enzyme Digestion of Genomic DNA

Approximately 10 or 20 µg of genomic DNA, extracted from the test and andcontrol and regime. substances, were digested overnight in a total volumeAG of approximately 500 µl using 100 units of the restriction enzyme. For positive hybridization controls, approximately one or half genomic equivalent of PV-ZMIR245 was spiked into conventional corn DNA prior to digestion. Bayer property publishing protectioncontents of its A.8. Agarose Gel Electrophoresis parties.data therefore and/oror

affiliates. Approximately 10 µg of digestedproperty DNA intellectualwere thirdseparated using 0.8% mayagarose gels. For its as insert number, copy number,the andof cassetteand in tactness experiments, a ‘long run’ and a ‘short run’ were performedis during the gel electrophoresis.regulatory Approximatelyowner. 20 µg of a document digested test and control substanceany such DNA was divided in half to loadits approximately 10 µg reproductiondocument on the long run gel and approximatelyowner 10 µg onthis the short runof gel. The long run under of electrophoresis enabled greaterrightsthe separation of the higherthis molecular weight restriction documentand/or rights fragments, while the shortto runof allowed fall the smalleruse molecularof weight restriction fragments to be retained on the gel. the This may distribution,and rights owner A.9. Probe Preparationsubject be the violate copy of Approximately 12.5-27 ng of each probe andtempla te were prepared by PCR amplification and radiolabeledmay withdocument publication,32P-dCTP (6000 Ci/mmol) using the random priming method It exploitation (except probe 10 which was labeled by PCR). this any

permission A.10. Southern Blot Analyses prohibited the be Digested genomiccommercial DNA isolat ed from test and control materials was evaluated using SouthernFurthermore, blotany analyses (Southern, 1975) with modifications. Consequently,without A.11. PCR and Sequence Analyses

Overlapping PCR products were generated that span the insert in MON 89034. These products were sequenced to determine the nucleotide sequence of the insert in MON 89034 as well as the nucleotide sequence of the genomic DNA flanking the 5' and 3' ends of the insert.

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The PCR analyses were conducted using 50 ng of genomic DNA template in a 50 µl reaction volume containing a final concentration of 2 mM MgSO4, 0.2 µM of each primer, 0.2 mM each dNTP, and 1 unit of DNA polymerase mix. The specific DNA polymerase mix used to amplify the products was Accuprime Taq (Invitrogen). The amplification of Product A was performed under the following cycling conditions: 94°C for 3 minutes; 30 cycles at 94°C for 15 seconds, 57°C for 30 seconds, 68°C for 3 minutes and 30 seconds; 1 cycle at 68°C for 5 minutes. The amplification of Product B was performed under the following cycling conditions: 94°C for 3 minutes; 35 cycles at 94°C for 15 seconds, 66°C for 30 seconds, 68°C for 3 minutes and 30 seconds; 1 cycle at 68°C for 5 minutes. The amplification of Products C, D, and E was performed under the following cycling conditions: 94°C for 3 minutes; 30 cycles at 94°C for 15 seconds, 60°C and for 30 seconds, 68°C for 3 minutes; 1 cycle at 68°C for 5 minutes.and The amplificationregime. of Products F and G was performed under the followingAG cycling conditions: 94°C for 3 minutes; 30 cycles at 94°C for 15 seconds, 60°C for 30 seconds, 68°C for 1 minute and 30 seconds; 1 cycle at 68°C for 5 minutes. The products A-G are illustrated in Figure A- Bayer property protectionpublishing 1. of contents its parties.data therefore Aliquots of each PCR product were separated on 1.0 % (w/v) agaroseand/oror gels and visualized affiliates. by ethidium bromide staining toproperty verify that intellectual the productsthird were of the expectedmay size prior to its sequencing. The PCR products wereas sequenced with multiple primers used for PCR the of and amplification in additionis to those designed internalregulatory to the amplifiedowner. sequences. All a document sequencing was performedany by thesuch Monsanto Genomics Sequencingits Center using dye- reproductiondocument terminator chemistry. owner this of under of this A.12. PCR Analysisand/or Resultrights acrossthe the Insert in MON 89034 document to fall of rights of use the The organizationThis of the elementsmay within distribution, andthe insert in MON 89034 was confirmed using rights owner PCR analysis bysubject amplifying seven overlapping regions of DNA that span the entire length of the insert. The location of the PCR products generated in relation to the insert, be the violate as well as the resultscopy of the PCR analyses,of are shown in Figure A-1. The control and reactionsmay containing document nopublication, template DNA (lanes 2, 5, 8, 12, 16, 20, and 23) and the conventionalIt corn control reactionsexploitation (lanes 3, 6, 9, 13, 17 and 21) did not generate PCR products with anythis of any the primer sets, as expected. The conventional corn control reaction in lane 24 produced a productpermission of equal size to that of MON 89034 (lane 25) because both primer sequences are located in prohibitedthe corn ge nomic flanking sequence adjacent to the 3' end the of the insert incommercial MON 89034. be Additionally, the products generated using plasmid PV-ZMIR245 DNA as a template (lanes 11 and 15) appear overloaded in comparison to Furthermore,any the Consequently,MON 89034without genomic DNA samples which likely contributes to the intensity of the products observed in these lanes.

MON 89034 DNA generated the expected size PCR products of approximately 2.5 kb for Product A (lane 4); approximately 3.3 kb for Product B (lane 7); approximately 2.6 kb for Product C (lane 10); approximately 2.6 kb for Product D (lane 14); approximately 3.2 kb for Product E (lane 18), approximately 1.1 kb for Product F (lane 22) and approximately 0.8 kb for Product G (lane 25). The generation of the predicted size PCR products from MON 89034 establishes that the arrangement or linkage of elements in the insert are the

Monsanto Company 06-CR-166U Page 186 of 262 CBI Deleted Version same as those in plasmid PV-ZMIR245 and that the elements within each gene cassette are arranged as depicted in the schematic of the insert in Figure V-4 (see Section V). The results of DNA sequencing on the amplified DNA fragments confirmed that the sequence of the DNA insert in MON 89034 matched the designed, corresponding sequences in PV-ZMIR245 with one exception. This exception is that the e35S promoter that regulates expression of the cry1A.105 gene has been modified and that the Right Border sequence present in PV-ZMIR245 was replaced by a Left Border sequence in MON 890343 (see Section V for more discussion).

A.13. References

Fulton, T.M., J. Chunwongse, and S.D. Tanksley. 1995. Microprep protocol for regime.and extraction of DNA from tomato and other herbaceous plants.and Plant Mol. Biol. Rep. AG 13:207-209. Rogers, S.O. and A.J. Bendich. 1985. Extraction of DNA from milligram amounts of Bayer property protectionpublishing fresh herbarium and mummified plant tisofsue. Plant Mol Biol Sci, 5:69-76. contents its Southern, E.M. 1975. Detection of specific sequencesparties. amongdata DNA fragmentstherefore separated and/oror by gel electrophoresis. J. Mol. Biol. 98:503-517. affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Product A Product B Product C Product D Product E Product F Product G

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

7.1 4.0 3.0 2.0 1.6 1.0 0.5

regime.and and AG Product B: ~ 3.3 kb Product D: ~ 2.6 kb Product F: ~ 1.1 kb

Product A: ~ 2.5 kb Product C: 2.6 kb Product E: ~ 3.2 kb Product G: ~ 0.8 kb Bayer property publishing protectioncontents 5 ' Flank of 3 ' Flank its parties.data therefore CTP - and/oror cry2Ab2 cry1A.105 SSU

affiliates. - nos Left Border Left Hsp17 Cab FMV e35S may -Left Border Hsp70 - Ract1 property intellectualthird B T CS- P- TS I- CS- T- B- P- L- I- its as the of and Figure A-1. Overlappingis PCR analysis acrossregulatory the insert in MON 89034owner. a document any such its PCR analyses demonstrating the linkage of the individualreproduction geneticdocument elementsof within the insert in MON 89034 were performed on MON owner89034 genomic DNAthis extracted from grain (Lanes 4, 7, 10, under of 14, 18, 22, and 25). Lanes 3,rights 6, 9,the 13, 17, 21, and 24 containthis reactions with conventional corn documentand/or rights control DNA while lanes to2, 5, 8,of 12, 16,fall 20, and use23 are ofreactions containing no template DNA. Lanes 11, 15, and 19 contain reactions with PV-ZMIR245the control DNA. Lane 1 contains InvitrogenThis 1 kb DNA ladder. Lanesmay aredistribution, markedand to show which product has been loaded and is rights owner visualized on the subjectagarose gel. The gel side arrow symbols denote the sizes of DNA (kb) based on the reference DNAbe size markers on the ethidiumthe brviolateomide stained gel. The expected product size for each amplicon copyis provided in the illustrationof of the insert in MON 89034 that appears below the image. and may documentpublication, Lane It 1: Invitrogen 1 kb DNAexploitation ladder 13: Conventional corn control DNA 2: No templatethis any DNA control 14: MON 89034 genomic DNA 3: Conventional corn control DNA 15: PV-ZMIR245 plasmid permission 4: MON 89034 genomic DNAprohibited 16: No template DNA control 5: No template DNA control 17: Conventional corn control DNA the 6: Conventionalcommercial corn control be DNA 18: MON 89034 genomic DNA 7: MON 89034 genomic DNA 19: PV-ZMIR245 plasmid Furthermore,8: Noany template DNA control 20: No template DNA control Consequently,9: Conventionalwithout corn control DNA 21: Conventional corn control DNA 10: MON 89034 genomic DNA 22: MON 89034 genomic DNA 11: PV-ZMIR245 plasmid 23: No template DNA control 12: No template DNA control 24: Conventional corn control DNA 25: MON 89034 genomic DNA

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Appendix B. Materials, Methods, and Results for Characterization of Cry1A.105 Protein

The Cry1A.105 protein was purified from the grain of MON 89034 as well as from a recombinant E. coli culture. A panel of analytical tests was used to identify, characterize and compare the MON 89034-produced and E. coli-produced Cry1A.105 protein including: (1) Western blot analysis; (2) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry; (3) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS); (4) glycosylation analysis; and (5) insect activity bioassay. The materials, methods and the results are described below. regime.and and AG B.1. Materials and Methods

B.1.1. Purification of MON 89034-ProducedBayer Cry1A.105 property Protein protection publishing of contents its The MON 89034 produced Cry1A.105 protein was parties.purifieddata from grain of thereforeMON 89034. The identity of the MON 89034 grain was confirmed by event-specificand/oror PCR. The affiliates. purified MON 89034-producedproperty Cry1A. 105intellectual proteinthird was stored in a 4ºCmay refrigerator in a its buffer solution containing 50 mM CAPS,as 1 mM PMSF, 2 mM benzamidine-HCl, 1 mM the of and EDTA, 0.8 M NaCl, andis 30% (v/v) ethylene glycol,regulatory pH 10. owner. a document any such its reproductiondocument The purification procedure was conductedowner at 4°C fromthis an extractof of ground grain using a combination of ammonium sulfate fractionation, ofanion exchange chromatography, and rights under this immunoaffinity chromatography.and/or the document to of fall of rights use the The groundThis grain (10 kg) was maymixed distribution, in PBSand (p hosphate buffered saline) extraction buffer rights owner (1 mM KH2PO4subject, 10 mM Na2HPO4, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) for 2.5 h at approximatelybe a 1:10 ratio of sample weightthe (g)violate to buffer volume (ml). The slurry was filtered using an Ertelcopy Alsop filter pressof (Kingston, NY) and the PBS-washed filter cakes and were retained.may Thedocument cakespublication, were resuspende d in approximately 100 L of CAPS extraction bufferIt [50 mM CAPS, 1 mMexploitation EDTA, 10 mM DTT, 1 mM PMSF, 2 mM benzamidine- this HCl, and 1% PVPPany (w/v), pH 10.8] and stirred for 2 h. To remove lipids from the extract, CelPure P65 diatomaceouspermission earth (Advanced Minerals Corp, Goleta, CA) was added to the slurry at 7.5% (w/v)prohibited and a llowed to mix for 10 min. The extract was the clarified by filtrationcommercial using thebe Ertel Alsop filter press and concentrated using a hollow fiber cartridge (Amersham Biosciences, Piscataway, NJ). The plant genomic DNA was Furthermore,any removedConsequently, by withouta combination of benzonase treatment and precipitation with polyethyleneimine. Benzonase was added to a final concentration of 7 U/ml in the presence of 5 mM MgCl2 and allowed to mix overnight. A polyethyleneimine solution [10% (w/v)] was added to the concentrated extract to a final concentration of 0.05% (w/v) and the extract was clarified by centrifugation to remove the remaining DNA. An ammonium sulfate pellet (35% saturation) was prepared by the addition of ammonium sulfate salt to the clarified extract and was allowed to settle overnight. After centrifugation, the ammonium sulfate pellet was dissolved over the weekend in 15 L of resuspension buffer (50 mM CAPS, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, and 2 mM

Monsanto Company 06-CR-166U Page 189 of 262 CBI Deleted Version benzamidine-HCl, pH 10.8). The resuspended sample was clarified by centrifugation, diafiltrated against fresh buffer (same as the resuspension buffer) to remove any residual ammonium sulfate, and then concentrated to final volume of 8 L, and CHAPS was added to a final concentration of 0.5 mM.

The concentrated sample was loaded onto a 2.1 L (6.7 cm x 20 cm) Q Sepharose Fast Flow anion exchange column, which was equilibrated with a loading buffer (50 mM CAPS, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, 2 mM benzamidine-HCl, and 0.5 mM CHAPS, pH 10.8). The bound proteins were eluted with a linear salt gradient that increased from 0 M to 0.20 M NaCl over 12 L and then maintained a 0.20 M NaCl for 4 L. Next, the salt gradient increased to 0.65 M NaCl over 21 L, and finally elevated to 1.0 M NaCl instantly. Fractions containing the Cry1A.105 protein (based on stained SDS- regime.and PAGE gel and Western blot analysis of all fractions) were pooledand to a final volume of 10 AG L. Salt was removed from these pooled fractions by diafiltration with a fresh buffer (50 mM EPPS, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, and 2 mM benzamidine-HCl, pH 7.5). Bayer property protectionpublishing of contents its The buffer-exchanged sample was loaded onto a 2.1 parties.L (13.7data cm x 14.0 cm)therefore Q Sepharose XL anion exchange column, which was equilibrated with anotherand/or loadingor buffer (50 mM affiliates. EPPS, 1 mM EDTA, 10 mM DTT,property 1 mM intellectual PMSF,third 2 mM benzamidine-HCl,may and 0.5 mM its CHAPS, pH 7.5). The bound proteinsas were eluted with a linear salt gradient that the of and increased from 0 M to 0.25is M NaCl over 4 L regulatoryand then held at 0.25 owner.M NaCl for 10 L. a document Next, the salt gradient increasedany tosuch 0.65 M NaCl over 21 L and finallyits increased to 1.0 M reproductiondocument NaCl over 4 L and held constantly ownerfor 8 L. The fractionsthis containingof Cry1A.105 protein were pooled to a final volume of 10 underL. Thes e fractionsof were diafiltrated into a fresh rightsthe this buffer (50 mMdocument EPPS,and/or 1 mM EDTA, 10 mM DTT, 1mM PMSF,rights and 2 mM benzamidine- to of fall use of HCl, pH 7.5) to remove salt, and then concentrated to 2the L using a hollow fiber cartridge. This may distribution,and rights owner Prior to the affinitysubject purification step, the sample was diafiltrated into fresh buffer (50 mM EPPS, 1mM EDTA, 1 mM PMSF, 2 mM thebenzam violateidine-HCl, and 150 mM NaCl, pH 7.6), be copy and concentrated to 1 L. The solutionof c ontaining Cry1A.105 protein was re-circulated and over themay 9.3 ml (1.75document cmpublication, x 2.6 cm) protein A agarose column (Sigma) conjugated with It exploitation monoclonal anti-Cry1Acthis antibody (Strategic Biosolutions, Newark, DE). Bound proteins were eluted using 50any mM CAPS, 1 mM EDTA, 1 mM PMSF, 2 mM benzamidine-HCl, permission 0.8 M NaCl, and 30% (v/v) ethyleneprohibited glycol, pH 10. After analysis of fractions by lateral flow test strips, SDS-PAGE gel and Western blot, fraction #25 was determined to contain the be the majority ofcommercial the full-length Cry1A.105. This fraction was used for further characterizationFurthermore,any tests. Consequently,without B.1.2. Escherichia coli-Produced Cry1A.105 Protein

E. coli-produced Cry1A.105 protein which was produced and purified previously was used as a reference standard for determination of protein concentration of the MON 89034-produced Cry1A.105 protein. This protein was also used as a reference standard to evaluate equivalence between MON 89034- and E. coli-produced Cry1A.105 protein for immunoreactivty (Western blot), molecular weight (SDS-PAGE) and functional activity assay. The E. coli-produced Cry1A.105 protein was stored in a -80°C freezer in

Monsanto Company 06-CR-166U Page 190 of 262 CBI Deleted Version a buffer solution (25 mM CAPS, 1 mM benzamidine-HCl, 0.1 mM EDTA, and 0.2 mM DTT, pH 10.3) at a total protein concentration of 1.2 mg/ml.

B.1.3. Determination of MON 89034-Produced Cry1A.105 Protein Concentration

The concentration of the full-length Cry1A.105 protein in the purified sample from MON 89034 grain was estimated using quantitative immunoblot analysis. The E. coli-produced Cry1A.105 protein was used to create a standard curve. Aliquots of the MON 89034- produced Cry1A.105 protein and reference standard were diluted in deionized water and 5x Laemmli buffer (5x LB), heated at approximately 95.5°C for 5 min, and applied to a pre-cast Tris-glycine 4→20% polyacrylamide gradient gel. Three different amounts of the MON 89034-produced protein were loaded in duplicate. Electrophoresisregime.and was performed at a constant voltage of 125 V for 15 min followedand by a constant voltage of AG 170 V for 75 min. Pre-stained molecular weight markers (Bio-Rad Precision Plus Dual Color, Hercules, CA) were used to verify the completeness of electrotransfer of protein to the membrane and estimate the size ofBayer the immunoreactive property bandspublishing observed. The protectioncontents electrotransfer to a 0.45 µm PVDF membraneof (Invitrogen, Carlsbad, CA) was performed its for 90 min at a constant voltage of 25 V. parties.data therefore and/or or affiliates. The membrane was blocked forproperty 1 h with 5%intellectual (w/v)third NFDM (non-fat drymay milk) in 1×PBST its (phosphate buffered saline with Tweenas 20). The membrane was probed with a 1:1000 the of and dilution of rabbit anti-Cry1Ais .105 antibody in regulatory1% (w/v) NFDM in owner.PBST for 60 min. a document Excess antibody was removedany usinsuchg four washes (5 min each) withits PBST. Finally, the reproductiondocument membrane was probed with HRP owner(horseradish peroxidase)-conjugatedthis of goat anti-rabbit under of IgG (Sigma, St. Louis, MO)rights at thea dilution of 1:7500 in this1% (w/v) NFDM in PBST for 60 documentand/or rights min. Excess HRP-conjugateto ofwas removedfall usiuseng threeof washes (5 min each) with PBST. All incubations were performed at room temperature.the Immunoreactive bands were visualizedThis using the ECL detectionmay systemdistribution,and (Amersham Biosciences, Piscataway, NJ) and rights owner exposed (5 min,subject 10 min, 20 min) to Hyperfilm ECL high performance the violate chemiluminescencebe copy film (Amersham Biosciences). Films were developed using a Konica SRX-101A automated film processor (Tokyo,of Japan). and may documentpublication, It exploitation The immunoreactivethis band of the MON 89034-produced Cry1A.105 protein in each lane migrating at the sameany distance as the full-length reference standard Cry1A.105 protein of permission E. coli was quantitated relative prohibitedto the standard curve. Quantitation was performed using the volume tool and thethe linear be regression method in the Quantity One software (version 4.4.0) after scanningcommercial with a Bio-Rad GS-800 densitometer. The concentration of the MONFurthermore, 89034-producedany Cry1A.105 was determined by dividing the amount of protein in eachConsequently, lane by thewithout volume of protein loaded in the respective lanes.

Monsanto Company 06-CR-166U Page 191 of 262 CBI Deleted Version

B.1.4. Western Blot Analysis

Western blot or immunoblot analysis was performed to confirm the identity of the Cry1A.105 protein, the intactness of its N-terminus, and compare immunoreactivity of the MON 89034- and E. coli-produced Cry1A.105 protein.

Western blot analysis using anti-Cry1A.105 antibody

The Western blot (see above Section B.1.3) was used to establish the identity of the MON 89034-produced protein and to compare the immunoreactivity of the MON 89034- produced and E. coli-produced Cry1A.105 protein. Equivalence was demonstrated for regime.and bands representing full-length Cry1A.105 protein that wasand identified by the anti- Cry1A.105 antibody and showed similar mobilityAG with the E. coli-produced reference standard on the gel/blot. Bayer property protectionpublishing Western blot analysis using the anti-N-terminalof peptide antibody contents its parties.data therefore Western blot analysis using the anti-N-terminal peptide antibodyand/oror was performed to affiliates. confirm the intactness of theproperty N-terminus intellectual ofthird the MON 89034-producedmay Cry1A.105 its protein. The anti-N-terminal peptideas anti body was produced with a synthetic peptide the of and consisting of the first 14 isamino acids of the Cry1A.105regulatory protein N-termiowner.nus as the antigen. a document The trypsin resistant core ofany Cry1A.105 such was used as a negative controlits because its N- reproductiondocument terminus was removed by trypsin. Theowner MON 89034-producedthis Cry1Aof .105 protein, the E. coli-produced Cry1A.105 reference standard,under andof the Cry1A.105 trypsin-resistant core rightsthe this were each loadeddocument onand/or gels at 20 ng and 40 ng per lane. Eachrights protein was mixed with 5× to of fall use of LB, heated at 96°C for 5 min, and applied to thea pre-cast Tis-glycine 4→20% polyacrylamideThis gradient gel. mayThe eletrophoresisdistribution,and was performed at a constant voltage of rights owner 150 V for 5 minsubject followed by a constant voltage of 200 V for 60 min. Pre-stained molecular weightbe markers (Bio-Rad Precisionthe violate Plus Dual Color) were used to verify electrotransfer of copyprotein to the membraneof and estimate the size of the immunoreactive and bands. mayElectrotransfer document topublication, a 0.45 µm PVDF membrane (Invitrogen) was performed for 90 It exploitation min at a constantthis voltage of 25 V. any permission The membrane was blocked overnightprohibited with 5% (w/v) NFDM in 1×PBST. The membrane was probed with a rabbit anti-N-terminal peptide antibody in 1% (w/v) NFDM in PBST the be for 60 min. Excesscommercial antibody was removed using three washes (5 min each) with PBST. Finally,Furthermore, the anymembrane was probed with HRP-conjugated anti-rabbit IgG (Sigma) at a dilutionConsequently, of 1:7500without in 1% (w/v) NFDM in PBST for 60 min. Excess HRP-conjugate was removed using three washes (5 min each) with PBST. Immunoreactive bands were visualized using the ECL detection system and exposed for 1 min, 2 min, 5 min, and 10 min to ECL high performance chemiluminescence film. Films were developed using a Konica SRX-101A automated film processor.

Monsanto Company 06-CR-166U Page 192 of 262 CBI Deleted Version

B.1.5. Molecular Weight and Purity Estimation by SDS-PAGE

Aliquots of the E. coli-produced reference standard and MON 89034-produced Cry1A.105 protein were mixed with 5× LB to a final protein concentration of 10 ng/µl and 2.4 ng/µl, respectively. The MON 89034-produced protein was analyzed in duplicate at 48, 72, and 96 ng of total protein per lane. The E. coli-produced Cry1A.105 reference standard was analyzed at 96 ng of purity-corrected for the full-length protein. All samples were heated in a thermo-block at 99°C for 3 min and applied to a pre-cast Tris- glycine 4→20% polyacrylamide gradient gel (Invitrogen). Electrophoresis was performed at a constant voltage of 125 V for 10 min followed by a constant voltage of 170 V for 70 min. and and regime. AG The gel was stained using the SilverXpress® silver staining kit (Invitrogen, Carlsbad, CA). The gel was fixed for 10 min in 200 ml of fixing solution (90 ml ultra pure water, 100 ml methanol, and 20 ml acetic acid).Bayer This wasproperty followed by publishing10 min in 100 ml protectioncontents sensitizing solution (105 ml ultra pure wateof r, 100 ml methanol, and 5 ml sensitizer) and its repeated once. The excess of the sensitizing solutionparties. was dataremoved using twotherefore washes (5 and/oror min each) in 200 ml ultra pure water. The gel was stained for 15 min in 100 ml staining affiliates. solution (5 ml Stainer A, 5 mlproperty Stainer B,intellectual andthird 90 ml ultra pure water).may The stain was its as removed using two washesthe (5 of min each)and of 200 ml ultra pure water. Development occurred in 100 ml of solutionis (5 ml developer regulatoryand 95 ml ultra pure waowner.ter) for 3-15 min, a document and was stopped by additionany of 5such ml stopping solution for 10 min. its The gel was washed reproductiondocument three times for 10 min each with 200owner ml of ultra purethis water. ofAnalysis of the gel was under of performed using a Bio-Radrights GS-800the densitometer withthis the supplied Quantity One documentand/or rights software (version 4.4.0,to Hercules, of fallCA). Molecularuse of weight markers (Bio-Rad, broad- range) were used to estimate the apparent molecular weightthe of the MON 89034-produced This may distribution,and Cry1A.105 protein. Forrights the purity evaluation,owner all visible bands within each lane were quantified. Stainedsubject bands corresponding to immunoreactive bands identified by anti- Cry1A.105 antibodybe and migrating fromthe approximatelyviolate 56 kDa to 130 kDa were copy of included in the purity calculation for theand prot ein of interest as they represent various lengths may(proteolytic document fragmentspublication, to full-length) of the Cry1A.105 protein. It exploitation

this any B.1.6 Glycosylation Analysis permission prohibited Glycosylation analysisthe was be used to determine whether the MON 89034-produced Cry1A.105 proteincommercial was post-tran slationally modified with covalently bound carbohydrate moieties.Furthermore, Aliquotsany of the MON 89034-produced Cry1A.105 protein, the E. coli-produced Cry1A.105Consequently, referencewithout standard, and the positive control glycoproteinss, transferrin (Amersham Biosciences) and horseradish peroxidase (Sigma), were each mixed with 5× LB. These samples were heated at 95 °C for 4 min, cooled, and loaded on a Tris-glycine 4→20% polyacrylamide gradient gel. Each sample was loaded at 48 and 96 ng (purity corrected for the full length protein) per lane. Precision Plus Dual Color pre-stained protein molecular weight markers (Bio-Rad) were loaded to verify electrotransfer of the proteins to the membrane, and the CandyCane™ glycoprotein molecular weight standards (Molecular Probes, Eugene, OR) were loaded as positive/negative controls and

Monsanto Company 06-CR-166U Page 193 of 262 CBI Deleted Version markers for molecular weight. Electrophoresis was performed at a constant voltage of 125 V for 10 min followed by a constant voltage of 170 V for 70 min. Electrotransfer to a 0.2 µm PVDF membrane was performed for 90 min at a constant voltage of 25 V.

Carbohydrate detection was performed directly on the PVDF membrane using the Pro- Q® Emerald 488 glycoprotein gel and blot stain kit (Molecular Probes). The manufacturer’s protocol was followed. All steps were performed at room temperature. The PVDF membrane was fixed in 25 ml of a solution containing 50% methanol and 5% glacial acetic acid for 1 h, and then the solution was changed once and the membrane was incubated overnight. Two 15 min washes (50 ml each) of 3% (v/v) glacial acetic acid (wash solution), were followed by a 20 min oxidation in 25 ml of the kit supplied oxidizing solution. After oxidation, three 15 min washes (50 ml each) prepared the regime.and membrane for staining. The blot was incubated in 25 ml ofand Pr o-Q Emerald Staining AG Solution that was prepared using the kit reagents. After 1 h of staining in the dark, two 30 min, 50 ml wash cycles were followed by two 45 min, 50 ml wash cycles. The final wash cycles included two 25 ml, 1 min deioniBayerzed waterproperty washes protection followedpublishing by three 25 ml, 5 min methanol washes. The blot was thenof scanned using the Bio-Rad Molecularcontents Imager its FX with the Alexa 488 illumination setting (Qunatityparties. Onedata software; versiontherefore 4.6, build 036) in order to visualize the fluorescing glycosylated proteins.and/or or affiliates. property intellectualthird may its B.1.7. Tryptic Peptide Mapping Analysisas by MALDI-TOF MS the of and is regulatory owner. a document Matrix assisted laser desorption/ionizationany such time-of-flight mass spectrometryits (MALDI- reproductiondocument TOF MS) was used to further confirmowner the identitythis of the ofMON 89034-produced Cry1A.105 protein. under of rightsthe this documentand/or rights to of fall use of The purified MON 89034-produced protein (4.5 ml)the was diluted to 9.0 ml with a concentrationThis buffer [25 mM CAPS,may distribution, 0.02%and (w /v) SDS] followed by concentration to 64 rights owner µl using Amiconsubject Ultrafree CL concentrators (Millipore Corporation, Bedford, MA). Sixty microliters of 5× LB was used to washthe theviolate concentrator walls and membranes. The be copy membranes were heated with 5× LB forof 5 min at 95°C. Sixteen microliters of this 5× and LB wasmay then used asdocument thepublication, loading buffer for the Cry1A.105 sample for SDS-PAGE. The It exploitation broad- range molecularthis weight markers (Bio-Rad) were used to estimate molecular weights. MON 89034-producedany Cry1A.105 protein and molecular weight markers were permission heated at 95°C for 5 min andprohibited then applied to a pre-cast Tris-glycine 4→20% polyacrylamide gradient gel. Electrophoresis was performed at constant voltage (125 V the be for 10 min followedcommercial by 170 V for 70 min). Proteins were stained with Bio-Rad CoomassieFurthermore, any1 × stain for 2 h, and destained by washing with 1× destain solution (Bio- Rad)Consequently, for 2 h withwithout one change of the destain solution.

The bands representing the full-length Cry1A.105 protein were excised from two gel lanes, destained, reduced, alkylated, and subjected to an in-gel trypsin digestion (Williams et al., 1997). Briefly, each gel band was individually destained for 30 min by incubation in 100 µl of 40% (v/v) methanol and 10% (v/v) glacial acetic acid in a microfuge tube. This was repeated two additional times. Following destaining, the gel bands were incubated in 100 µl of 100 mM ammonium bicarbonate buffer for 30 min at

Monsanto Company 06-CR-166U Page 194 of 262 CBI Deleted Version room temperature. The protein was reduced in 100 µl of 10 mM dithiothreitol solution for 2 h at 37°C. The protein was alkylated by the addition of 100 µl of 200 mM iodoacetic acid. The alkylation reaction was allowed to proceed at room temperature for 20 min in the dark. The gel bands were then incubated in 100 µl of 100 mM ammonium bicarbonate buffer for 30 min at room temperature at which time 100 µl of acetonitrile was added and the incubation was continued for an additional 30 min. The ammonium bicarbonate/acetonitrile incubations were repeated two additional times to remove the reducing and alkylating reagents and other salts from the gel. The gel slices were dried in a SpeedVac concentrator, rehydrated with 40 µl 25 mM ammonium bicarbonate containing 33 µg/ml trypsin, and the protein contained in the gel band was digested for 16 h at 37°C. Digested peptides were extracted for 60-80 min at room temperature with 50 µl 70% (v/v) acetonitrile containing 0.1% (v/v) TFA per gel band. Bothregime. extractionand supernatants were combined into a single tube and dried in anda SpeedVac concentrator. AG This process of extracting the peptides was repeated two more times. The final dried materials were reconstituted in 5 µl of 0.1% (v/v) TFA. Bayer property publishing protectioncontents An aliquot (4 µl) of the digested sampleof was desalted (Bagshaw et al., 2000) using its Millipore (Bedford, MA) ZipTip C18 tips. Prior to parties.desalting,data the tips weretherefore wetted with and/oror methanol and equilibrated with 0.1% (v/v) TFA. The sample was applied to a ZipTip and affiliates. eluted with 4 µl of Wash 1 [0.1%property (v/v) TFA],intellectualthird followed by 4 µl of Washmay 2 [20% (v/v) its acetonitrile containing 0.1% (v/v) TFA],as followed by 4 µl of Wash 3 [50% (v/v) the of and acetonitrile containing 0.1%is (v/v) TFA], and regulatoryfinally with 4 µl of Washowner. 4 [90% (v/v) a document acetonitrile containing 0.1%any (v/v) such TFA]. its reproductiondocument owner this of under of Mass spectral analyses wererights performedthe as follows. Massthis calibration of the instrument documentand/or rights was performed using anto externalof fall peptide mixtureuse of from a Sequazyme peptide mass standards kit (Applied Biosystems, Foster City, CA). theSamples (0.5 µl) from each of the desaltingThis steps, as well as amay sample distribution, ofand solution taken prior to desalting, were co- rights owner crystallized withsubject 0.8 µ l α-cyano-4-hydroxy cinnamic acid on the analysis plate. The the violate sample was beanalyzed copy in the 500 to 5000 Da range using 100 shots at a laser intensity setting of 2781 (a unit-less MALDI-TOFof instrument specific value). Protonated (MH+) and peptide maymasses weredocument measuredpublication, monoisotopical ly in reflector mode (Aebersold, 1993; It exploitation Billeci and Stults,this 1993). GPMAW32 software (Applied Biosystems, version 4.23) was used to generate expectedany tryptic peptide masses based on the expected cleavage sites in permission Cry1A.105 protein sequence. Theprohibited in silico generated masses were compared to the raw + experimental masses obtained.the be Experimental masses (MH ) were assigned to peaks in the 500 to 1000 commercialDa range if there were two or more isotopically resolved peaks, and in theFurthermore, 1000 to any5000 Da range if there were three or more isotopically resolved peaks in the spectra.Consequently, Peaks withoutwere not assessed if the peak heights were less than approximately twice the baseline noise, or when a mass could not be assigned due to overlap with a stronger signal ±2 Da from the mass analyzed.

Monsanto Company 06-CR-166U Page 195 of 262 CBI Deleted Version

B.1.8. Functional Activity Biossay

Aliquots of the MON 89034-produced and E. coli-produced Cry1A.105 protein were tested for biological activity in a diet-incorporation insect bioassay to compare the equivalence of the biological activity of the Cry1A.105 protein from the two sources.

The total protein concentration of the E. coli-produced Cry1A.105 protein aliquots was 1.2 mg/ml, with a purity of 92%, and a purity corrected concentration of 1.1 mg Cry1A.105 mg/ml. The Cry1A.105 protein concentration of the purified MON 89034- produced Cry1A.105 protein aliquots was 3 µg/ml. The control substances used in the bioassays were buffers of the same composition used for formulatingregime. the andE. coli - produced and MON 89034-produced proteins. The E. coli-producedand reference standard AG was suspended in 25 mM CAPS, pH 10.3, 1 mM benzamidine-HCl, 0.1 mM EDTA, 0.2 mM DTT buffer solution. The buffer composition for the MON 89034-produced protein was 50 mM CAPS, 1.0 mM PMSF, 2.0 BayermM benzamidine-HCl, property protection 1 mMpublishing EDTA, 0.8 M NaCl, 30% (v/v) ethylene glycol, pHof 10. The MON 89034-producedcontents and E. coli- its produced Cry1A.105 proteins were stored at 4°C andparties. -80data°C, respectively.therefore The control and/oror buffers for both proteins were stored at 4°C. affiliates. property intellectualthird may its as The test system was corn theearworm of (CEW,and Helicoverpa zea) which was obtained from Benzon Research Inc (Carlisle,is PA). Insect regulatoryeggs were incubated owner.at a temperatures a document ranging from 10oC to 27oanyC, tosuch achieve the desired hatch time.its The bioassay was reproductiondocument replicated three times on separate ownerdays with separatethis batches ofof insects. The MON under of 89034-produced and E. colirights-producedthe protein treatmentsthis we re run in parallel during each documentand/or rights bioassay. Each bioassayto replicof ate fallconsisted ofuse a seriesof of five protein levels yielding a dose series ranging from 0.00048 – 0.039 µg Cry1A.105the protein/ml diet with a 3-fold This may distribution,and separation factor betweenrights dose levels. Thisowner dose series was chosen to adequately characterize thesubject dose-effect relationship for the proteins from both sources. Each dose level, includingbe the control, had an equivathelent violate volume of buffer added to the dosing copy of solution. The Cry1A.105 protein dosing solutiand ons were prepared by diluting the protein with purifiedmay waterdocument andpublication, incorporating the diluted solution into an agar-based insect diet It exploitation (Southland Corp., Lake Village, AR). Diet mixture was then dispensed in 1 ml aliquots this any into a 128 well tray (CD International, Pitman, NJ). Insect larvae were placed on these permission diets using a fine paintbrush, withprohibited a target number of 16 insects per treatment. The infested wells were coveredthe be by a ventilated adhesive cover (CD International, Pitman, NJ) and the insectscommercial were allowed to feed for a period of 6-7 days in an environmental chamberFurthermore, programmedany at 27°C, ambient relative humidity and a lighting regime of 14 h:10Consequently, h (light:dark).without The combined weight of the surviving insects at each dose level for each source of protein was recorded at the end of the 6-7 day incubation period.

The following three-parameter logistic model, with an extra parameter for the change in variation with the expected weight (equation below), was used to model the dose- response curves for each protein source and each replicate under the PROC NLMIXED procedure in SAS (Statistical Analysis System):

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Equation: W0 Wt = B + e ⎛ DietDose ⎞ 1 + ⎜ ⎟ ⎝ EC50 ⎠

2 ⎧ ⎫ ⎪ ⎪ ⎪ ⋅Ws 0 ⎪ ()eVar = ⎨ B ⎬ ⎪ ⎛ DietDose ⎞ ⎪ 1 + ⎜ ⎟ ⎩⎪ ⎝ EC50 ⎠ ⎭⎪

and and regime. where Wt is the average CEW larvae weight andAG DietDose is the Cry1A.105 protein diet dose level. The residual variation was assumed to be proportional to the expected mean weight. The parameters that are includedBayer in the modelproperty are W0 whichpublishing represents the protectioncontents expected weight at DietDose = 0.0, ECof50 which represents the concentration needed to its inhibit the growth of the target insect by 50%, B whichparties. reflectsdata the rate of thetherefore weight loss as DietDose increases, s which represents the proportion of theand/or standardor deviation to the affiliates. expected weight, and e which denotesproperty the intellectualresidualthird (error). may its as the of and B.2. Results is regulatory owner. a document any such its reproductiondocument B.2.1 Cry1A.105 Protein Immunoreactivityowner this of under of rightsthe this documentand/or rights Western blot analysis usingto ofa polyclonafall l anti-Cry1A.105use of antibody was conducted to determine the relative immunoreactivity of the thepurified, MON 89034-produced Cry1A.105This protein and the E. maycoli -produceddistribution,and Cry1A.105 reference standard. The results rights owner demonstrated thatsubject the anti-Cry1A.105 antibody recognized the full-length MON 89034- produced Cry1A.105 protein that migratedthe similarly violate to the full-length E. coli-produced be copy reference Cry1A.105 protein (Figure ofB-1 ). As expected, the immunoreactive signal and increasedmay with increasingdocumentpublication, levels of loading for both MON 89034- and E. coli-produced It exploitation proteins. The immunoreactivethis band with lower molecular weight most likely represents a a proteolytic fragmentany of the Cry1A.105 protein. It is common to observe such permission proteolytic fragments of Cry1 prohibitedproteins due to the cleavage by proteases in vivo or in vitro. A faint immunoreactive band with molecular weight above 250 kDa was observed the be in the samples ofcommercial the MON 89034-produced and E. coli-produced Cry1A.105 protein. ThisFurthermore, band mostany likely represents the aggregate of the Cry1A.105 protein. Consequently,without The Western blot analysis confirmed the identity of the MON 89034-produced Cry1A.105 protein and demonstrated that the MON 89034- and E. coli-produced Cry1A.105 protein had equivalent immunoreactive properties for the antibody used for the antibody used.

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1 2 3 4 5 6 7 8 9 10 11 12 kDa

250 250

150 100 Full-length Cry1A.105 Proteolytic fragment 75 of Cry1A.105 50 37 regime.and 25 and AG 20 15 10 Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and Figure B-1. Western blotis analysis of MON 89034-producedregulatory and E.owner. coli-produced a documentits Cry1A.105 protein any such reproduction this documentof Aliquots of the purified, MON 89034-producedowner and E. coli-produced Cry1A.105 protein under of were separated by SDS-PAGE,rightsthe and electrotransferredthis to a PVDF membrane. The documentand/or rights membrane was incubatedto withof rabbitfall polycl onaluse anti-Cry1A.105 of antibody and developed using an ECL system. Approximate molecular weightsthe (kDa) are shown on the left side This may distribution,and of the blot, which correspondrights to the markers loadedowner in lanes 1 and 12. subject be the violate Lane copy Sample of Amount Amount and Loaded (ng) Loaded (µl) may documentpublication, 1 It Precision Plus Dualexploitation Color molecular weight markers ⎯ ⎯ 2 E. coli-thisproduced any Cry1A.105 standard 1 ⎯ 3 E. coli-produced Cry1A.105permission standard 2 ⎯ 4 E. coli-produced Cry1A.105prohibited standard 4 ⎯ the 5 E. coli-commercialproduced Cry1A.105 be standard 6 ⎯ 6 MON 89034-produced Cry1A.105 protein ⎯ 7.5 Furthermore,any 7Consequently, MON without89034-produced Cry1A.105 protein ⎯ 7.5 8 MON 89034-produced Cry1A.105 protein ⎯ 10 9 MON 89034-produced Cry1A.105 protein ⎯ 10 10 MON 89034-produced Cry1A.105 protein ⎯ 20 11 MON 89034-produced Cry1A.105 protein ⎯ 20 12 Precision Plus Dual Color molecular weight markers ⎯ ⎯

Monsanto Company 06-CR-166U Page 198 of 262 CBI Deleted Version

B.2.2. Cry1A.105 Protein Molecular Weight

The equivalence in apparent molecular weight of the purified MON 89034- and the E. coli-produced Cry1A.105 protein was demonstrated using SDS-PAGE stained using the SilverXpress® silver staining kit (Invitrogen). The full-length MON 89034-produced Cry1A.105 protein migrated to a position similar to that of the E. coli-produced protein standard which was analyzed concurrently (Figure B-2). Based on the comparable electrophoretic mobility, the MON 89034- and E. coli-produced Cry1A.105 protein was determined to have equivalent molecular weight.

B.2.3. Confirmation of the N-terminus Intactness Using Immunoblot Analysisregime.and and AG The N-terminus of the plant-produced proteins could be blocked by chemical modifications. Therefore, the intactness of the N-terminus of the MON 89034-produced Cry1A.105 was examined using WesternBayer blot analysisproperty with protection an N-terminalpublishing peptide- specific antibody. The anti-N-terminalof pep tide antibody was raised againstcontents a synthetic its peptide consisting of the first 14 amino acids (MDNNPNINECIPYN)parties.data at thetherefore N-terminus of the Cry1A.105 protein. and/oror affiliates. property intellectualthird may its The E. coli-produced Cry1A.105 containingas the intact N-terminal sequence, the the of and Cry1A.105 trypsin-resistantis core lacking the N-terminusregulatory of the full-lengthowner. protein were a document used as positive and negativeany suchreference standards, respectively.its As expected, no reproductiondocument immunoreactive bands were observedowner in the lanesthis loaded withof Cry1A.105 trypsin- resistant core (Figure B-3, lanes 6 andunder 7). The ofband corresponding to the full-length rightsthe this Cry1A.105 proteindocument (approximateand/or ly 130 kDa) was observedrights in the lanes loaded with both to of fall use of the MON 89034- and E. coli-produced Cry1A.105 protein.the Additionally, a fragment of lower molecularThis weight (approximatelymay distribution,and 85 kDa) was observed in both protein rights owner preparations (Figuresubject B-3 , lanes 2-5). This fragment represents a proteolytic product of C-terminal degradation of Cry1A.105 protein.the violate As expected , the intensity of the bands be copy increased in a manner dependent of theof loading quantities. These results are consistent and with publishedmay literaturedocumentpublication, that the intermediate proteolytic fragments of Cry1 proteins It exploitation have intact N-terminus,this while the N-terminal peptide (approximately 25-30 amino acids) was cleaved for the anytrypsin-resist ant core (Gao et al., 2006). permission prohibited In conclusion, the intactness of the N-terminus of the MON 89034-produced Cry1A.105 the be protein was confirmed.commercial Furthermore,any Consequently,without

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kDa

Full-length Cry1A.105

Proteolytic fragment of Cry1A.105

regime.and and AG

Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. Figure B-2. SDS-PAGE of MONproperty 89034-produced intellectualthird and E. coli-producedmay Cry1A.105 its protein as the of and is regulatory owner. Aliquots of the MON 89034-produced Cry1A.105a proteindocument and its the E. coli-produced Cry1A.105 reference standardany weresuch separated byreproduction a Tris-glycine 4→20% polyacrylamide this documentof gradient gel and stained with an Invitrogenowner SilverXpress silver staining kit. Approximate under of molecular weights (kDa) rightsare shownthe on the left side thisof the gel and correspond to the documentand/or of rights markers loaded in lanes to1 andof 9. fall use the This may distribution,and Lane rights Sample owner Amount (ng) subject the violate 1be Broad Range molecular weight markers (Bio-Rad) ⎯ copy of 2 E. coli-produced Cry1A.105and reference standard 96 may documentpublication, It 3 MON 89034-producedexploitation Cry1A.105 protein 48 4 this any MON 89034-produced Cry1A.105 protein 48 5 MON 89034-produced Cry1A.105 protein 72 permission 6 MON 89034-producedprohibited Cry1A.105 protein 72 7 MONthe 89034-produced Cry1A.105 protein 96 commercial be 8 MON 89034-produced Cry1A.105 protein 96 Furthermore,any 9 Broad Range molecular weight markers (Bio-Rad) ⎯ Consequently,without

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kDa

Full-length Cry1A.105

Proteolytic fragment of Cry1A.105

regime.and and AG

Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. Figure B-3. Examination of theproperty intactness intellectual of thirdN-terminus by immunoblotmay analysis its using anti-N-terminal peptide antibodyas the of and is regulatory owner. Aliquots of the MON 89034–produced Cry1A.105a protein,document E. coli-its produced Cry1A.105 reference standard, and Cry1A.105any such trypsin-resireproductionstant core standard were separated by this documentof SDS-PAGE and electrotransferred ownerto a PV DF membrane. The membrane was probed under of with the anti-N-terminal peptiderightsthe antibody, and immunoreactivethis bands were visualized documentand/or rights using an ECL system to(5 minof exposure).fall Approximateuse of molecular weights (kDa) are shown on the left side of the blot and correspond to thethe markers loaded in lanes 1 and 8. This may distribution,and rights owner Lane subject Sample Amount (ng) be the violate copy of 1 Precision Plus Dual Colorand molecular weight markers ⎯ may documentpublication, It 2 MON 89034-producedexploitation Cry1A.105 protein 20 3 this MONany 89034-produced Cry1A.105 protein 40 4 E. coli-produced Cry1A.105 reference standard 20 permission 5 E. coli-producedprohibited Cry1A.105 reference standard 40 the 6 commercial Cry1A.105 be trypsin-resistant core standard 20 7 Cry1A.105 trypsin-resistant core standard 40 Furthermore, 8 any Precision Plus Dual Color molecular weight markers ⎯ Consequently,without

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B.2.4. Tryptic Peptide Mapping by MALDI-TOF MS

The MON 89034-produced, full-length Cry1A.105 protein was further identified by tryptic peptide mapping analysis using MALDI-TOF MS. The protein sample was heat- denatured, chemically reduced, alkylated and digested with trypsin, and the masses of the tryptic peptides were measured.

There were 52 protein peptide masses identified that matched the expected tryptic peptides generated in silico based on the predicted cleavage sites in the sequence. The identified masses were used to assemble a coverage map that displays those matched peptide sequences for the protein (Figure B-4). Overall, the confirmed sequence regime.and accounts for 43.8% (516 out of 1177 amino acids) of theand full-length sequence of AG Cry1A.105 protein. In general, a protein identification made by peptide mapping is considered to be reliable if the measured coverage of the sequence is 15% or higher with a minimum of five matched peptides (JenseBayern et al.,property 1997). protectionIn thepublishing current case, the detected peptide coverage was 43.8% withof 52 matched peptides, therefore,contents the protein its identity of MON 89034-produced Cry1A.105 proteinparties. was confirmed.data therefore and/oror affiliates. property intellectualthird may B.2.5. Lack of Glycosylation for Cry1A.105its Protein as the of and is regulatory owner. Some eukaryotic proteins are post-translationallya modifieddocument with carbohydrateits moieties (Rademacher et al.; 1988).any These such carbohydra tereproduction moieties may be complex, branched this documentof polysaccharide structures or simpleowner oligosac charides to monosaccharides. In contrast, under of prokaryotic organisms suchrights asthe non-virulent E. colithis strains used for cloning and documentand/or rights expression purposes lackto theof necessaryfall biochemicaluse of synthetic capacity required for protein glycosylation. the This may distribution,and rights owner To examine whethersubject potential post-translational glycosylation of the MON 89034- be the violate produced Cry1A.105copy protein occurred,of the purified protein sample was subjected to glycosylation analysis. The E. coli-producedand Cry1A.105 reference standard represented a may documentpublication, negativeIt control. The positiveexploitation control was represented by transferrin and horseradish peroxidase (HRP)this anyproteins that are known to have multiple covalently-linked carbohydrate modifications. The transferrin, HRP, as well as the purified Cry1A.105 permission protein isolated from MON 89034prohibited and E. coli were separated on SDS-PAGE, and the glycosylation analysiscommercial was beperformed to detect oxidized carbohydrate moieties on the proteins. The results of this analysis are shown in Figure B-5. The positive controls wereFurthermore, detectedany at the expected molecular weights in a concentration-dependent manner (FigureConsequently, B-5, laneswithout 2-5). No detectable signal was observed for the MON 89034- produced and E. coli-produced Cry1A.105 protein (Figure B-5, lanes 6-9).

Therefore, the MON 89034-produced protein is not glycosylated and, thus is equivalent to the E. coli-produced Cry1A.105 reference standard with respect to the lack of glycosylation.

Monsanto Company 06-CR-166U Page 202 of 262 CBI Deleted Version

0001 MDNNPNINEC IPYNCLSNPE VEVLGGERIE TGYTPIDISL SLTQFLLSEF 0051 VPGAGFVLGL VDIIWGIFGP SQWDAFLVQI EQLINQRIEE FARNQAISRL 0101 EGLSNLYQIY AESFREWEAD PTNPALREEM RIQFNDMNSA LTTAIPLFAV 0151 QNYQVPLLSV YVQAANLHLS VLRDVSVFGQ RWGFDAATIN SRYNDLTRLI 0201 GNYTDHAVRW YNTGLERVWG PDSRDWIRYN QFRRELTLTV LDIVSLFPNY 0251 DSRTYPIRTV SQLTREIYTN PVLENFDGSF RGSAQGIEGS IRSPHLMDIL 0301 NSITIYTDAH RGEYYWSGHQ IMASPVGFSG PEFTFPLYGT MGNAAPQQRI 0351 VAQLGQGVYR TLSSTLYRRP FNIGINNQQL SVLDGTEFAY GTSSNLPSAV 0401 YRKSGTVDSL DEIPPQNNNV PPRQGFSHRL SHVSMFRSGF SNSSVSIIRA 0451 PMFSWIHRSA EFNNIIASDS ITQIPLVKAH TLQSGTTVVR GPGFTGGDIL 0501 RRTSGGPFAY TIVNINGQLP QRYRARIRYA STTNLRIYVT VAGERIFAGQ 0551 FNKTMDTGDP LTFQSFSYAT INTAFTFPMS QSSFTVGADT FSSGNEVYIDand 0601 RFELIPVTAT LEAEYNLERA QKAVNALFTS TNQLGLKTNVand TDYHIDQVSNregime. 0651 LVTYLSDEFC LDEKRELSEK VKHAKRLSDEAG RNLLQDSNFK DINRQPERGW 0701 GGSTGITIQG GDDVFKENYV TLSGTFDECY PTYLYQKIDE SKLKAFTRYQ 0751 LRGYIEDSQD LEIYSIRYNA KHETVNVPGT GSLWPLSAQS PIGKCGEPNR Bayer property protectionpublishing 0801 CAPHLEWNPD LDCSCRDGEK CAHHSHHFSLof DIDVGCTDLN EDLGVWVIFKcontents 0851 IKTQDGHARL GNLEFLEEKP LVGEALARVK RAEKKWRDKR EKLEWETNIVits 0901 YKEAKESVDA LFVNSQYDQL QADTNIAMIH AADKRVHSIRparties.data EAYLPELSVItherefore and/oror 0951 PGVNAAIFEE LEGRIFTAFS LYDARNVIKN GDFNNGLSCW NVKGHVDVEE affiliates. 1001 QNNQRSVLVV PEWEAEVSQEproperty VRVCPGRGYI intellectualthird LRVTAYKEGY GEGCVTIHEImay its 1051 ENNTDELKFS NCVEEEIYPN NTVTCNDYTVas NQEEYGGAYT SRNRGYNEAP the of and 1101 SVPADYASVY EEKSYTDGRRis ENPCEFNRGYregulatory RDYTPLPVGY VTKELEYFPEowner. 1151 TDKVWIEIGE TEGTFIVDSV ELLLMEE a document any such its reproductiondocument owner this of

under of Figure B-4. Sequence coveragerightsthe in the tryptic peptidethis mapping analysis of MON documentand/or of rights 89034-produced full-lengthto ofCry1A.105 fall withuse MALDI-TOF MS the Shaded regionsThis correspond to 52may fragments distribution,and of tryptic peptide masses that were identified rights owner from the full-lengthsubject protein band. Overall, 43.8% of the protein sequence was identified. be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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1 2 3 4 5 6 7 8 9 10 kDa kDa 7

250 180 7 150 100 82 7 75 50 42 37 25

20 regime.and 18 and15 AG 10

Bayer property protectionpublishing Figure B-5. Glycosylation analysis of theof MON 89034-produced Cry1A.105contents protein its parties.data therefore Aliquots of the MON 89034-produced Cry1A.105 protein, E.and/or coli-producedor Cry1A.105 reference standard (negative control),affiliates. horseradish peroxidase (positive control) and property intellectualthird may transferrin (positive control) wereits separated by SDS-PAGE (4→20% gradient) and as electrotransferred to a PVDFthe membrane.of and Where present, periodate-oxidized proteinis regulatory owner. bound carbohydrate moieties reacted with Pra o-Q Emeralddocument 488 glycoproteinits stain and emitted a fluorescent signalany at such488 nm. The reproductionsignal was captured using a Bio-Rad this documentof Molecular Imager FX. Approximateowner mo lecular weights (kDa) correspond to the under of CandyCane glycoprotein markersrightsthe loaded in lane 1 and thisthe Precision Dual Color markers documentand/or of rights in lane 10. to of fall use the This may distribution,and Lane rightsSample owner Amount (ng) subject be the violate 1 copy CandyCane glycoproteinof molecular weight standards ⎯ 2 Horseradish Peroxidaseand (positive control) 48 may documentpublication, It 3 Horseradishexploitation Peroxidase (positive control) 96 4 this any Transferrin (positive control) 48 5 Transferrin (positive control) 96 permission 6 MON 89034prohibited-produced Cry1A.105 48 the 7 commercial MON 89034 be -produced Cry1A.105 96 8 E. coli-produced Cry1A.105 (negative control) 48 Furthermore,9 any E. coli-produced Cry1A.105 (negative control) 96 Consequently, 10 without Precision Plus Dual Color molecular weight markers ⎯

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B.2.6. Cry1A.105 functional activity

The biological activities of E. coli-produced and MON 89034-produced Cry1A.105 protein were estimated by determining EC50 values in a corn earworm (CEW) diet- incorporation bioassay. The EC50 value is defined as the level of Cry1A.105 protein in the diet that results in 50% inhibition to larval growth.

The EC50 values for each replicate bioassay are summarized in Table B-1, and the dose response relationships for MON 89034- and E. coli-produced Cry1A.105 are illustrated in Figure B-6. The ranges of the estimated EC50 values overlapped for the protein from the two host sources. The EC values for the MON 89034-produced protein ranged from 50 regime.and 0.0055 to 0.0089 µg Cry1A.105/ml diet and the EC valuesand for the E. coli-produced AG 50 protein ranged from 0.0053 to 0.0170 µg Cry1A.105/ml diet. Figure B-6 shows an equivalent slope for the dose-response relationship for the E. coli- and MON 89034- produced Cry1A.105 protein in the CEWBayer bioassa y. property These results clearlypublishing demonstrate protectioncontents that the Cry1A.105 protein derived fromof MON 89034 and E. coli has equivalent its functional activity. parties.data therefore and/oror

affiliates. property intellectualthird may its Table B-1. EC values of E. coli-producedas and MON 89034-produced Cry1A.105 50 the of and protein in a corn earwormis diet-incorporationregulatory bioassay owner. a document any such its reproductiondocument owner this 1 of EC50 (µg Cry1A.105/ml diet) under of rightsthe this document and/or E. coli-produced MON 89034-producedrights to of fall use of 1 0.0150 ± 0.0025 0.0055 ± 0.0014 the ReplicateThis2 2 0.0053may ± 0.0022distribution,and 0.0089 ± 0.0018 rights owner subject 3 0.0170 ± 0.0021 0.0077 ± 0.0012 be the violate Overall copy 0.0120 ± 0.0062of 0.0074 ± 0.0017 1 and EC50 (mean ± standarddocument error) represents the concentration needed to inhibit the growth may publication,exploitation It of the target insect by 50%. this 2 Each bioassay replicateany consisted of a series of five protein levels yielding a dose series ranging from 0.00048permission – 0.039 µg Cry1A.105 protein/ml diet with a 3-fold prohibited separation factorthe between dose levels. Insect larvae were placed on the diets with 16 insectscommercial per treatment. be The combined weight of the surviving insects at each dose level was recorded at the end of the 6-7 day incubation period. EC was Furthermore,any 50 Consequently,calculatedwithout with SAS software.

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250

200

150

100 E. coli Plant 50 and and regime. Mean Insect Mass (mg) 0 AG Controls 2 3 4 5 LogBayer Cry1A.105 property Dose Levelprotectionpublishing of (pg/mL diet) contents its parties.data therefore and/oror affiliates. property intellectualthird may its Figure B-6. Functional equivalence ofas the E. coli-produced and the MON 89034- the of and produced Cry1A.105 proteinis against corn earwormregulatory (CEW) owner. a document Bioassay results from threeany replicates such have been combined for illustrationits purpose only reproductiondocument of and are shown on a log concentrationowner scale. Each datathis point represents the mean of the under of three bioassays along withrights the standardthe error of the mean.this Equivalent slope was seen for documentand/or rights dose-response relationshipsto forof the E.fall coli - anduse MON of 89034-produced Cry1A.105 protein in the CEW bioassay. The data demonstrate that thethe test insect body weight decreases This may distribution,and with the increase of therights Cry1A.105 dose level,owner indicating the greater growth inhibitory effect on the testsubject insects at higher doses. be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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B.3. References

Aebersold, R. 1993. Mass spectrometry of proteins and peptides in biotechnology. Curr. Opin. Biotechnol. 4:412-419. Bagshaw, R.D., J.W. Callahan, and D.J. Mahuran. 2000. Desalting of in-gel-digested protein sample with mini-C18 columns for matrix-assisted laser desorption ionization time of flight peptide mass fingerprinting. Anal. Biochem. 284:432- 435. Billeci, T.M. and J.T. Stults. 1993. Tryptic mapping of recombinant proteins by matrix- assisted laser desorption/ionization mass spectrometry. Anal. Chem. 65:1709- 1716. and and regime. Crickmore, N., D.R. Ziegler, J. Feitelson, E. Schnepf,AG J. Van Rie, D. Lereclus, J. Baum, and D.H. Dean. 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:807-813. Bayer property protectionpublishing Gao, Y., K.J. Fencil, X. Xu, D.A. ofSchwedler, J.R. Gilbert, R.A. Herman.contents 2006. Purification and characterization of a chimeric Cry1F δ-endotoxinits expressed in parties.data therefore transgenic cotton plants. J. Agric. Food Chem. 54:829-835.and/or or affiliates. Jensen, O.L., A.V. Podtelejnikov,property and M.intellectual Mann.third 1997. Identification mayof the components its of simple protein mixtures by high-accuas racy peptide mass mapping and database the of and searching. Anal. isChem. 69:4741-4750. regulatory owner. a document Rademacher, T.W., R.B. anyParekh, such and R.A. Dwek. 1988. Glycobiology.its Annu. Rev. reproductiondocument of Biochem. 57:785-838. owner this under of this Williams, K., M. LoPresti,rights andthe K. Stone. 1997. Internal protein sequencing of SDS- documentand/or of rights PAGE-separatedto proteins: of Optimizationfall use of an in-gel digest protocol. Tech. Protein Chem. 8:79-98. the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Appendix C. Materials, Methods, and Results for Characterization of Cry2Ab2 Protein

The Cry2Ab2 protein was purified from the grain of MON 89034 as well as from a recombinant E. coli culture. A panel of analytical tests was used to identify, characterize and compare the MON 89034-produced and E. coli-produced Cry2Ab2 protein including: (1) Western blot analysis; (2) SDS-PAGE and densitometry; (3) MALDI-TOF MS; (4) N-terminal sequence analysis; (5) glycosylation analysis; and (6) insect activity bioassay. The materials, methods and the results are described below.

C.1. Materials and Methods regime.and and AG C.1.1. Purification of MON 89034-Produced Cry2Ab2 Protein

The MON 89034 produced Cry2Ab2 proteinBayer was purifiedproperty from protection grainpublishing of MON 89034. The identity of the grain containing MONof 89034 was confirmed by event-specificcontents PCR. its The isolated MON 89034-produced Cry2Ab2 proteinparties. was datastored in a –80°Ctherefore freezer in a buffer solution containing 50 mM CAPS, 2 mM DTT (pHand/or 11)or at a total protein affiliates. concentration of 0.25 mg/ml. property intellectualthird may its as the of and The purification was performedis using a combinregulatoryation of ammonium sulfateowner. fractionation, a document anion exchange chromatography,any such and immunoaffinity chromatography.its reproductiondocument owner this of The isolation of Cry2Ab2 protein fromunder ground cornof grainthis was started from two 10 kg and/orrights the batches. Eachdocument batch was extracted with PBS buffer (1 mMrights KH 2PO4, 10 mM Na2HPO4, to of fall use of 137 mM NaCl, 2.7 mM KCl, pH 7.4) to remove contaminant proteins. The PBS the extractionThis procedure consistedmay of soaki distribution,ngand ground grain in PBS for 2 h in a 4°C cold room rights owner at approximatelysubject 1:10 ratio of sample weight (g) to buffer volume (ml). The slurry was clarified by befiltration using an Ertel Alsopthe filterviolate press (Kingston, NY), and the PBS washed solid particlecopy (cake) was retained.of Subsequently, the Cry2Ab2 protein in the cake and was extractedmay withdocument extractionpublication, buffer containing 50 mM CAPS, 1 mM EDTA, 2.5 mM It exploitation DTT, 1 mM PMSF,this 2 mM benzamidine-HCl, 0.5 mM CHAPS, 1% (w/v) PVPP, pH 10.8 at approximately 1:10any ratio of sample weight (g) to buffer volume (ml) for 2-3 h. During permission extraction, lipids were removedprohibited from the extract by adding CelPure P65 diatomaceous earth (Advanced Minerals Corp, Goleta, CA) to the homogenate at approximately 7.5% the be (w/v) and allowedcommercial to mix for 10 min. The slurry was clarified by filtration using the filter pressFurthermore, and theany resultant extract from both batches was pooled for a total volume of 230 L. TheConsequently, pooled extractwithout was concentrated using a 30,000 NMWC (nominal molecular weight cutoff) hollow fiber cartridge (Amersham Biosciences, Piscataway, NJ) to a final volume of 35 L. To remove plant genomic DNA, 10% (w/v) polyethyleneimine, was added to a final concentration of 0.05% (w/v), the extract was clarified by centrifugation to remove precipitated DNA, and the supernatant was retained. The Cry2Ab2 protein in the supernatant was precipitated with 35% ammonium sulfate saturation. The precipitate was pelleted by centrifugation and the pellet was dissolved in 20 L of resuspension buffer containing 50 mM CAPS, 1 mM EDTA, 2.5 mM DTT, 1 mM PMSF, 2 mM

Monsanto Company 06-CR-166U Page 208 of 262 CBI Deleted Version benzamidine-HCl, pH 10.8 by mixing in 4°C cold room. The suspension was clarified by centrifugation, and 21 L supernatant was retained, concentrated, and buffer-exchanged by diafiltration against a buffer containing 50 mM CAPS, 1 mM EDTA, 2.5 mM DTT, 1 mM PMSF, 2 mM benzamidine-HCl, pH 10.8 (loading buffer) to remove any residual ammonium sulfate salt. The concentrated sample of 13 L was loaded onto an anion exchange column in two batches – Run 1 (6 L) and Run 2 (7 L). The elution parameters were identical for both runs and thus only the Run 1 column parameters are described below.

A portion of concentrated sample was loaded onto a 4.5 L (20 cm x 14.4 cm column) Q Sepharose Fast Flow anion exchange column, which was equilibrated with the loading buffer. The bound proteins were eluted with step gradients as follows: 0-20%regime. bufferand B (loading buffer plus 1M NaCl) in 10 column volumes (CV), thenand the gradient was held at AG 20% buffer B for 4 CV, and then the gradient was increased to 65% buffer B over 10 CV, and finally the gradient was stepped up to 100% buffer B and held at 100% buffer B for 2 CV. Fractions, each of 4 L, containingBayer Cry2Ab2 propertyprotein were identifiedpublishing by Cry2A protectioncontents QuickStix™ lateral flow test kit (EnviroLogix,of Portland, ME) for further analysis. Based its on SDS-PAGE and Western blot analysis results, fractionparties. data7 from Run 2 wastherefore selected for and/oror affinity purification. affiliates. property intellectualthird may its The fraction 7 was concentrated to 400as ml using a 30,000 NMWC hollow fiber cartridge. the of and Approximately one half isof the aforementioned regulatorysample was buffer-exchangedowner. into EPPS a document buffer (50 mM EPPS, 2 mMany DTT,such pH 7.2) using centrifuge concentratorsits (30 kDa reproductiondocument MWCO), resulting in a final volumeowner of 200 ml. Thisthis sample, ofin two separate batches, under of was applied to an affinityrights columnthe (1.0 cm × 2.7 cm)this containing Protein G agarose documentand/or rights conjugated with Cry2Aa-specificto of mAbfall and equilibrateduse of with EPPS buffer (50 mM EPPS, 2 mM DTT, pH 7.2). The sample was re-circulated throughthe the column for 2 h at 100 ml/h. TheThis column was then washedmay distribution,with and17-20 CV of EPPS buffer (50 mM EPPS, 2 mM rights owner DTT, pH 7.2), followedsubject by 5-7 CV of an EPPS buffer with a different pH (50 mM EPPS, the violate 2 mM DTT,be pH copy 8.5). The bound Cry2Ab2 protein was then eluted with 100 mM triethylamine, pH 11.4, in five fractionsof (1.5 ml each). Based on SDS-PAGE analysis, and fractionsmay containing document Cry2Ab2publication, protein from batches one and two were concentrated and It exploitation buffer-exchangedthis into a storage buffer containing 50 mM CAPS, 2 mM DTT, pH 11.0, using centrifuge concentratorsany (30 kDa MWCO). Concentrated samples were then permission pooled, resulting in a final volumeprohibited of 2 ml. the be The affinity chromatographycommercial procedure was repeated beginning with 100 ml of the fractionFurthermore, 7 fromany Run 2. As previously described, the sample was concentrated and buffer- exchangedConsequently, intowithout EPPS buffer (50 mM EPPS, 2 mM DTT, pH 7.1) to a final volume of 50 ml using centrifuge concentrators (30 kDa MWCO). This sample was re-circulated for 2 h (60 ml/h) through the affinity column equilibrated with an EPPS buffer (50 mM EPPS, 2 mM DTT, pH 7.1). The column was then washed with 15 CV of EPPS buffer (50 mM EPPS, 2 mM DTT, pH 7.1) followed by 5 CV of another EPPS buffer with a different pH (50 mM EPPS, 2 mM DTT, pH 8.5). The bound Cry2Ab2 protein was eluted with 100 mM triethylamine, pH 11.4, in five fractions (1.5 ml each).

Monsanto Company 06-CR-166U Page 209 of 262 CBI Deleted Version

Based on SDS-PAGE analysis, Cry2Ab2 enriched fractions were individually concentrated and buffer-exchanged into a storage buffer containing 50 mM CAPS, 2 mM DTT, pH 11, using a centrifuge concentrator (30 kDa MWCO). The concentrated samples were pooled into a final volume of 2.4 ml. The sample and the sample from the first runs (2 ml) were pooled, resulting in a final volume of 4.4 ml. This sample was a clear, colorless solution and was used for further characterization tests.

C.1.2. Escherichia coli-Produced Cry2Ab2 Protein

E. coli-produced Cry2Ab2 protein produced and purified previously was used as a reference standard for determination of protein concentration of the MON 89034- produced Cry2Ab2 protein. This protein was also used as a reference standard to regime.and evaluate equivalence between MON 89034-produced and E.and coli -produced Cry2Ab2 proteins for immunoreactivity (Western blot),AG molecular weight (SDS-PAGE) and functional activity assay. Bayer property protectionpublishing C.1.3. Determination of MON 89034-Producedof Cry2Ab2 Protein Concentrationcontents its parties.data therefore The total protein concentration of the purified MON 89034-produand/orcedor Cry2Ab2 protein affiliates. was estimated using a Bio-Radproperty protein assay.intellectual thirdThe E. coli-produced Cry2Ab2may reference its standard protein (concentrations rangeas from 0.05 to 0.5 mg/ml) was used to prepare a the of and standard curve. The MONis 89034-produced Cry2Ab2regulatory total protein owner.concentration was a document estimated by comparison ofany absorbance such values obtained for the sampleits to the values of reproductiondocument the standard curve. owner this of under of rightsthe this C.1.4. Westerndocument Blotand/or Analysis rights to of fall use of

the AliquotsThis of the stock solutionsmay of distribution,the andMON 89034-produced Cry2Ab2 and E. colirights owner produced referencesubject standard were diluted to a final concentration of 2 ng/µl in dilution buffer (50 mMbe CAPS, 2 mM DTT, thepH 11)violate and 5× sample loading buffer [5x concentrated Laemmlicopy buffer [312 mMof Tris-HCl, 20% (v/v) 2-mercaptoethanol, 10% and (w/v) SDS,may 0.025%document (w/v)publication, bromophenol blue, 50% (v/v) glycerol, pH 6.8)]. Samples It exploitation were then heatedthis to 100°C for 5 min and applied to a pre-cast Tris-glycine 4→20% polyacrylamide gradientany gel. The MON 89034-produced Cry2Ab2 protein was loaded in permission duplicate at three different loadingsprohibited of 20, 30, and 40 ng per lane. The E. coli-produced Cry2Ab2 reference standard was loaded at 20 ng per lane. Electrophoresis was the be performed at a constantcommercial volta ge of 140 V for 20 min followed by a constant voltage of 200Furthermore, V for 43any min. The pre-stained MW markers (Bio-Rad Precision Plus Dual Color, Hercules,Consequently, CA) withoutwere used to verify electrotransfer of protein to the membrane and to estimate the molecular weight of the immunoreactive bands. Samples were electrotransferred to a 0.45 µm PVDF membrane (Invitrogen, Carlsbad, CA) for 60 min at a constant current of 300 mA.

The membrane was blocked for one hour with 5% (w/v) NFDM in PBST. The membrane was probed with a 1:3000 dilution of goat anti-Cry2Ab2 antibody in 2% (w/v) NFDM in PBST for one hour. Excess antibody was removed using three 10 min washes

Monsanto Company 06-CR-166U Page 210 of 262 CBI Deleted Version with PBST. The membrane was probed with HRP-conjugated rabbit anti-goat IgG (Sigma, St. Louis, MO) at a dilution of 1:10,000 in 2% (w/v) NFDM in PBST for one hour. Excess HRP-conjugated antibody was removed using three washes (10 min each) with PBST. All procedures, including blocking, and all other incubations were performed at room temperature. Immunoreactive bands were visualized using the ECL detection system (Amersham Biosciences, Piscataway, NJ) and exposed (30 sec, 1 min, 2 min, 3 min, and 7 min) to Hyperfilm ECL high performance chemiluminescence film (Amersham Biosciences, Piscataway, NJ). Films were developed using a Konica SRX- 101A automated film processor.

C.1.5. Molecular Weight and Purity Estimation by SDS-PAGE

regime.and Aliquots of the test substance and reference standard proteinand were each diluted with AG sample dilution buffer and 5× sample loading buffer to a final protein concentration of 0.2 µg/µl. MW markers (Bio-Rad broad-range, Hercules, CA) that were used to estimate the apparent MW of the test protein wereBayer diluted toproperty a final concentration protectionpublishing of 0.9 µg/µl. of contents The MON 89034-produced Cry2Ab2 protein was analyzed in duplicate at 1, 2, and 3 µg its parties.data therefore total protein loads per lane. The E. coli-produced Cry2Ab2and/or referenceor standard was analyzed at 1 µg total protein per lane.affiliates. All samples were heated at approximately 102°C property intellectualthird may for 5 min and applied to a pre-castits Tris-glycine 4→20% polyacrylamide gradient gel (Invitrogen, Carlsbad, CA).the El ectrophoresisas was performed at a constant voltage of 150 of and owner. V for 82 min. Proteins wereis fixed by placing theregulatory gel in a solution of 40% (v/v) methanol a documentits and 7% (v/v) glacial aceticany acid such for 30 min, stained for 16 h with Brilliant Blue G- reproductiondocument of Colloidal stain (Sigma, St. Louis, MO),owner de stained 30this sec with a solution containing 10% under of (v/v) acetic acid and 25% (v/v)rights themethanol, and finally destainethis d with 25% (v/v) methanol documentand/or rights for 6 h. to of fall use of the This may distribution,and Analysis of the proteinsrights in the stained gel ownerwas performed using a Bio-Rad GS-800 densitometer withsubject the supplied Quantity One software (version 4.4.0, Hercules, CA). the Values for thebe markers supplied by the manufactviolateurer were used to estimate the apparent copy of MW of each observed band. All visible andbands within each lane were quantified using may documentpublication, QuantityIt One software. Forexploitation the MON 89034-produced Cry2Ab2 protein, purity was calculated as thethis percent any optical density of the subject protein band relative to all bands combined a given lane. permission prohibited C.1.6 Glycosylation Analysisthe commercial be

GlycosylationFurthermore,any analysis was used to determine whether the MON 89034-produced Cry2Ab2Consequently, proteinwithout was post-translationally modified with covalently bound carbohydrate moieties. Aliquots of the MON 89034-produced Cry2Ab2 protein, the E. coli-produced Cry2Ab2 reference standard (a negative control), and the positive control transferrin (Amersham Biosciences, Piscataway, NJ) were each diluted in dilution buffer and in 5× sample loading buffer to a final concentration of 50 ng/µl. These samples were heated at 100°C for 5 min, and loaded along with Precision Plus Dual Color pre-stained protein MW markers (Bio-Rad, Hercules, CA) on a pre-cast Tris-glycine 4→20% polyacrylamide gradient gel. All three samples were loaded at 0.5 and 1 µg protein per

Monsanto Company 06-CR-166U Page 211 of 262 CBI Deleted Version lane. Electrophoresis was performed at a constant voltage of 140 V for 20 min followed by a constant voltage of 200 V for 47 min. After electrophoresis, proteins were electrotransfered to a 0.45 µm PVDF membrane for one hour at a constant current of 300 mA.

Carbohydrate detection was performed directly on the PVDF membrane using the ECL detection system (Amersham Biosciences, Piscataway, NJ). After the electrotransfer of the proteins, the PVDF membrane was incubated in PBS for 10 min, and transferred to a solution of 100 mM acetate buffer, pH 5.5, containing the oxidation reagent – 10 mM sodium metaperiodate. The membrane was incubated in the dark for 20 min. The oxidation solution was removed from the membrane by two brief rinses followed by three sequential washes (10 min each) in PBS. The membrane was transferred to a solution of regime.and 100 mM acetate buffer, pH 5.5, containing 25 nM biotin hydrazideand and incubated for 60 AG min. Biotin hydrazide solution was removed by washing in PBS as described above. The membrane was blocked with 5% blocking agent in PBS for 60 min. The blocking solution was removed by washing in PBSBayer as previously property described. protection Thepublishing membrane was incubated with streptavidin-HRP conjugateof (diluted 1:6000) in acetate buffercontents for 30 min its to detect carbohydrate moieties bound to biotin. Excessparties. streptavidin-HRPdata thereforewas removed by washing in PBS as described above. Bands were visualizedand/or usingor the ECL detection affiliates. system (Amersham Biosciences,property Piscataway, intellectual NJthird). Films were exposedmay (1 min, 3 min, its and 6 min) to Hyperfilm ECL high performaas nce chemiluminescence film. Films were the of and developed using a Konicais SRX-101A automatedregulatory film processor. owner. a document any such its reproductiondocument C.1.7. N-terminal Sequencing owner this of under of rightsthe this documentand/or rights An aliquot of the MONto 89034-produced of fall Cry2Ab2use ofprotein was diluted with 5× sample loading buffer to a final concentration of 80 ng/µl. Molecularthe weight markers (Bio-Rad PrecisionThis Plus Dual Color, Hercules,may distribution, CA)and were used to confirm the transfer of protein to rights owner the PVDF membrane.subject The MON 89034-produced Cry2Ab2 protein was electrophoresed in eight lanesbe at 2 µg per lane. The theCry2Ab2 violate contai ning samples were heated to copy of approximately 99°C for 5 min prior to electrophoreand sis on a pre-cast Tris-glycine 4→20% SDS polyacrylamidemay document gelpublication, at 140 V for 20 min followed by 200 V for 43 min. The gel was It exploitation then electroblotted to a 0.2 µm PVDF membrane for 60 min at a constant current of 300 this any mA in a solution containing 10 mM CAPS, 10% (v/v) methanol, pH 11. Protein bands permission were stained by briefly soakingprohibited the membra ne with Coomassie Blue R-250 stain (Bio- Rad) and visualized by thedestaining be with a Coomassie R-250 destain solution (Bio-Rad). commercial TheFurthermore, proteinany bands of interest were excised from the membrane. N-terminal sequence analysisConsequently, was performedwithout using automated Edman degradation chemistry (Hunkapillar et al., 1983). An Applied Biosystems 494 Procise Sequencing System with 140C microgradient system and 785 programmable absorbance detector and Procise Control Software (version 2.1) was used. Chromatographic data were collected using Atlas software (version 2003R1.1, LabSystems, Altrincham, Cheshire, England). A PTH- amino acid standard mixture (Applied Biosystems, Foster City, CA) was used to calibrate the instrument for each analysis. This mixture served to verify system suitability criteria such as percent peak resolution and relative amino acid chromatographic retention times.

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A control protein (10 picomole β-lactoglobulin from Applied Biosystems) was analyzed before and after the analysis of each different protein band to verify that the sequencer met performance criteria for repetitive yield and sequence identity.

C.1.8. Tryptic Peptide Mapping Analysis by MALDI-TOF MS

MALDI-TOF mass spectrometry was used to confirm the identity of the MON 89034- produced Cry2Ab2 protein. An aliquot of the MON 89034-produced Cry2Ab2 protein was diluted with 5× sample loading buffer to a final concentration of 80 ng/µl, and 2 µg was electrophoresed in each of five lanes. The broad-range MW markers (Bio-Rad, Hercules, CA) were used to estimate molecular weights. Samples were heated to approximately 99°C for 5 min prior to electrophoresis on a pre-cast Tris-glycine and4→20% and regime. SDS polyacrylamide gel at 140 V for 20 min followedAG by a constant voltage of 200 V for 46 min. Proteins were fixed by placing the gel in a solution of 40% (v/v) methanol and 7% (v/v) glacial acetic acid for 30 min, stained 2 h with Brilliant Blue G-Colloidal stain (Sigma, St. Louis, MO), destained 30 sec wBayerith a solution property containing 10%publishing (v/v) acetic acid protectioncontents and 25% (v/v) methanol, and followed byof 25% (v/v) methanol for 1 h. its parties.data therefore and/oror Two protein bands, identified as band-1 and band-2, were subjected to the tryptic peptide affiliates. mapping analysis. The proteinproperty bands intellectualof interestthird were excised, destained,may reduced, its alkylated, and subjected to an in-gel trypsinas digestion (Williams et al., 1997). Each gel the of and band was individually isdestained for 30 min regulatoryby incubation in 100owner. µl of 40% (v/v) a document methanol and 10% (v/v) anyglacial such acetic acid in its own microfugeits tube. Following reproductiondocument of destaining, the gel bands were incubatedowner in 100 µl thisof 100 mM ammonium bicarbonate under of buffer for 30 min at roomrights temperature.the Proteins werethis reduced in 100 µl of 10 mM documentand/or rights dithiothreitol solution forto 2 hof at 37fall°C. Proteinsuse wereof then alkylated by the addition of 100 µl of buffer containing 200 mM iodoacetic acid.the The alkylation reaction was This may distribution,and allowed to proceed at roomrights temperature for 20owner min in the dark. The gel bands were then incubated in 100subject µl of 100 mM ammonium bicarbonate for 30 min at room temperature be the violate at which time 100copy µl of acetonitrile wasof added and the incubation was continued for an additional 30 min. The ammonium bicarbonateand /acetonitrile incubations were repeated may documentpublication, two additionalIt times to removeexploitation the reducing and alkylating reagents, and salts from the gel. The gel bandsthis any were dried in a SpeedVac concentrator (Savant, Holbrook, NY), rehydrated with 40 µl 25 mMpermission ammonium bicarbonate containing 33 µg/ml trypsin, and the protein contained in the gelprohibited band was digested for 16 h at 37°C. Digested peptides the were extracted commercialfor one hour be at room temperature with 50 µl 70% (v/v) acetonitrile containing 0.1% (v/v) TFA per gel band. Supernatants for each extraction were Furthermore,any combinedConsequently, into withouta single tube and dried in a SpeedVac concentrator. This process of extracting the peptides was repeated two more times. The final dried materials were reconstituted in 10 µl of 0.1% (v/v) TFA.

A portion (5 µl) of the digested samples was desalted (Bagshaw et al., 2000) using Millipore (Bedford, MA) ZipTip C18 tips. Prior to desalting, the tips were wetted with methanol and equilibrated with 0.1% (v/v) TFA. Each sample was applied to a ZipTip C18 and eluted with 5 µl of Wash 1 [0.1% (v/v) TFA], followed by 5 µl of Wash 2 [20%

Monsanto Company 06-CR-166U Page 213 of 262 CBI Deleted Version

(v/v) acetonitrile containing 0.1% (v/v) TFA], followed by 5 µl of Wash 3 [50% (v/v) acetonitrile containing 0.1% (v/v) TFA], and finally with 5 µl of Wash 4 [90% (v/v) acetonitrile containing 0.1% (v/v) TFA].

Mass spectral analyses were performed as follows. Mass calibration of the instrument was performed using an external peptide mixture from a Sequazyme™ peptide mass standards kit (Applied Biosystems). Samples (0.3 µl) from each of the desalting steps, as well as a sample of the solution taken prior to desalting, were co-crystallized with 0.75 µl α-cyano-4-hydroxy cinnamic acid (Waters, Milford, MA) on the analysis plate. All samples were analyzed in the 500 to 5000 dalton range using 100 shots at a laser intensity setting of 2318-2460 (a unit-less MALDI-TOF instrument specific value). Protonated + and (MH ) peptide masses were observed monoisotopically in reflectorand moderegime. (Aebersold, 1993; Billeci and Stults, 1993). GPMAW32 softwareAG (Applied Biosystems, version 4.23) was used to generate the peptide masses, in silico, based on the trypsin cleavage sites in the Cry2Ab2 protein sequence. Experimental peptide masses (MH+) were assigned to Bayer property protectionpublishing peaks in the 500 to 1000 Da range if thereof were two or more isotopically contentsresolved peaks, and in the 1000 to 5000 Da range if there were three or more isotopicallyits resolved peaks parties.data therefore in the spectra. Peaks were not assessed if the peak heights wereand/or lessor than approximately twice the baseline noise, or when a affiliates.mass could not be assigned due to overlap with a property intellectualthird may stronger signal ±2 Da from the massits analyzed. as the of and is regulatory owner. C.1.9. Functional Activity Biossay a documentits any such reproduction this documentof The purpose of this assay was to comparowner e the biological activity of the MON 89034- under of produced and the E. coli-producedrightsthe Cry2Ab2 proteins thisby determining EC values in a documentand/or rights 50 corn earworm (CEW, Helicoverpato of zea)fall diet-incorporationuse of bioassay. the This may distribution,and The test system was cornrights earworm (CEW) whichowner was obtained from Benzon Research Inc (Carlisle, PA).subject Insect eggs were incubated at temperatures ranging from 10oC to o be the violate 27 C, to achieve copythe desired hatch time.of The bioassay was replicated three times on separate days with separate batches of insects.and The MON 89034-produced and E. coli- may documentpublication, producedIt proteins were run inexploitation parallel during each bioassay. Each bioassay replicate for the E. coli-producedthis any and MON 89034-produced Cry2Ab2 proteins consisted of a series of seven dilutions and a buffer control yielding a dose series with a 2-fold separation permission factor ranging from 0.016 – 1.0prohibited µg Cry2Ab2 protein/ml diet. The dose-response curves the for each protein commercialincluded a bebuffer control. The Cry2Ab2 protein dosing solutions were prepared by diluting the protein with purified water and incorporating the dilution into an agar-basedFurthermore, insectany diet (Southland Corp. Lake Village, AR). This dose series in diet was chosenConsequently, to adequatelywithout characterize the dose-effect relationship on CEW weight gain for the proteins from both sources. The diet mixture was then dispensed in 1 ml aliquots into a 128 well tray (CD International, Pitman, NJ). Insect larvae were placed on these diets using a fine paintbrush, with a target number of 16 insects per treatment. The infested wells were covered by a ventilated adhesive cover (CD International, Pitman, NJ) and the insects were allowed to feed for a period of 7 days in an environmental chamber programmed at 27° C, ambient relative humidity and a lighting regime of 14 h:10 h (light:dark). The combined weight of the surviving insects at each dose level for each

Monsanto Company 06-CR-166U Page 214 of 262 CBI Deleted Version source of protein was recorded at the end of the 7-day incubation period. Dose response modeling was the same as described in Section B.1.8 of Appendix B.

C.2. Results

C.2.1. Cry2Ab2 Protein Immunoreactivity

Western blot analysis using a polyclonal, goat anti-Cry2Ab2 antibody was conducted to confirm the identity and determine the relative immunoreactivity of the MON 89034- produced Cry2Ab2 protein and the E. coli-produced Cry2Ab2 reference standard. Results indicated that the anti-Cry2Ab2 antibody recognized the MON 89034-produced Cry2Ab2 protein which migrated comparably to the E. coli-produced reference standard regime.and protein (Figure C-1, band-1). The immunoreactive signal andincreased with increasing AG levels of the Cry2Ab2 protein. Besides the expected band, an immunoreactive band with lower molecular weight (Figure C-1, band-2) was observed in the partially purified MON 89034-produced Cry2Ab2 sample, whichBayer representsproperty a proteolyticprotectionpublishing fragment of the Cry2Ab2 protein. of contents its parties.data therefore The above Western blot result confirmed the identity of Cry2Ab2and/or orprotein produced by affiliates. MON 89034, and demonstratedproperty that MON intellectual 89034-third and E. coli-producedmay Cry2Ab2 had its equivalent immunoreactivity for the antibodyas used for the antibody used. the of and is regulatory owner. a document C.2.2. Cry2Ab2 Protein Molecularany such Weight Equivalence its reproductiondocument owner this of The equivalence in apparent molecularunder weight ofof the purified MON 89034- and the E. rightsthe this coli-produceddocument Cry2Ab2and/or protein was further demonstrated rightsusing SDS-PAGE stained with to of fall use of Brilliant Blue G-Colloidal stain. The MON 89034-produced,the full-length Cry2Ab2 protein (band-1)This migrated to a mayposition distribution, comparableand to that of the E. coli-produced protein rights owner standard which subjectwas anal yzed concurrently (Figure C-2). Based on the comparable electrophoretic mobility, the MON 89034-the andviolate E. coli -produced, full-length Cry2Ab2 be copy protein (band-1) was determined to haveof equivalent molecular weight. document and may publication,exploitation It this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 215 of 262 CBI Deleted Version

and and regime. AG

Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. Figure C-1. Western blot analysis of MON a89034-produced document andits E. coli-produced Cry2Ab2 protein any such reproduction this documentof owner under of Samples of the partially purifiedrightsthe MON 89034-producedthis and E. coli-produced Cry2Ab2 documentand/or of rights protein were separated byto SDS-PAGEof fall (4→20%use gradient), electrotransferred to a PVDF membrane. The membrane was then detected usingthe goat anti-Cry2Ab2 antibody and This may distribution,and developed using an ECLrights system. Amounts ownerloaded correspond to subject protein after normalization withsubject purity. The approximate molecular weights (kDa) correspond to the be the violate markers loaded incopy Lanes 2 and 10. of and Amount of may documentpublication, Lane It exploitationSample Cry2Ab2 (ng) this any 1 Empty Lane...... N/A permission 2 Precision Plus Dual Colorprohibited molecular weight markers (MWM) ...... N/A the 3 E. coli-producedcommercial Cry2Ab2 be protein ...... 20 4 MON 89034-produced Cry2Ab2 protein ...... 20 Furthermore, 5 MONany 89034-produced Cry2Ab2 protein ...... 20 6Consequently, MON 89034-producedwithout Cry2Ab2 protein ...... 30 7 MON 89034-produced Cry2Ab2 protein ...... 30 8 MON 89034-produced Cry2Ab2 protein ...... 40 9 MON 89034-produced Cry2Ab2 protein ...... 40 10 Precision Plus Dual Color molecular weight markers (MWM) ...... N/A

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1 234 5678910 MWM (kDa) MWM (kDa)

200 200 116.3 116.3 97.4 97.4 66.2 66.2 Band-1 45 45 Band-2

31 31 21.5 21.5 regime.and and 14.4 14.4 AG 6.5 6.5

Bayer property protectionpublishing of contents its Figure C-2. SDS-PAGE of the MON 89034- and E.parties. coli-produceddata Cry2Ab2therefore protein and/oror Samples of the partially purified MONaffiliates. 89034-produced Cry2Ab2 protein, and the E. coli- property intellectualthird may produced Cry2Ab2 reference standardits were separated by a Tris-glycine 4→20% SDS as polyacrylamide gel and stainedthe of with Brilliantand Blue G-Colloidal stain. Amounts loaded is regulatory owner. correspond to total protein loaded per lane.a Approximadocumentte molecularits weights (kDa) correspond to the markers loadedany such in Lanes 2 and 10.reproduction this documentof owner under of Lane Sample rightsthe this Amount (µg) documentand/or of rights to of fall use 1 Empty Lane...... the N/A This may distribution,and 2 MWM (molecularrights weight markers, Bio-Rad,owner broad range) ...... 4.5 3 E. coli-producedsubject Cry2Ab2 reference standard...... 1 be the violate 4 MON 89034-producedcopy Cry2Ab2of protein ...... 1 5 MON 89034-produced Cry2Ab2 proteinand ...... 2 may documentpublication, 6 It MON 89034-producedexploitation Cry2Ab2 protein ...... 2 7 MON 89034-producedthis any Cry2Ab2 protein ...... 3 8 MON 89034-produced Cry2Ab2 protein ...... 3 permission 9 MON 89034-produced prohibitedCry2Ab2 protein ...... 1 the 10 MWM (molecularcommercial weight be markers, Bio-Rad, broad range) ...... 4.5

Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 217 of 262 CBI Deleted Version

C.2.3. Analysis of the N-terminal Sequence

N-terminal sequencing analysis demonstrated that the MON 89034-produced, full-length Cry2Ab2 (band-1) was blocked at the N-terminus with no definitive sequence obtained in the sequencing analysis using Edman degradation chemistry. A minor portion of the protein co-migrating with the full-length protein was proteolytically degraded and the sequence was determined to start from amino acid residue No. 24. N-terminal sequence analysis of the lower molecular weight proteolytic fragment (band-2) revealed that this fragment starts from amino acid residue No. 145. With E. coli-produced Cry2Ab2 (band- 1) the N-terminus sequence was determined as MQAMDN, as expected. This result further confirms that band-1 is the full-length Cry2Ab2 protein. regime.and and AG In summary, the N-terminal sequencing results indicate that MON 89034-produced, full- length Cry2Ab2 protein is blocked at its N-terminus, but N-terminal sequencing result of the lower molecular weight proteolytic fragmentBayer (band-2)property confirms protection thepublishing Cry2Ab2 identity. In addition, the N-terminus of E. coli-produced,of full-length Cry2Ab2 was confirmed.contents its parties.data therefore C.2.4. Tryptic peptide mapping by MALDI-TOF MS and/oror affiliates. property intellectualthird may its The MON 89034-produced, full-lengthas Cry2Ab2 (band-1) and the proteolytic fragment the of and (band-2) were characterizedis by tryptic peptide regulatorymapping analysis withowner. MALDI-TOF MS a document to further confirm their identity.any such For band-1, a total of 32 observedits peptide masses reproductiondocument matched the theoretical tryptic peptideowner masse s of Cry2Ab2this protein.of These identified peptides were used to assemble a coverageunder map in ofthe Cry2Ab2 protein sequence (Figure rightsthe this C-3). The overalldocument and/orpeptide sequence coverage was 44.4%rights out of the 637 amino acid to of fall use of residues of the full-length Cry2Ab2 protein. For band-2,the a total of 24 observed peptide masses matchedThis the expected trypticmay distribution, peptideand masses of Cry2Ab2 protein, which yielded a rights owner coverage map equalsubject to 47.7% out of the 493 amino acid residues of this proteolytic fragment (Figure C-4). the violate be copy of and In general,may a proteindocument identificationpublication, made by peptide mapping is considered to be reliable It exploitation if the measuredthis coverage of the sequence is 15% or higher with a minimum of five matched peptides (Jensenany et al., 1997). In the current case, the detected peptide coverage permission was 44.4% with 32 matched peptidesprohibited for the full-length Cry2Ab2 protein, and 47.7% with 24 matched peptides for the proteolytic fragment of Cry2Ab2, therefore, the identity the be of MON 89034-producedcommercial Cry2Ab2 protein is confirmed. Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 218 of 262 CBI Deleted Version

1 MQAMDNSVLN SGRTTICDAY NVAAHDPFSF QHKSLDTVQK EWTEWKKNNH SLYLDPIVGT 61 VASFLLKKVG SLVGKRILSE LRNLIFPSGS TNLMQDILRE TEKFLNQRLN TDTLARVNAE 121 LTGLQANVEE FNRQVDNFLN PNRNAVPLSI TSSVNTMQQL FLNRLPQFQM QGYQLLLLPL 181 FAQAANLHLS FIRDVILNAD EWGISAATLR TYRDYLKNYT RDYSNYCINT YQSAFKGLNT 241 RLHDMLEFRT YMFLNVFEYV SIWSLFKYQS LLVSSGANLY ASGSGPQQTQ SFTSQDWPFL 301 YSLFQVNSNY VLNGFSGARL SNTFPNIVGL PGSTTTHALL AARVNYSGGI SSGDIGASPF 361 NQNFNCSTFL PPLLTPFVRS WLDSGSDREG VATVTNWQTE SFETTLGLRS GAFTARGNSN 421 YFPDYFIRNI SGVPLVVRNE DLRRPLHYNE IRNIASPSGT PGGARAYMVS VHNRKNNIHA 481 VHENGSMIHL APNDYTGFTI SPIHATQVNN QTRTFISEKF GNQGDSLRFE QNNTTARYTL 541 RGNGNSYNLY LRVSSIGNST IRVTINGRVY TATNVNTTTN NDGVNDNGAR FSDINIGNVV 601 ASSNSDVPLD INVTLNSGTQ FDLMNIMLVP TNISPLY and Figure C-3. Peptide mass coverage map of the MON 89034-producedand full-lengthregime. Cry2Ab2 protein in MALDI-TOF MS analysisAG Shaded regions correspond to 32 fragments of tryptic peptide masses that were identified from the band-1 of the isolated Cry2Ab2Bayer protein fromproperty MON protection89034.publishing The N-terminal of contents MQA sequence (underlined) originates from the chloroplast transit peptideits (CTP). parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document 145 AVPLSITSSV NTMQQLFLNRany LPQFQMQGYQsuch LLLLPLFAQA ANLHLSFIRDits VILNADEWGI reproductiondocument 205 SAATLRTYRD YLKNYTRDYS NYCINTYQSAowner FKGLNTRLHDthis MLEFRTYMFLof NVFEYVSIWS 265 LFKYQSLLVS SGANLYASGS GPQQTQSFTSunder QDWPFLYSLFof QVNSNYVLNG FSGARLSNTF rightsthe this 325 PNIVGLPGSTdocument TTHALLAARVand/or NYSGGISSGD IGASPFNQNFof rightsNCSTFLPPLL TPFVRSWLDS 385 GSDREGVATV TNWQTESFETto of TLGLRSGAFTfall useARGNSNYFPD YFIRNISGVP LVVRNEDLRR 445 PLHYNEIRNI ASPSGTPGGA RAYMVSVHNR KNNIHAVHENthe GSMIHLAPND YTGFTISPIH 505 ATQVNNQTRTThis FISEKFGNQGmay DSLRFEQNNT distribution,and TARYTLRGNG NSYNLYLRVS SIGNSTIRVT rights owner 565 INGRVYTATNsubject VNTTTNNDGV NDNGARFSDI NIGNVVASSN SDVPLDINVT LNSGTQFDLM 625 NIMLVPTNIS PLY be the violate copy of Figure C-4. Peptide mass coverage mapand of the MON 89034-derived proteolytic may documentpublication, fragmentIt of Cry2Ab2 proteinexploitation in MALDI-TOF MS analysis this Shaded regions correspondany to 24 tryptic peptide masses that were identified from the permission band-2 (proteolytic fragment ofprohibited Cry2Ab2) from MON 89034. The amino acid residue number was assigned thebased be on the respective position in the full-length sequence of Cry2Ab2 protein.commercial N-terminal sequencing result showed that band-2 started from amino acidFurthermore, residueany No. 145. Consequently,without

Monsanto Company 06-CR-166U Page 219 of 262 CBI Deleted Version

C.2.5. Lack of Glycosylation of MON 89034-Produced Cry2Ab2

To test whether post-translational glycosylation of the MON 89034-produced Cry2Ab2 protein occurred, the isolated MON 89034-produced Cry2Ab2 protein was analyzed for the presence of covalently bound carbohydrate moieties. The E. coli-produced Cry2Ab2 reference standard (negative control) and transferrin (positive control) were analyzed concurrently with the MON 89034-produced Cry2Ab2 protein.

The result showed that positive glycoprotein transferrin was detected, as expected, in a concentration-dependent manner at loadings of 0.5 and 1.0 µg/lane (Figure C-5, lanes 3- 4). No detectable signal was observed for the MON 89034-producedregime. and andE. coli - produced Cry2Ab2 protein at the positions of the expected molecularand weights between 50 AG to 75 kDa (Figure C-5, lanes 5-8).

Since no positive signal was seen at the bandBayer position property for Cry2Ab2 protection protein,publishing it was evident that the MON 89034-produced Cry2Ab2of protein is not glycosylated and contentsis equivalent to its the E. coli-produced Cry2Ab2 protein regarding to theparties. lackdata of glycosylation. therefore and/or or affiliates. C.2.6. Functional activity of propertythe Cry2Ab2 intellectual proteinthird may its as the of and The functional activity isof the MON 89034-producedregulatory and E. coli-producedowner. Cry2Ab2 a document protein was compared by determiningany such EC50 values in a corn earwormits diet-incorporation reproductiondocument of bioassay. The EC50 value is definedowner as the level ofthis Cry2Ab2 protein in the diet that under of results in 50% inhibition torights larvalthe growth. this documentand/or rights to of fall use of The EC50 values for each replicate bioassay are summarizedthe in Table C-1 and the dose responseThis relationships for themay Cry2Ab2 distribution, andprotei n from the two sources are illustrated in rights owner Figure C-6. Thesubject mean EC50 values for the E. coli- and the MON 89034-produced proteins werebe similar and were determinedthe to beviolate 0.16 µg Cry2Ab2/ml diet, with standard copy of deviations of 0.04 and 0.01 µg Cry2Ab2/mland diet, respectively. Figure C-6 shows an equivalentmay slope fordocument thepublication, dose- response relationship for the E. coli- and MON 89034- It exploitation produced Cry2Ab2 protein in the CEW bioassay. These results clearly showed that this any MON 89034- and E. coli-produced Cry2Ab2 protein has equivalent functional activity. permission prohibited the commercial be Furthermore,any Consequently,without

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12 345678910 MWM (kDa) MWM (kDa) 250 250 150 150 100 100 75 75 50 50 37 37

25 25 20 20

15 15 regime.and and AG 10 10

Bayer property protectionpublishing of contents its parties.data therefore Figure C-5. Glycosylation analysis of the MON-89034-producedand/oror Cry2Ab2 Protein affiliates. property intellectualthird may Samples of the MON 89034-producedits Cry2Ab2 protein, E. coli-produced Cry2Ab2 as reference standard (negativethe control) of and tranandsferrin (positive control) were separated by is regulatory owner. a Tris-glycine 4→20% SDS polyacrylamidea gel, anddocument electrotransferredits to PVDF membrane. Where present,any protein-bound such carbohydratereproduction moieties were labeled with this documentof biotin, and detected with streptavidinowner -horseradish peroxidase and enhanced under of chemiluminescence. Amountrights refersthe to total protein loadedthis per lane, except for the E. coli documentand/or rights and the MON 89034-producedto of proteinsfall whoseuse concentrations of were normalized based on Cry2Ab2 purity. the This may distribution,and rights owner Lane subjectSample Amount (µg) be the violate copy of 1 Empty Lane...... and N/A may documentpublication, 2 It MWM (molecular weightexploitation markers, Precision Plus Dual Color)...... N/A 1 3 Transferrinthis any (positive control) ...... 0.5 4 Transferrin (positive control)1 ...... 1 permission 5 E. coli-produced Cry2Ab2prohibited protein (negative control)...... 0.5 the 6 E. coli-producedcommercial Cry2Ab2 be protein (negative control)...... 1 7 MON 89034-produced Cry2Ab2 protein ...... 0.5 Furthermore, 8 MONany 89034-produced Cry2Ab2 protein ...... 1 9Consequently, Empty withoutLane...... N/A 10 Empty Lane...... N/A

1 Part of the transferrin appeared to be degraded. But this did not affect the utility of this protein as a positive control for glycoproteins in this test.

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Table C-1. EC50 values of E. coli-produced and MON 89034-produced Cry2Ab2 proteins in a corn earworm diet-incorporation bioassay

1 EC50 (µg Cry2Ab2/ml diet) E. coli-produced MON 89034-produced 1 0.13 ± 0.03 0.17 ± 0.03 Replicate2 2 0.16 ± 0.02 0.16 ± 0.03 3 0.20 ± 0.02 0.16 ± 0.02 Overall 0.16 ± 0.04 0.16 ± 0.01 and and regime. 1 EC50 (mean ± standard error) represents the concentrationAG needed to inhibit the growth of the target insect by 50%. 2 Each bioassay replicate for the E. coli-produced and MON 89034- Bayer property protectionpublishing produced Cry2Ab2 proteins consisof ted of a series of seven contents dilutions yielding a dose series with a 2-fold separation factorits parties.data therefore ranging from 0.016 – 1.0 µg Cry2Ab2 protein/ml diet.and/or Insector affiliates. larvae were placed on theproperty diets wi thintellectual 16 insectsthird per treatment. Themay its combined weight of the survivingas insects at each dose level was the of and recorded at the endis of the 7 day incubationregulatory period. EC50 wasowner. a document calculated with SASany software. such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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200

150

100 and E. coli and regime. AG 50 Plant Bayer property publishing protectioncontents

Mean Insect Mass (mg) of 0 its parties.data therefore Controls 1 2 3 and/oror 4 affiliates. Log Cry2Ab2property intellectualDosethird Level (ng/mL diet)may its as the of and is regulatory owner. a document any such its reproductiondocument Figure C-6. Functional equivalenceowner of the E. coli-producedthis andof the MON 89034- produced Cry2Ab2 protein against cornunder earworm of (CEW) rightsthe this documentand/or rights Bioassay results from threeto replicatesof fall have beenuse combinedof for illustration purpose only and are shown on a log concentration scale. Each datathe point represents the mean of the This may distribution,and three bioassays along withrights the standard error ofowner the mean. Equivalent slope was seen for dose-response relationshipssubject for the E. coli- and MON 89034-produced Cry2Ab2 protein the in the CEW bebioassay. The data demonstrate thatviolate the test insect body weight decreases copy of with the increase of the Cry2Ab2 dose level,and indicating the greater growth inhibitory may documentpublication, effectIt on the test insects at higherexploitation doses. this any permission prohibited the commercial be Furthermore,any Consequently,without

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C.3. References

Aebersold, R. 1993. Mass spectrometry of proteins and peptides in biotechnology. Curr. Opin. Biotechnol. 4:412-419. Bagshaw, R.D., J.W. Callahan, and D.J. Mahuran. 2000. Desalting of in-gel-digested protein sample with mini-C18 columns for matrix-assisted laser desorption ionization time of flight peptide mass fingerprinting. Anal. Biochem. 284:432- 435. Billeci, T.M. and J.T. Stults. 1993. Tryptic mapping of recombinant proteins by matrix- assisted laser desorption/ionization mass spectrometry. Anal. Chem. 65:1709- 1716. and and regime. Hunkapillar, M.W., R.M. Hewick, W.J. Dreyer, AGand L.E. Hood. 1983. High-sensitivity sequencing with gas-phase sequenator. Methods Enzymol. 91:399-413. Jensen, O.L., A.V. Podtelejnikov, and M. Mann. 1997. Identification of the components Bayer property protectionpublishing of simple protein mixtures by high-accuof racy peptide mass mappingcontents and database searching. Anal. Chem. 69:4741-4750. its parties.data therefore Williams, K., M. LoPresti, and K. Stone. 1997. Internal proteinand/or orsequencing of SDS- affiliates. PAGE-separated proteins:property Optimization intellectual thirdof an in-gel digest protocol.may Tech. Protein its Chem. 8:79-98. as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Appendix D. Materials and Methods for the Determination of Cry1A.105 and Cry2Ab2 Proteins

D.1. Test, Control, and Reference Materials

The test material was MON 89034 grown in 2005 U.S. field trials. The control material was a conventional corn with a similar genetic background to the MON 89034 plants. The identities of the test and control corn seeds were confirmed by event-specific PCR analyses. In addition, the identities of grain samples harvested from the field were also verified by PCR analyses. regime.and The protein reference standards of Cry1A.105 and Cry2Ab2and were produced with AG recombinant E. coli. The Cry1A.105 standard (lot 20-100086) had a purity-corrected concentration of 1.0 mg/ml by amino acid composition analysis. The purity was 80% as determined SDS-PAGE and densitometry. Bayer The Cry2Ab2 property standard protection (lotpublishing 20-100071) had a purity-corrected concentration of 0.4 mg/mlof by amino acid composition contentsanalysis. The its purity was 87% as determined by SDS-PAGE and densitometry.parties.data therefore and/oror affiliates. D.2. Generation of Plant Samplesproperty intellectualthird may its as the of and D.2.1. Field Design is regulatory owner. a document any such its reproductiondocument The field production of the test materialsowner wa s initiatedthis during theof 2005 planting season to generate test and control materials at undervarious corn-growingof locations in the U.S. The rightsthe this field sites weredocument as follows:and/or Jefferson County, IA; Warrenrights County, IL; Clinton County, to of fall use of IL; York County, NE; and Fayette County, OH. Thesethe field sites were located within the major corn-growingThis regions mayof thedistribution, U.S.and and provided a variety of environmental rights owner conditions. At subjecteach site, three replicated plots of MON 89034 and the conventional control corn were planted using a randomizedthe violatecomplete block design. Over season leaf be copy (OSL 1-4), over season root (OSR 1-4),of over season whole plant (OSWP 1-4), forage, and stover, mayforage root,document senescentpublication, root, pollen, silk, and grain were collected from each It exploitation replicated plot thisat all field sites. All samples, except grain, were stored and shipped on dry ice to the Monsantoany processing facility in Creve Coeur, Missouri. Grain samples permission were stored and shipped at ambientprohibited temperature.

the be D.2.2. Descriptioncommercial of the Collected Tissues Furthermore,any Pollen.Consequently, Approximatelywithout 10 ml of pollen was collected from multiple tassels in each plot at pollination, approximately 60-74 days after planting.

Silks. Silks were collected from the ears of five plants in each plot, approximately 60-74 days after planting. Silks were only collected from ears of plants that were covered with shoot bags to preserve their genetic identity.

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Forage. Two whole plants in each plot were cut above the soil surface at an early dent stage, at approximately 100-120 days after planting, and then combined to form the forage sample.

Grain. Grain was harvested at maturity from all plants in each plot and dried to a moisture content of 12-15%.

Stover. Following harvest, approximately 130-160 days after planting, two whole plants in each plot were cut above the soil surface and combined to form the stover sample.

Leaf. The youngest immature whorl leaf was collected from 15 plants in each plot, which were combined to form the leaf sample. Overseason leaf samples were collected regime.and as follows: and AG

Overseason leaf (OSL) Corn development stage Days after planting (DAP) OSL-1 V2-V4Bayer property protection 21-29publishing OSL-2 V6-V8of 28-43contents its OSL-3 V10-V12 parties.data 41-53 therefore OSL-4 pre-VT (pre-tasseling) and/oror56-68 affiliates. property intellectualthird may its Whole plant. The aerial portion of theas plant without the root was collected from four the of and plants in each plot at theis V2-V4 stage and combinedregulatory to form the wholeowner. plant sample. a document Two plants were collected anyand combinedsuch to form the whole plantits samples for the later reproductiondocument growth stages. Overseason whole plantowner samples were thiscollected asof follows: under of rightsthe this Overseasondocument wholeand/or plant (OSWP) Corn developmentrights stage DAP to of fall use of OSWP-1 V2-V4the 21-29 This OSWP-2 may distribution,and V6-V8 28-43 rights owner subjectOSWP-3 V10-V12 41-53 OSWP-4 the pre-VTviolate (pre-tasseling) 56-68 be copy of and Root. Rootsmay remainingdocument afterpublication, collection of whole plants from each plot were combined to It exploitation form the root sample.this Overseason root samples were collected as follows: any permission Overseason root (OSR)prohibited Corn development stage DAP OSR-1 V2-V4 21-29 the be commercialOSR-2 V6-V8 28-43 Furthermore,any OSR-3 V10-V12 41-53 Consequently,withoutOSR-4 pre-VT (pre-tasseling) 56-68 Forage root early dent stage (R4-R6) 100-120 Senescent root after harvest 130-160

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D.3. Tissue Processing and Protein Extraction

All tissue samples produced at the field sites were shipped to Monsanto’s processing facility. Processed tissue samples were stored in a -80°C freezer until shipped on dry ice to Monsanto’s analytical facility. All processed tissue samples were stored in a -80°C freezer during the study.

For all tissue types except pollen, Cry1A.105 protein was extracted with a Tris-borate buffer (pH 7.8) containing 100 mM Tris, 100 mM Na2B4O7·10 H2O, 5 mM MgCl2·6 H2O, 0.05% (v/v) Tween 20, 0.2% (w/v) L-ascorbic acid, 2 M urea, and 100 mM dithiothreitol. For pollen, Cry1A.105 protein was extracted with 50 mM Na2B4O7·10 H2O, 750and mM and regime. KCl, 0.075% (v/v) Tween 20, 0.2% (w/v)AG L-ascorbic acid, and 10 mM N- lauroylsarcosine, pH 10.5.

For all tissue types except pollen, Cry2Ab2Bayer pr oteinproperty was extracted publishingwith a Tris-borate protectioncontents buffer (pH 7.8) containing 100 mM Tris,of 100 mM Na B O ·10 H O, 5 mM MgCl ·6 H O, 2 4 7 2 its 2 2 and 0.05% (v/v) Tween 20. For pollen, Cry2Ab2 parties.proteindata was extracted thereforewith a buffer and/oror with the same compositions except the pH of 7.4. affiliates. property intellectualthird may its All tissue samples were extracted usingas a Harbil mixer, and tissue debris was removed the of and from the extracts by usingis a Serum Filter Systemregulatory (Fisher Scientific, Pittsburgh,owner. PA), or a document by centrifugation. The extractsany such were aliquoted and stored in aits -80 °C freezer until reproductiondocument analysis. owner this of under of rightsthe this documentand/or rights D.4. ELISA Reagents andto Methodsof fall use of the This may distribution,and D.4.1 Cry1A.105 Antibodiesrights and ELISA. owner subject Goat polyclonalbe antibodies (lot 7509175)the specificviolate for the Cry1A.105 protein were copy of purified using Protein-G Agarose affinityand ch romatography. The concentration of the purifiedmay IgG was determineddocumentpublication, to be 0.93 mg/ml by spectrophotometric methods. The It exploitation purified antibody was stored in a phosphate buffered saline (1X PBS) buffer (pH 7.4) this any containing 1 mM KH2PO4, 10 mM Na2HPO4·7H2O, 137 mM NaCl, and 2.7 mM KCl. permission The purified Cry1A.105 antibodiesprohibited were coupled with biotin (Sigma, St. Louis, MO) according to the manufacturer’sthe be instructions and assigned lot 7509180. The detection reagent was NeutrAvidincommercial (Pierce, Rockford, IL) conjugated to horseradish peroxidase (HRP).Furthermore, any Consequently,without The goat anti-Cry1A.105 antibody was diluted in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, and 150 mM NaCl, pH 9.6) and immobilized onto 96-well microtiter plates at 5.0 µg/ml followed by incubation in a 4°C refrigerator for >8 h. Prior to each step in the assay, plates were washed with 1X PBS containing 0.05% (v/v) Tween-20 (1X PBST). For grain tissue only, plates were blocked with the addition of 100 to 150 µl per well of 1X PBST with 9% non-fat dried milk (NFDM) for 30 to 90 minutes at 37oC. Cry1A.105 protein standard or sample extract was added at 100 µl per well and incubated for 1 h at

Monsanto Company 06-CR-166U Page 227 of 262 CBI Deleted Version

37°C. The captured Cry1A.105 protein was detected by the addition of 100 µl per well of biotinylated goat anti-Cry1A.105 antibodies and NeutrAvidin-HRP (Pierce). Plates were developed by adding 100 µl per well of HRP substrate, 3,3',5,5'-tetramethyl- benzidine (TMB; Kirkegaard & Perry, Gaithersburg, MD). The enzymatic reaction was terminated by the addition of 100 µl per well of 6 M H3PO4. Quantitation of the Cry1A.105 protein was accomplished by interpolation from a Cry1A.105 protein standard curve that ranged from 0.438 – 14 ng/ml.

D.4.2. Cry2Ab2 Antibodies and ELISA

Mouse monoclonal antibody (lot G-800601) specific for the Cry2Ab2 protein was purified using Protein-A Agarose affinity chromatography. The concentrationand of the and regime. purified IgG was determined to be 1.0 mg/mlAG by spectrophotometric methods. Production of the Cry2Ab2 monoclonal antibody was performed by Strategic Biosolutions (Newark, DE). The purified antibody was stored in a buffer (pH 7.2) containing 20 mM Na HPO ·7H O and 150Bayer mM NaClproperty with 0.05%publishing NaN added as a 2 4 2 protectioncontents 3 preservative. Purified Cry2Ab2 antibodiesof (lot 7381862) were coupled with biotin its (Sigma) according to the manufacturer’s instructionsparties. anddata assigned lot 7381898.therefore The and/oror detection reagent was NeutrAvidin-HRP. affiliates. property intellectualthird may its The mouse anti-Cry2Ab2 antibody wasas d iluted in coating buffer (15 mM Na CO and 35 the of and 2 3 mM NaHCO , pH 9.6) isand immobilized ontoregulatory 96-well microtiter platesowner. at 5.0 µg/ml 3 a document followed by incubation in anya 4° Csuch refrigerator for >8 h. Prior to eachits step in the assay, reproductiondocument of plates were washed with 1X PBST.owner Cry2Ab2 proteinthis standard or sample extract was under of added at 100 µl per well andrights incubatedthe for 1 h at 37°C.this The captured Cry2Ab2 protein documentand/or rights was detected by the additionto of of fall100 µl peruse well ofof biotinylated goat anti-Cry2Ab2 antibodies and NeutrAvidin-HRP. Plates were developedthe by adding 100 µl per well of This may distribution,and TMB. The enzymatic rightsreaction was terminatedowner by the addition of 100 µl per well of 6 M H PO . Quantitationsubject of the Cry2Ab2 protein was accomplished by interpolation from a 3 4 the Cry2Ab2 proteinbe standard curve that ranged fromviolate 0.219 – 7 ng/ml. copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Appendix E: Materials, Methods and Individual Site Results for Seed Dormancy and Germination

E.1. Materials

The MON 89034, the conventional control corn, and reference corn seeds were produced in Dayton, Webster Co., IA (IA); Jerseyville, Jersey Co., IL (JR); and Monmouth, Warren Co, IL (MN) in 2004.

Material Material Type Genotype Locations MON 89034 Test Lepidopteran-protected IA, JR, MNand and regime. H1325023 Control ConventionalAG IA, JR, MN HEXP1184 Reference Conventional IA, JR, MN H4242304 Reference Conventional IA, JR, MN H1198600 Reference Bayer Conventional property protectionpublishingIA, JR, MN of contents H1200203 Reference Conventional IA, JR, MN its parties.data therefore and/oror E.2. Characterization of the Materialsaffiliates. property intellectualthird may its as The presence or absence theof MONof 89034and inse rt was verified by event-specific PCR is regulatory owner. analysis for the MON 89034 and control corna seeds. Thedocument results of these verifications were as expected. any such its reproductiondocument of owner this under of E.3. Performing Facility andrights Experimentalthe Methods this documentand/or rights to of fall use of Dormancy and germination evaluations were conductedthe at BioDiagnostics, Inc. in River This may distribution,and Falls, WI. The principalrights investigator was ownercertified to conduct seed dormancy and germination testingsubject consistent with the standards established by the Association of Official Seedbe Analysts (AOSA), a seed tradethe associationviolate (AOSA, 2000; AOSA, 2002). copy of and may documentpublication, SevenIt germination chambersexploitation were used in the study and each chamber was maintained dark under one of the following seven temperature regimes: constant temperature of this any approximately 5, 10, 20 or 30oC or alternating temperatures of approximately 10/20, o permission 10/30, or 20/30 C. The alternatingprohibited temperature regimes were maintained at the lower temperature for 16 h andthe the higher temperature for 8 h. The temperature inside each commercial be germination chamber was monitored and recorded throughout the duration of the study. Furthermore,any GerminationConsequently, towelswithout for MON 89034, control, and reference materials were prepared per the facility SOPs. Each germination towel represented one replication. The types of data collected depended on the temperature regime. Each rolled germination towel in the AOSA-recommended temperature regime (i.e., 20/30°C) was assessed periodically during the study for normally germinated, abnormally germinated, hard (viable and nonviable), dead, and firm swollen (viable and nonviable) seed as defined by AOSA guidelines (AOSA, 2002). Each rolled germination towel in the additional temperature regimes (i.e., 5, 10, 20, 30, 10/20 and 10/30oC) was assessed periodically during the

Monsanto Company 06-CR-166U Page 229 of 262 CBI Deleted Version study for germinated, hard (viable and nonviable), dead, and firm swollen (viable and nonviable) seed.

E.4. Statistical Analysis

Statistical analyses were performed by the Monsanto Statistics Technology Center. Analysis of variance was conducted according to a completely randomized design using the Statistical Analysis System (SAS® Version 9.1, SAS Institute, Inc. 2002-2003) to compare the MON 89034 to the control material for each temperature regime.

For each temperature regime, test material was separately compared to the control material at each site (individual-site or by-site analysis) and pooled acrossregime. andall sites (referred to as combined-site). No statistical comparisons wereand made between the test AG and reference materials. Minimum and maximum values for the reference materials were determined. An analysis of variance was conducted according to a completely randomized design with four replications usingBayer SAS. property The level protection of statisticalpublishing significance was predetermined to be 5% (p≤0.05). of Dormancy and germination characteristicscontents that its were analyzed included: percent germinated seed (categorizedparties.data as normallytherefore germinated and/or and abnormally germinated seed for AOSA-recommended temperatureor regimes), percent affiliates. viable hard seed, percent dead propertyseed, and percentintellectualthird viable firm swollen seed.may its as the of and E.5. Individual Site Seedis Dormancy and Germinationregulatory Results owner. a document any such its reproductiondocument MON 89034, the control, and referenceowner seed materialsthis were producedof at three sites to under of assess germination characteristrightsicsthe of seed grown under thisvariou s environmental conditions. documentand/or rights No viable hard seed wereto detectedof fall in any temperatureuse of regime with seed produced from any location, indicating no increased weediness potentialthe of MON 89034 from changes in seed dormancy.This Furthermore, mayof the distribution, 87 compand arisons over the germination characteristics rights owner from the three subjectseed production locations, only two differences were detected between MON 89034 and the control. MON 89034the fromviolate the JR site had higher percentage of be copy germinated seed and a lower percentageof of dead seed at the 10oC temperature regime than and the controlmay (Table documentE-1).publication, No differences were detected between MON 89034 and the It exploitation control at the thisIA or MN site in any temperature regime. The significant differences detected in the by-siteany analysis were not detected in the combined-site analyses (see permission Section VII for the combined-siteprohibited result). Therefore, the differences detected in the by- site analysis were not indicative of a consistent trend and are not likely to be biologically the be meaningful in termscommercial of altered seed dormancy or germination of MON 89034 compared toFurthermore, the conventionalany corn. Consequently,without E.6. References

AOSA. 2000. Tetrazolium Testing Handbook. Association of Official Seed Analysts. Lincoln, NE.

® SAS is a registered trademark of SAS Institute, Inc.

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AOSA. 2002. Rules for Testing Seeds. Association of Official Seed Analysts. Lincoln, NE. 166 pp.

Table E-1. Germination for MON 89034

Mean (%) AOSA Viable Temp. Normally Abnormally Firm Reg.1 Prod. Germinated Germinated Viable Hard Dead Swollen (°C) Site2 T3 C4 T C T C T C T C IA 99.5 99.0 0.0 0.0 0.0 0.0 0.5 1.0 0.0and 0.0 20/30 JR 83.3 87.0 0.3 0.5 0.0 0.0 and16.5 12.5regime. 0.0 0.0 MN 99.8 100.0 0.0 0.0 0.0AG 0.0 0.3 0.0 0.0 0.0

Mean (%) Additional Bayer property protectionpublishing Temp. Prod. of contentsViable Germinated Viable Hard Dead its Firm Regime Site parties.data therefore (°C) and/oror Swollen T C T C T C T C affiliates. IA 0.0 property0.0 0.0intellectual third0.0 3.5 2.8may 96.5 97.3 its 5 JR 0.0 0.0 as0.0 0.0 13.0 12.8 87.0 87.3 the of and MN 0.0is 0.0 0.0 regulatory0.0 0.5 2.5owner. 99.5 97.5 IA 98.0 98.5 0.0 a 0.0 document2.0 1.5 0.0 0.0 any such its 10 JR 90.5* 84.5 0.0 reproduction 0.0 9.5*document of 15.5 0.0 0.0 MN 99.5 100.0 owner0.0 0.0 this 0.5 0.0 0.0 0.0 under of IA 99.3 rights98.5the 0.0 0.0 this 0.8 1.5 0.0 0.0 20 document JR and/or89.3 87.5 0.0 0.0 rights10.8 12.5 0.0 0.0 to of fall use of MN 99.3 99.5 0.0 0.0 0.8 0.5 0.0 0.0 the This IA 98.3 may98.5 distribution,0.0and 0.0 1.8 1.5 0.0 0.0 30 JR 88.8rights 88.0 0.0 owner 0.0 11.3 12.0 0.0 0.0 MNsubject 99.8 100.0 0.0 0.0 0.3 0.0 0.0 0.0 the violate be IA copy99.5 98.8 0.0 0.0 0.5 1.3 0.0 0.0 10/20 JR 88.8 90.0 of0.0 0.0 11.3 10.0 0.0 0.0 and may MN document99.5publication, 100.0 0.0 0.0 0.5 0.0 0.0 0.0 It IA 98.8 exploitation99.5 0.0 0.0 1.3 0.5 0.0 0.0 10/30 JRthis any87.5 90.8 0.0 0.0 12.5 9.3 0.0 0.0 MN 99.8 100.0 0.0 0.0 0.3 0.0 0.0 0.0 permission * Indicates that a significant differenceprohibited between MON 89034 and the control was detected at p≤0.05 for seed produced at a given site withinthe a givenbe temperature regime. 1 In alternating temperaturecommercial regimes the lower temperature was maintained for 16 hours and the higher temperature for eight hours. 2Furthermore,any Prod.Consequently, Site = site wherewithout seed lot was produced; IA = Dayton, Iowa; JR = Jerseyville, Illinois; MN = Monmouth, Illinois. 3 T = MON 89034 4 C = Control corn (H1325023)

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Appendix F: Phenotypic and Agronomic Assessment: Individual Site Results from 2004 and 2005 Field Trials

The methods, test sites, and the combined-site results for the phenotypic and agronomic assessments are described in Section VII. This appendix provides the details of the test, control, and reference corn materials and the results for each individual test site.

F.1. Materials

The materials for phenotypic, agronomic, and ecological interaction assessments studies are included in Tables F-1, F-2, and F-3. The presence or absence of MON 89034 insert regime.and in the test and control materials was confirmed by event-specificand PCR analyses. AG

F.2. Test Sites Bayer property protectionpublishing 2004 field trials included nine (9) test sitesof (site codes in parenthesis): Hendrickscontents County, its IN (IN-DA); Freeborn County, MN (MN-GE); Shelbyparties. County,data MO (MO-BEtherefore and MO- CL); Jefferson County, IA (IA); Jersey County, IL (IL-1); Warrenand/oror County, IL (IL-2); affiliates. York County, NE (NE); and Fayetteproperty County, intellectual OHthird (OH). may its as the of and Study-1 of the 2005 fieldis trials included four regulatory(4) test sites: Ottawa owner.County, MI (MI); a document Freeborn County, MN (MN);any Wayne such County, NY (NY); and Walworthits County, WI (WI). reproductiondocument owner this of Study-2 of the 2005 field trials includedunder five (5)of test sites: Jefferson County, IA (IA); rightsthe this Warren County,document IL and/or(IL-N); Clinton County, IL (IL-S); Shelbyrights County, MO (MO), and to of fall use of York County, NE (NE). the This may distribution,and rights owner F.3. Individual subjectField Site Results the violate be copy Table F-4 shows the results of the 14 phenotypicof or agronomic characteristics evaluated and at 9 sitesmay in 2004. Nodocument differencespublication, between MON 89034 and the control were detected for It exploitation seedling vigor, thisear height, plant height, dropped ears, root lodged plants, and final stand count. A total of any13 out of 123 site-by-characteristic comparisons were significantly permission different between MON 89034prohibited and the control. The significant differences were distributed among eight of the fourteen phenotypic characteristics. Except for plant the be height and numbercommercial of stalked lodged plants, all of the significant differences detected in theFurthermore, individual-siteany analysis were not detected in the combined-site analyses (see Section VII Consequently,for combined-sitewithout analysis result). Therefore, the differences detected in the individual-site analysis were not indicative of a consistent, trait-induced response, and are not likely to be biologically meaningful in terms of increased weediness potential of MON 89034 compared to the control.

Table F-5 shows the results of 14 phenotypic or agronomic characteristics for the four test sites in Study-1 of the 2005 trials. No differences were detected between MON 89034 and the control for the following ten characteristics: seedling vigor, early stand

Monsanto Company 06-CR-166U Page 232 of 262 CBI Deleted Version count, days to 50% pollen shed, stay green, ear height, dropped ears, stalk lodged and root lodged plants, grain moisture and test weight. A total of four differences out of 56 site-by-characteristic comparisons were significantly different between MON 89034 and the control. The significant differences were distributed among four of the fourteen phenotypic characteristics. None of the significant differences detected in the individual- site analysis were detected in the combined-site analyses (see Section VII for combined- site analysis result). Therefore, the differences detected in the individual-site analysis were not indicative of a consistent, trait-induced response, and are not likely to be biologically meaningful in terms of increased weediness potential of MON 89034 compared to the control.

Table F-6 shows the results of the 14 phenotypic or agronomic characteristics for the five regime.and test sites in Study-2 of the 2005 trials. For the individual-siteand analyses, no differences AG were detected between MON 89034 and the control for the following nine characteristics: seedling vigor, days to 50% pollen shed and 50% silking, plant height, dropped ears, stalk and root lodged plants, final stand count Bayerand grain moisture.property protectionA totapublishingl of five out of 65 site-by-characteristic comparisons wereof significantly different between MONcontents 89034 and its the control. The significant differences were distributedparties.data among five oftherefore the fourteen phenotypic characteristics. None of the significant differences and/ordetectedor in the individual- affiliates. site analysis were detected in theproperty combined-site intellectualthird analyses (see Section mayVII for combined- its site analysis result). Therefore, the differeas nces detected in the individual-site analysis the of and were not indicative of isa consistent, trait-inducedregulatory response, and areowner. not likely to be a document biologically meaningful inany terms such of increased weediness potentialits of MON 89034 reproductiondocument compared to the control. owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 233 of 262 CBI Deleted Version

Table F-1. Test, control, and reference corn used in the 2004 field trials

Corn Hybrid Names Seed Types Site MON 89034 Test All H1325023 Control All Golden Harvest-H8751 Reference IA Golden Harvest-H9231 Reference IA Northrup King-N60-N2 Reference IA Burris-590 Reference IL-1 Mycogen-2784 Reference IL-1 Reference IL-1 and DKC 62-15 and regime. Pfister-2730 Reference AG IL-2 Mycogen-2E685 Reference IL-2 DKC 61-42 Reference IL-2 Bayer property protectionpublishing DK611 Reference of IN-DA contents its DKC59-08 Reference parties.IN-DAdata therefore and/oror RX708 Reference IN-DA affiliates. property intellectualthird may DKC51-45 Referenceits MN-GE as DKC57-01 the Referenceof and MN-GE is regulatory owner. DKC60-15 Reference a MN-GEdocument any such its reproductiondocument DKC61-42 Referenceowner thisMO-BE of DKC62-15 Referenceunder of MO-BE rightsthe this RX690 documentand/or Reference MO-BErights to of fall use of DKC60-17 Reference MO-CLthe This may distribution,and RX742RR2 rights Reference owner MO-CL RX772 subject Reference MO-CL be the violate DKC 60-15 copy Referenceof NE Mycogen-2P682 document Reference and NE may publication,exploitation It Reference NE Mycogen-2A791this Seed Consultants-SC1124Aany Reference OH permission Crows-4908 Referenceprohibited OH RX708 the Reference OH commercial be

Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 234 of 262 CBI Deleted Version

Table F-2. Test, control and reference corn used in the Study-1 of 2005 field trials

Corn hybrid Names Seed Types Sites MON 89034 Test All DKC51-43 Control All DKC48-15 Reference MI DK537 Reference MI Pioneer 36K67 Reference MI DKC51-45(RR2) Reference MN DKC53-33(RR2) Reference MN Pioneer 36N70 Reference MN DKC51-88 Reference NY regime.and Garst 8779 Reference NY and Garst 8715 Reference NYAG RX508 Reference WI N45-T5 Reference WI Bayer property protectionpublishing N48-K2 Reference of WI contents its parties.data therefore and/oror Table F-3. Test, control, and referenceaffiliates. corn used in the Study-2 of 2005 field trials property intellectualthird may its as Corn Hybrid Names Seedthe Types of and Sites is regulatory owner. MON 89034 Test a All document any such its H1325023 Control Allreproduction document owner this of DKC59-08 Reference under IAof rightsthe this DKC60-15 documentand/or Reference IA rights to of fall use of H-8991 Reference IA the DKC61-42This Referencemay distribution,and IL-S rights owner RX740 subject Reference IL-S the Mycogen 2E685be Reference violateIL-S copy of RX708 Reference and IL-N may documentpublication, DKC61-50It Referenceexploitation IL-N Pioneer 34H31this any Reference IL-N DKC61-44(RR2) Referencepermission MO prohibited Pfister 2760 Reference MO the be Pioneer 34B23 commercial Reference MO Furthermore,DKC60-17(RR2)any Reference NE RX715Consequently, without Reference NE Mycogen 2E685 Reference NE

Monsanto Company 06-CR-166U Page 235 of 262 CBI Deleted Version

Table F-4. Phenotypic comparison of MON 89034 to the control at each site in 2004 field trials and and regime. Phenotypic CharacteristicAG (units) Early stand count Days to 50% Days to 50% Ear height Seedling vigor (#/plot) pollen shed silking Stay green1 (in) Bayer property protectionpublishing Site Test Control Test Control Test Controlof Test Control Test contentsControl Test Control its IA 6.7 6.3 65.7 64.3 59.0 59.3 58.3 parties. 58.7data 4.7 therefore 4.7 45.3 45.7 and/oror affiliates. IL-1 5.3 5.0 60.0 56.0 53.3 property 53.7 intellectual50.7*third 50.0 — may — 38.9 42.3 its as the of and IL-2 8.0 8.5 68.3 68.3 is59.4 58.9 56.2regulatory 56.7 7.6* owner. 6.6 44.5 41.0 a document any such its reproductiondocument IN-DA 5.0 6.0 72.3* 76.7 66.0* owner 64.3 66.0* this 64.3 of— — 31.9 36.1 under of rightsthe this MN-GE 8.0 7.7 75.0 document 77.3and/or 73.3 73.3 73.0 73.3rights 6.0 6.0 42.5 41.2 to of fall use of MO-BE 7.3 7.3 75.3 72.3 58.3 58.3 57.3 the 57.0 5.0 4.7 40.3 40.1 This may distribution,and rights owner MO-CL 6.0 5.7 70.7 subject 68.0 59.0 58.3 57.7 57.3 6.3 6.3 39.1 38.8 be the violate copy of NE 8.0 8.0 63.7 63.0 67.0 66.7 and67.0 66.3 6.7* 5.7 40.1 41.1 may documentpublication, It exploitation OH 7.7 7.7 63.7 64.0 61.0 60.0 61.0 60.0 6.0 7.0 37.1 37.5 this any permission * Indicates a statistically significant difference between theprohibited test and control at p ≤ 0.05. 1 Dashes indicate data that are missing or excludedthe from be the statistical analysis. commercial Furthermore,any Table F-4 continues on the next page. Consequently,without

Monsanto Company 06-CR-166U Page 236 of 262 CBI Deleted Version

Table F-4 (continued). Phenotypic comparison of MON 89034 to the control at each site in 2004 field trials

Phenotypic Characteristic (units) and and regime. Plant height Dropped ears Stalk lodged RootAG lodged Final stand count Grain moisture (in) (#/plot) plants (#/plot)1 plants (#/plot) (#/plot) (%) Site Test Control Test Control Test Control Test Control Test Control Test Control Bayer property protectionpublishing IA 85.2 84.8 0.0 0.0 0.0 0.3 of 0.0 0.0 56.0 contents59.0 28.5 28.0 its parties.data therefore IL-1 81.8 83.1 0.0 0.0 — — 0.0 0.0 and/or 54.3or 52.7 14.1 13.1 affiliates. property intellectualthird may its IL-2 90.6 91.7 0.0 0.0 2.3* 8.3as 11.1 4.6 66.2 64.2 25.3 23.5 the of and is regulatory owner. a document IN-DA 69.6 74.8 0.0 0.0 0.0any such1.3 0.3 0.3 65.7its 61.7 40.1 38.4 reproductiondocument owner this of MN-GE 102.1 102.8 0.0 0.0 0.0 0.3under 0.0 of 0.0 62.7 63.7 30.5 30.5 rightsthe this documentand/or of rights MO-BE 83.8 86.1 1.0 1.0 to 1.7of fall1.7 use 0.0 0.0 63.7 64.7 16.8* 19.6 the This may distribution,and MO-CL 85.7 85.6 0.0 0.0 rights1.0 1.3 owner 0.0 0.0 53.3 57.3 15.9 16.1 subject the NE 80.2 81.0 0.0 be 0.0 0.0 1.3 violate 0.0 0.0 62.0 62.0 19.3* 17.6 copy of and OH 78.6 79.1 0.0may 0.7document publication, 1.0* 4.7 0.0 0.0 60.7 59.3 20.2 18.9 It exploitation this * Indicates a statistically significant differenceany between the test and control at p ≤ 0.05. 1 permission Dashes indicate data that are missing or excluded from theprohibited statistical analysis. the be commercial Table F-4 continues on the next page. Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 237 of 262 CBI Deleted Version

Table F-4 (continued). Phenotypic comparison of MON 89034 to the control at each site in 2004 field trials and and regime. Phenotypic characteristic (units) AG Test weight Yield (lbs/bu) (bu/ac) Bayer property protectionpublishing Site Test Control Test Control of contents its IA 54.3 54.7 258.8*234.8 parties.data therefore and/oror affiliates. IL-1 54.4 54.8 207.0 197.0 property intellectualthird may its as the of and IL-2 48.6 52.0 224.9 205.8 is regulatory owner. a document any such its reproductiondocument IN-DA 43.3 44.2 96.7 100.2 owner this of under of rightsthe this MN-GE 53.0 53.0 171.1 document187.0and/or rights to of fall use of MO-BE 53.7 53.1 236.3 235.4 the This may distribution,and rights owner MO-CL 55.0 55.0 177.5 202.0subject be the violate copy of NE 55.1* 57.2 205.3*185.5 and may documentpublication, It exploitation OH 56.5 57.0 159.3 172.0 this any permission * Indicates a statistically significant difference between prohibited the test and control at p ≤ 0.05. the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 238 of 262 CBI Deleted Version

Table F-5. Phenotypic comparison of MON 89034 to the control at each site in Study-1 of 2005 field trials

Phenotypic Characteristic Means and and regime. Early stand Days to 50% AG Days to 50% Seedling Vigor count (#/plot) pollen shed silking Stay green MON MON MON MON MON Bayer Site 89034 Control 89034 Control 89034 Control 89034property Control protection publishing 89034 Control of contents its MI 6.7 6.7 76.7 79.7 69.3 68.7 parties.69.3 data 70.0 therefore 5.7 6.0 and/oror affiliates. MN 6.0 5.7 76.0 72.0 64.7property 64.3intellectual third 66.3 66.3 may 5.0 5.0 its as NY 6.0 6.0 79.3 80.0 the 71.0of 71.0and 62.0 62.0 9.0 9.0 is regulatory owner. a document any such its WI 8.0 8.0 76.3 78.0 67.0 66.3 reproduction70.0* document 67.0 5.7 4.7 owner this of under of rightsthe this documentand/or rights Phenotypicto of Characteristicfall use Means of Dropped ears theStalk lodged Root lodged plants This may distribution,and Ear height (in) Plant height (in)rights (#/plot) owner plants (#/plot) (#/plot) MON MON subject MON MON MON Site 89034 Control 89034be Control 89034 Controlthe violate 89034 Control 89034 Control copy of and MI 38.7 37.3 may82.1 81.7document publication, 0.0 0.3 0.0 0.7 0.0 0.0 It exploitation

MN 36.0 38.8 82.7 this82.7 any 0.0 0.0 0.3 0.3 0.0 0.0 permission NY 48.7 48.7 103.9* 100.5 prohibited0.0 0.0 0.0 0.0 0.0 0.0 the be commercial WI 44.2 43.6 97.7 94.1 0.0 0.0 0.0 0.3 0.0 0.0 Furthermore,any * Indicates a statistically significantConsequently, differencewithout between MON 89034 and the control at p ≤ 0.05. Table F-5 continues on the next page.

Monsanto Company 06-CR-166U Page 239 of 262 CBI Deleted Version

Table F-5 (continued). Phenotypic comparison of MON 89034 to the control at each site in Study-1 of 2005 field trials and and regime. Phenotypic Characteristic Means AG Final stand count Grain moisture Test weight (#/plot) (%) (lbs/bu) Yield (bu/ac) Bayer MON MON MON propertyMON protection publishing of contents Site 89034 Control 89034 Control 89034 Control 89034 Controlits parties.data therefore MI 64.0 64.0 21.7 21.2 55.7 55.3 205.8* and/or175.6or affiliates. property intellectualthird may its MN 61.7 61.3 17.5 17.8 56.5 as 56.4 204.3 197.7 the of and is regulatory owner. NY 58.7 58.7 19.0 18.7 55.5 55.7a 174.1document 175.6 any such its reproductiondocument owner this of WI 60.7* 63.3 17.4 16.7 55.8 56.8 237.7 231.6 under of rightsthe this documentand/or rights * Indicates a statistically significant difference betweento MON of 89034 fall and the controluse atof p ≤ 0.05. the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 240 of 262 CBI Deleted Version

Table F-6. Phenotypic comparison of MON 89034 to the control at each site in Study-2 of 2005 field trials

Phenotypic Characteristic Means Early stand Days to 50% Days to 50% regime.and Seedling Vigor count (#/plot) pollen shed silkingand Stay green AG MON MON MON MON MON Site 89034 Control 89034 Control 89034 Control 89034 Control 89034 Control Bayer property protectionpublishing IA 6.0 4.7 67.0* 73.0 57.7 of58.3 58.3 59.0 contents6.7* 7.3 its IL-N 7.3 7.3 72.0 71.3 65.0 64.7 parties.64.3 data 63.7 therefore 6.3 6.3 and/or or IL-S 6.3 7.0 75.0 73.7 51.3 affiliates.50.3 52.0 51.3 3.3 3.3 property intellectualthird may its MO 6.0 6.5 64.7 70.5 65.0 as64.0 67.0 66.0 4.7 5.0 the of and is regulatory owner. NE 6.0 6.0 67.5 72.5 63.0 63.5 a 64.0document 64.0 3.5 3.0 any such its reproductiondocument owner this of Phenotypic Characteristic Means under of this and/orrights Droppedthe ears Stalk lodged Root lodged plants document to fall of rights Ear height (in) Plant height (in) of (#/plot) use plants (#/plot) (#/plot) the MON MONThis MONmay distribution,and MON MON rights owner Site 89034 Control 89034 subjectControl 89034 Control 89034 Control 89034 Control IA 39.9 39.8 83.0be 84.3 1.7 0.0the violate 0.0 0.3 0.0 0.0 copy of

IL-N 35.2 36.1 85.7 86.0document 0.0 0.7and 0.0 0.0 0.0 0.0 may publication,exploitation IL-S 42.3* 46.0 It 87.1 89.9 1.3 2.3 40.0 19.0 2.0 2.7 this any MO 35.9 37.2 68.9 69.8 3.3 1.0 4.3 3.0 1.0 0.0 permission prohibited NE 35.8 35.9 75.1 73.2 the 0.0 0.5 1.0 3.0 0.0 0.0 commercial be * Indicates a statistically significant difference between MON 89034 and the control at p ≤ 0.05. Furthermore,any Table F-6 continues on the next page. Consequently,without

Monsanto Company 06-CR-166U Page 241 of 262 CBI Deleted Version

Table F-6 (continued). Phenotypic comparison of MON 89034 to the control at each site in Study-2 of 2005 field trials

regime.and Phenotypic Characteristic Means and AG Final stand count Grain moisture Test weight (#/plot) (%) (lbs/bu) Yield (bu/ac) MON MON MON Bayer MONproperty protection publishing Site 89034 Control 89034 Control 89034 Controlof 89034 Control contents its parties.data therefore IA 58.3 58.3 26.0 26.2 55.9 55.3 164.3 154.9and/or or affiliates. IL-N 63.3 63.0 18.7 17.6 property59.1 59.4intellectual third 183.3 164.5 may its as IL-S 52.0 56.7 16.2 16.5 the 55.3of 55.7and 83.9 100.6 is regulatory owner. a document MO 60.0 60.0 19.3 17.7 any53.3* such 51.9 47.2 40.1its reproductiondocument of NE 62.0 62.0 16.4 18.0 56.5 owner55.1 169.7*this 146.9 under of rightsthe this * Indicates a statistically significant differencedocument betweenand/or MON 89034 and the control at p ≤ 0.05.rights to of fall use of

the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 242 of 262 CBI Deleted Version

Appendix G: Forage and Grain Compositional Analysis

G.1. Materials

MON 89034, control corn (LH198 × LH172), and commercial reference corns were grown at five U.S. locations in 2004. The control material, LH198 × LH172, has a genetic background similar to MON 89034 but does not possess the lepidopteran protection trait. In addition, 15 conventional corn hybrids produced along side of MON 89034 were included for the generation of 99% tolerance interval. The reference corn hybrid varieties, locations, and seed lot numbers are listed below:

Vendor/Hybrid Starting Seed Lot No. Field Site and and regime. Golden Harvest/ H8751 REF-0404-14931-SAG IA Golden Harvest/ H9231 REF-0404-14932-S IA Bayer property protectionpublishing Northrup King/ N60-N2 of REF-0404-14933-S IAcontents its Burrus/ 590 REF-0404-14934-Sparties.data IL-1 therefore and/oror Mycogen/ 2784 affiliates.REF-0404-14935-S IL-1 property intellectualthird may its Dekalb/ DKC62-15 asREF-0404-14936-S IL-1 the of and is regulatory owner. Pfister/ 2730 REF-0404-14937-Sa document IL-2 any such its reproductiondocument Mycogen/ 2E685 ownerREF-0404-14938-S this of IL-2 under of Dekalb/ DKC61-42 rightsthe REF-0404-14939-Sthis IL-2 documentand/or rights to of fall use of Dekalb/ DKC60-15 REF-0404-14940-Sthe NE This may distribution,and Mycogen/ 2P682 rights REF-0404-14941-Sowner NE subject Mycogen/be 2A791 REF-0404-14942-Sthe violate NE copy of Seed Consultantsdocument / SC1124A REF-0404-14943-Sand OH may publication,exploitation Crow’s/It 4908 REF-0404-14944-S OH this any Asgrow/ RX708 REF-0404-14945-S OH permission prohibited the The identities ofcommercial the MON 89034, be the control, and reference corn hybrids were verified prior to the study by examination of the chain-of-custody documentation supplied with theFurthermore, samplesany collected from the field. Additionally, the grain samples of the test and without controlConsequently, corn were further characterized by an event-specific PCR analysis to confirm the presence or absence of the MON 89034 insert.

Monsanto Company 06-CR-166U Page 243 of 262 CBI Deleted Version

G.2. Field Trial Description

Seed was planted in a randomized complete block design with three replicates per block. All the samples at the field sites were grown under normal agronomic field conditions for their respective geographic regions. The five sites were: Site 1-Jefferson County, IA; Site 2-Jersey County, IL; Site 3-Warren County, IL; Site 4-York County, NE; and Site 5- Fayette County, OH. Forage and grain samples were harvested from all plots and shipped on dry ice (forage) or ambient temperature (grain) to Monsanto Company, St. Louis, MO, USA. The samples were ground, stored in a -20°C freezer located at Monsanto Company (St. Louis, MO), and then shipped, overnight, on dry ice to Covance Laboratories, Inc. (Madison, WI) for analyses.

regime.and G.3. Analytical Methods and AG

A total of 90 ground forage and grain samples were analyzed by Covance Laboratories, Inc. Analyses were performed using methodsBayer that propertyare currently protection uspublishinged to evaluate the nutritional quality of food and feed. of Sp ecifically, standard AOAC (Associationcontents of its Official Analytical Chemists, 2000) methods wereparties. useddata to determinetherefore amino acid composition, fat, minerals, total dietary fiber, moisture, folicand/or acid,or niacin, pyridoxine affiliates. (Vitamin B6), riboflavin (Vitaminproperty B2), andintellectual thiaminthird (Vitamin B1). Themay protein content its was determined based on both AOACas (2000) methods and two literature methods the of and (Bradstreet, 1965; Kalthoffis and Sandell, 1948). regulatory Acid detergent fiber wasowner. analyzed based a document on the method of USDA Agricultureany such Handbook No. 379 (1970). its Carbohydrates were reproductiondocument measured based on an USDA Agricultureowner Handbookthis No. 74 (1973).of Fatty acids were analyzed based on an AOCS (Americanunder Oil Chemistsof Society, 1997) method. Neutral rightsthe this detergent fiberdocument wasand/or determined based on an AACC (Americanrights Association of Cereal to of fall use of Chemists, 1998) method and an USDA Agriculture Handbookthe No. 379 (1970) method. In addition,This other literature methodsmay distribution, wereand used to measure furaldehyde (Albala-Hurtado rights owner et al., 1997), p-coumaricsubject and ferulic acids (Hagerman and Nicholson, 1982). phytic acid (Lehrfeld 1989; 1994), raffinose (Mason andthe Slviolateover, 1971; Brobst, 1972), and Vitamin E be copy (Cort et al., 1983; McMurray et al., 1980;of Speek et al., 1985). and may documentpublication, It exploitation G.4. Statisticalthis Analysis any permission At the field sites, the test (MONprohibited 89034), control, and reference corns were grown in single plots randomly assigned within each of three replication blocks. The the be compositional componentscommercial for the test and control corn were statistically analyzed using a mixedFurthermore, modelany analysis of variance. The data from the five replicated sites were analyzed separatelyConsequently, and aswithout a combined data set. Individual replicated site analyses used the model

Yij = U + Ti + Bj + eij ,

where Yij = unique individual observation, U = overall mean, Ti = hybrid effect, Bj = random block effect, and eij = residual error.

Combined site analyses used the model:

Monsanto Company 06-CR-166U Page 244 of 262 CBI Deleted Version

Yijk = U + Ti + Lj + B(L)jk + LTij + eijk ,

where Yijk = unique individual observation, U = overall mean, Ti = hybrid effect, Lj = random location effect, B(L)jk = random block within location effect, LTij = random location by hybrid interaction effect, and eijk = residual error. For each compositional component, the forage and grain from MON 89034 was compared to the conventional control.

A range of observed values from the reference corns was determined for each analytical component. Additionally, the reference corn data were used to develop population tolerance intervals. A tolerance interval is an interval that one can claim, with a specified regime.and degree of confidence, contains at least a specified proportion,and p, of an entire sampled AG population for the parameter measured. For each compositional component, 99% tolerance intervals were calculated that are expected to contain, with 95% confidence, 99% of the quantities expressed in the populaBayertion ofproperty commercial protection referencespublishing (George et al., 2004; Ridley et al., 2002b). Each oftolerance interval estimate was basedcontents upon one its observation per unique reference substance. Individualparties.data substances withtherefore multiple observations were summarized within sites to obtain a singleand/or estimateor for inclusion in affiliates. tolerance interval calculations.property Because intellectualnegativethird quantities are not possible,may calculated its ® negative lower tolerance bounds were asset to zero. SAS software was used to generate the of and all summary statistics andis perform all analyses (SASregulatory Software Releaseowner. 9.1, 2002-2003). a document any such its reproductiondocument G.5. Compositional Analysis Resultsowner this of under of rightsthe this documentand/or rights The results of compositionalto ofan alysis,fall includinguse a summaryof of statistical differences, are discussed in Section VII. Tables G-1 to G-6 of this appendixthe provide a summary of the combinedThis site analysis, and Tablemay distribution,G-7 andlists the literature and historical ranges for the rights owner components analyzed.subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 245 of 262 CBI Deleted Version

Table G-1. Comparison of fiber, mineral, and proximate content in forage from MON 89034 and control corn in combined-site analysis and and regime. MON 89034 Control AG Difference (MON 89034 minus Control) Commercial Mean ± SE1 Mean ± SE Mean ± SE 95% CI1 Significance (Range) Analytical Component (units)1 2 (Range) (Range) Bayer(Range) (Lower,Upper) (p-Value) [99% Tolerance Int. ] Fiber property protectionpublishing of contents Acid Detergent Fiber (% DW) 28.95 ± 1.69 27.26 ± 1.69 1.69 ± 1.18 -0.81,4.19 0.17 (26.72 - 38.94) its (22.60 - 35.85) (19.93 - 35.59) (-6.22 - 10.45)parties. data therefore [16.76,43.76] and/oror Neutral Detergent Fiber (% DW) 39.69 ± 1.32 37.60 ± 1.32affiliates. 2.09 ± 1.40 -0.88,5.05 may 0.155 (33.70 - 46.74) (33.99 - 46.82) (31.44property - 43.96) intellectual (-3third.47 - 7.47) [25.94,55.67] its Mineral as the of and Calcium (% DW) 0.20 ± 0.019 is 0.19 ± 0.019 0.0066regulatory ± 0.011 -0.017,0.031 owner. 0.569 (0.11 - 0.29) a document (0.16 - 0.24) any (0.13 such - 0.28) (-0.036 - 0.063) its [0.016,0.38] reproductiondocument of Phosphorus (% DW) 0.25 ± 0.011 0.21 ± 0.011owner 0.040 ± 0.014this 0.011,0.069 0.01 (0.14 - 0.25) (0.22 - 0.32) (0.15 - 0.25)under (-0.0019 of - 0.13) [0.071,0.32] rightsthe this Proximate documentand/or rights to of fall use of Ash (% DW) 3.70 ± 0.27 3.90 ± 0.27 -0.20 ± 0.21 -0.65,0.25 0.356 (3.40 - 5.45) the This(2.51 - 4.67) (2.59may - 5.10)distribution, and (-1.72 - 0.97) [1.93,6.31] rights owner Carbohydrates (% DW) 86.90subject ± 0.43 86.69 ± 0.43 0.21 ± 0.53 -0.91,1.33 0.697 (84.88 - 88.39) (84.93 - 89.13) (84.36 - 89.57) the (-4violate.23 - 4.41) [83.05,90.74] be copy Moisture (% FW) 72.20 ± 1.35 71.53 ± 1.35of 0.67 ± 0.52 -0.44,1.77 0.22 (64.90 - 77.40) and may(68.50 - 75.40)document publication, (65.90 - 76.80) (-3.50 - 4.20) [57.62,86.45] It exploitation Protein (% DW) 7.82this ± 0.27 7.70 ± 0.27 0.13 ± 0.26 -0.43,0.68 0.635 (6.58 - 8.82) (6.34 - 8.98)any (6.06 - 8.87) (-2.32 - 2.35) [4.78,10.38] permission Total Fat (% DW) 1.57 ± 0.24 1.71prohibited ± 0.24 -0.13 ± 0.23 -0.59,0.32 0.558 (0.58 - 3.11) (0.63-3.17) the (0.77 - 2.91) (-2.28 - 1.95) [0,4.54] commercial be 1 DW = dry weight; FW = fresh weight; SE = standard error; CI = confidence interval. 2 With 95% confidence, interval Furthermore,contains 99%any of the values expressed in the population of commercial lines. Negative limits were set to zero. Consequently,without

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Table G-2. Comparison of amino acid content in grain from MON 89034 and control corn in combined-site analysis

MON 89034 Control Difference (MON 89034 minus Control)and Commercial and regime. Mean ± SE1 Mean ± SE1 Mean ± SE 95% CI1 Significance (Range) Analytical Component (units)1 AG (Range) (Range) (Range) (Lower,Upper) (p-Value) [99% Tolerance Int.2] Alanine (% DW) 0.77 ± 0.039 0.78 ± 0.039 -0.0070 ± 0.019 -0.046,0.032 0.709 (0.67 - 0.96) Bayer property protectionpublishing (0.64 - 0.89) (0.67 - 0.89) of (-0.13 - 0.089) contents [0.48,1.08] its Arginine (% DW) 0.48 ± 0.013 0.47 ± 0.013 0.011 ± 0.012parties. data -0.014,0.036 therefore 0.361 (0.37 - 0.49) (0.38 - 0.52) (0.41 - 0.51) (-0.090 - 0.062) and/or or [0.33,0.56] affiliates. Aspartic acid (% DW) 0.68 ± 0.029 0.67property ± 0.029 intellectual 0.0038third ± 0.015 -0.028,0.036may 0.804 (0.57 - 0.77) (0.56 - 0.78) (0.60 -its 0.76) (-0.11 - 0.078) [0.43,0.90] as the of and Cystine (% DW) 0.23 ± 0.0057 is 0.23 ± 0.0057 0.0023regulatory ± 0.0038 -0.0057,0.010owner. 0.554 (0.20 - 0.24) (0.20 - 0.26) (0.21 - 0.25) a(-0.022 - 0.023)document [0.18,0.27] any such its reproductiondocument Glutamic acid (% DW) 1.97 ± 0.097 1.99 ± 0.owner097 -0.012 ± 0.049this -0.11,0.091of 0.809 (1.71 - 2.41) (1.63 - 2.29) (1.70 - 2.26) (-0.33 - 0.24) [1.25,2.75] under of this and/orrights the Glycine (% DW) 0.38document ± 0.0087 to 0.38 ± 0.0087fall 0.0042 ± 0.0071of rights -0.011,0.019 0.566 (0.32 - 0.40) (0.32 - 0.41) of (0.36 - 0.41) (-0.067use - 0.035) [0.28,0.46] the Histidine (% DW) This0.31 ± 0.011 0.31may ± 0.011distribution, and 0.0027 ± 0.0055 -0.0090,0.014 0.632 (0.26 - 0.33) rights owner (0.25 subject- 0.35) (0.28 - 0.34) (-0.050 - 0.030) [0.22,0.38] Isoleucine (% DW) 0.36be ± 0.018 0.36 ± 0.018 the -0 .00003violate ± 0.0088 -0.019,0.019 0.997 (0.32 - 0.45) (0.30 - 0.43)copy (0.30 - 0.42)of (-0.056 - 0.041) [0.23,0.51] and Leucine (% DW) may1.31 ± 0.077document publication, 1.32 ± 0.077 -0.014 ± 0.036 -0.089,0.062 0.7 (1.14 - 1.68) It (1.09 - 1.57) exploitation (1.08 - 1.55) (-0.21 - 0.16) [0.77,1.92] this Lysine (% DW) 0.33 ± 0.0097any 0.32 ± 0.0097 0.0088 ± 0.0078 -0.0077,0.025 0.273 (0.24 - 0.34) (0.26 - 0.36) permission (0.29 - 0.36) (-0.056 - 0.033) [0.20,0.40] prohibited Methionine (% DW) 0.23 ± 0.0064 the 0.22 ± 0.0064 0.0038 ± 0.0047 -0.0061,0.014 0.427 (0.17 - 0.22) (0.20 -commercial 0.27) be (0.20 - 0.24) (-0.017 - 0.028) [0.14,0.25] 1 DW = dry weight; SE = standardFurthermore, error; CI = anyconfidence interval. 2 With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits wereConsequently, set to zero.without Table G-2 continues on the next page.

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Table G-2 (continued). Comparison of amino acid content in grain from MON 89034 and control corn in combined-site analysis and and regime. MON 89034 Control AGDifference (MON 89034 minus Control) Commercial Mean ± SE1 Mean ± SE1 Mean ± SE 95% CI1 Significance (Range) Analytical Component (units)1 (Range) (Range) (Range) (Lower,Upper) (p-Value) [99% Tolerance Int.2] Bayer property protectionpublishing Phenylalanine (% DW) 0.51 ± 0.028 0.52 ± 0.028 of -0.0012 ± 0.013 -0.029,0.026 contents 0.925 (0.45 - 0.65) (0.43 - 0.61) (0.43 - 0.60) (-0.080 - 0.067) its [0.32,0.73] parties.data therefore Proline (% DW) 0.93 ± 0.030 0.93 ± 0.030 0.0034 ± 0.019 and/or -0.037,0.044or 0.861 (0.83 - 1.11) (0.79 - 1.05) (0.83 - 1.01)affiliates. (-0.15 - 0.10) [0.68,1.21] property intellectualthird may Serine (% DW) 0.52 ± 0.022 0.52 ±its 0.022 -0.0046 ± 0.012 -0.030,0.021 0.703 (0.45 - 0.62) as (0.44 - 0.61) the (0.46of - 0.60) and(-0.087 - 0.058) [0.34,0.71] is regulatory owner. a document Threonine (% DW) 0.33 ± 0.010 any 0.33 such ± 0.010 0.00063 ± 0.0074 -0.015,0.016its 0.933 (0.29 - 0.37) (0.27 - 0.37) (0.29 - 0.36) (-0.052reproduction - 0.039) document [0.24,0.41] owner this of Tryptophan (% DW) 0.056 ± 0.0018 0.056 ± 0.0018under 0.00031of ± 0.0013this -0.0025,0.0031 0.817 (0.043 - 0.059) (0.048 - 0.064) rights (0.045the - 0.063) (-0.0055 - 0.0072) [0.032,0.072] documentand/or rights to of fall use of Tyrosine (% DW) 0.37 ± 0.015 0.36 ± 0.015 0.0088 ± 0.016 -0.026,0.043 0.596 (0.25 - 0.40) (0.22 - 0.43) (0.24 - 0.42) (-0.21 - 0.14)the [0.17,0.52] This may distribution,and rights owner Valine (% DW) 0.49 ±subject 0.020 0.49 ± 0.020 0.0034 ± 0.010 -0.019,0.026 0.748 (0.42 - 0.55) (0.40 - 0.55) (0.43 - 0.55) the (-0.084violate - 0.055) [0.35,0.62] be copy 1 DW = dry weight; SE = standard error; CI = confidence interval. of 2 With 95% confidence, interval contains 99% of the valuesdocument expressed in the populationand of commercial lines. Negative limits were set to zero. may publication,exploitation It this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table G-3. Comparison of fatty acid content in grain from MON 89034 and control corn in combined-site analysis

MON 89034 Control Difference (MON 89034 minus Control)and Commercial and regime. Mean ± SE1 Mean ± SE1 Mean ± SE 95% CI1 Significance (Range) Analytical Component (units)1 AG (Range) (Range) (Range) (Lower,Upper) (p-Value) [99% Tolerance Int.2] 16:0 Palmitic (% Total FA) 9.19 ± 0.060 9.12 ± 0.060 0.071 ± 0.049 -0.034,0.18 0.171 (9.10 - 12.55) Bayer property protectionpublishing (8.98 - 9.46) (8.91 - 9.34) of (-0.14 - 0.33) contents [6.12,15.67] 16:1 Palmitoleic (% Total FA) 0.13 ± 0.0058 0.12 ± 0.0058 0.0022 ± 0.0054 -0.0093,0.014its 0.696 (0.050 - 0.19) parties.data therefore (0.11 - 0.14) (0.048 - 0.14) (-0.012 - 0.079) and/or or [0,0.28] affiliates. 18:0 Stearic (% Total FA) 1.89 ± 0.021 1.82property ± 0.021 intellectual 0.072third ± 0.021 0.028,0.12may 0.002 (1.57 - 2.45) (1.79 - 2.03) (1.76 -its 1.87) (-0.055 - 0.18) [0.86,2.98] as 18:1 Oleic (% Total FA) 24.96 ± 0.34 the 24.84of ± 0.34 and 0.12 ± 0.20 -0.32,0.55 0.574 (21.17 - 35.33) is regulatory owner. (23.38 - 25.75) (23.62 - 26.66) a (-1.48 - 1.15) document [7.51,46.46] any such its reproductiondocument 18:2 Linoleic (% Total FA) 61.82 ± 0.40 62.07 ± owner0.40 -0.25 ± this0.23 -0.73,0.24of 0.292 (50.33 - 63.59) (60.85 - 63.61) (60.51 - 63.41)under (-1.62of - 1.24) [39.41,76.74] rightsthe this 18:3 Linolenic (% Total FA) 1.19document ± 0.027and/or 1.22 ± 0.027 -0.028 ± of0.016 rights -0.063,0.0061 0.099 (0.93 - 1.52) (1.12 - 1.23) to of (1.15 - fall1.43) use (-0.23 - 0.036) [0.63,1.77] the 20:0 Arachidic (% Total FA) This0.39 ± 0.0062 0.38may ± 0.0062 distribution, and 0.013 ± 0.0031 0.0063,0.019 <0.001 (0.32 - 0.47) (0.36 - 0.42) rights (0.36 - 0.40) owner(-0.019 - 0.032) [0.23,0.54] subject 20:1 Eicosenoic (% Total FA) 0.28be ± 0.0040 0.28 ± 0.0040 the violate 0 ± 0.0024 -0.0051,0.0051 0.999 (0.23 - 0.32) (0.26 - 0.29)copy (0.25 - 0.29)of (-0.014 - 0.011) [0.15,0.39] and 22:0 Behenic (% Total FA) may0.16 ± 0.0050document publication, 0.15 ± 0. 0050 0.0027 ± 0.0062 -0.010,0.016 0.665 (0.12 - 0.19) It (0.13 - 0.20) exploitation (0.13 - 0.18) (-0.019 - 0.029) [0.081,0.23] this any 1 FA = fatty acid; SE = standard error; CI = confidence interval. 2 With 95% confidence, interval contains 99% of the values expressedpermission in the popu lation of commercial lines. Negative limits were set to zero. prohibited the commercial be Furthermore,any Consequently,without

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Table G-4. Comparison of mineral content in grain from MON 89034 and control corn in combined-site analysis

MON 89034 Control Difference (MON 89034 minus Control)and Commercial and regime. Mean ± SE1 Mean ± SE1 Mean ± SE 95% CI1 Significance (Range) Analytical Component (units)1 AG (Range) (Range) (Range) (Lower,Upper) (p-Value) [99% Tolerance Int.2] - Calcium (% DW) 0.0050 ± 0.00034 0.0049 ± 0.00034 0.00016 ± 0.00011 0.18 (0.0031 - 0.0049) Bayer property 0.00008,0.00040protectionpublishing (0.0038 - 0.0066) (0.0040 - 0.0059)of (-0.00027 - 0.00090) contents [0.0016,0.0059] its Copper (mg/kg DW) 1.74 ± 0.38 2.07 ± 0.37 -0.33 parties.± 0.53 data -1.45,0.79 therefore0.547 (1.15 - 3.56) and/or (1.33 - 2.38) (1.26 - 4.54) (-2.96 - 0.78) or [0,4.20] affiliates. may Iron (mg/kg DW) 21.40 ± 1.00 22.20property ± 0.99 intellectualthird -0.80 ± 0.67 -2.22,0.62 0.25 (18.04 - 29.22) its (19.23 - 25.23) the (19.03 - 28.26)as (-6.50 - 5.90) [8.88,34.51] of and owner. Magnesium (% DW) 0.12 ± 0.0043 is 0.12 ± 0.0043 -0.regulatory00028 ± 0.0021 -0.0047,0.0041 0.893 (0.099 - 0.14) a document (0.10 - 0.14) any (0.11 such - 0.14) (-0.018 - 0.011) its [0.075,0.17] reproductiondocument of Manganese (mg/kg DW) 6.79 ± 0.29 6.51 ± 0.29owner 0.28 ±this 0.21 -0.18,0.73 0.213 (5.56 - 8.64) under of (5.43 - 9.32) rights (5.57the - 8.00) (-1.54 - 2.36)this [3.17,9.99] documentand/or rights Phosphorus (% DW) 0.33 ± 0.0095 to of 0.33 ± 0.0095fall use 0.00039 of± 0.0043 -0.0087,0.0095 0.929 (0.25 - 0.37) (0.27 - 0.36) (0.29 - 0.36) (-0.038 - 0.026)the [0.18,0.45] This may distribution,and Potassium (% DW) 0.36 ± 0.0065 rights 0.36 ± 0.0065 owner 0.0032 ± 0.0042 -0.0052,0.012 0.45 (0.32 - 0.40) (0.32 subject- 0.40) (0.34 - 0.40) (-0.030 - 0.035) [0.26,0.46] be the violate Zinc (mg/kg DW) 22.05 ±copy 1.14 21.91 ± 1.14of 0.14 ± 0.51 -0.94,1.22 0.788 (16.72 - 34.04) (18.91 - 26.89) (18.81 - 26.04) (-3.37 - 3.19) [7.16,38.55] document and 1 may publication, DW = dry weight; SE = standard error;It CI = confidence interval. exploitation 2 With 95% confidence, interval contains 99% of thisthe values any expressed in the population of commercial lines. Negative limits were set to zero.

permission prohibited the commercial be Furthermore,any Consequently,without

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Table G-5. Comparison of proximate and fiber content in grain from MON 89034 and control corn in combined-site analysis

MON 89034 Control Difference (MON 89034 minus Control)and Commercial and regime. Mean ± SE1 Mean ± SE1 Mean ± SE 95% CI1 Significance (Range) Analytical Component (units)1 AG (Range) (Range) (Range) (Lower,Upper) (p-Value) [99% Tolerance Int.2] Proximate Bayer property publishing Ash (% DW) 1.41 ± 0.036 1.39 ± 0.036 0.014 ± 0.041 -0.072,0.10protection contents 0.734 (1.12 - 1.62) (1.25 - 1.56) (1.28 - 1.51) of (-0.11 - 0.13) [0.74,1.96] its parties.data therefore Carbohydrates (% DW) 84.85 ± 0.42 84.96 ± 0.42 -0.11 ± 0.18 and/or -0.50,0.28or 0.562 (82.91 - 86.78) (83.29 - 86.52) (83.58 - 86.22) (-1.42 - 0.84) [81.08,88.80] affiliates. property intellectualthird may Moisture (% FW) 9.52 ± 0.77 9.50 ±its 0.77 0.021 ± 0.22 -0.44,0.48 0.923 (7.60 - 15.30) (7.89 - 12.80) (7.86 - 13.10)as (-1.00 - 0.87) [0.45,19.52] the of and is regulatory owner. Protein (% DW) 10.43 ± 0.42 10.36 ± 0.42 a 0.070 ± 0.19 document -0.34,0.48 0.725 (9.33 - 11.82) any such its (8.54 - 11.98) (9.22 - 11.52) (-1reproduction.26 - 1.28) document [7.54,13.13] owner this of Total Fat (% DW) 3.32 ± 0.069 3.29 ± 0.069under 0.025of ± 0.089 -0.16,0.21 0.784 (2.66 - 3.71) (3.05 - 3.89) rights (3.05the - 3.75) (-0.50 - 0.29)this [2.20,4.55] documentand/or rights Fiber to of fall use of Acid Detergent Fiber (% DW) 5.48 ± 0.19 5.27 ± 0.19 0.21 ± 0.25the -0.30,0.72 0.41 (4.11 - 6.33) This(3.82 - 7.24) (4.17may - 7.00)distribution, and (-3.18 - 3.07) [2.77,7.56] rights owner Neutral Detergent Fiber (% DW) 10.06subject ± 0.37 9.75 ± 0.37 0.31 ± 0.34 -0.41,1.03 0.37 (8.20 - 11.30) (8.59be - 12.08) (8.48 - 11.75) the (-2violate.26 - 2.05) [5.93,13.63] copy of Total Dietary Fiber (% DW) 15.17 ± 0.47 14.67 ± 0.47 and 0.50 ± 0.54 -0.66,1.65 0.375 (12.99 - 18.03) may(13.39 - 17.02)document publication, (12.82 - 17.62) (-3.61 - 4.20) [9.20,20.27] It exploitation 1 DW = dry weight; FW = fresh weight; SE = standardthis anyerror; CI = confidence interval. 2 With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero. permission prohibited the commercial be Furthermore,any Consequently,without

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Table G-6. Comparison of vitamin, antinutrient and secondary metabolite content in grain from MON 89034 and control corn in combined-site analysis and and regime. MON 89034 Control AGDifference (MON 89034 minus Control) Commercial Mean ± SE1 Mean ± SE1 Mean ± SE 95% CI1 Significance (Range) Analytical Component (units)1 2 (Range) (Range) Bayer(Range) (Lower,Upper) (p-Value) [99% Tolerance Int. ] Vitamin property protectionpublishing of contents Folic Acid (mg/kg DW) 0.35 ± 0.037 0.36 ± 0.037 -0.0080 ± 0.022 -0.054,0.038 0.717 (0.13 - 0.45) its (0.26 - 0.48) (0.23 - 0.53) (-0.11 - parties.0.11) data therefore [0.012,0.69] and/oror Niacin (mg/kg DW) 30.08 ± 1.11 29.59 ± 1.11affiliates. 0.48 ± 0.65 -0.82,1.79may 0.461 (16.17 - 29.19) (25.72 - 34.84) (24.93property - 35.75) intellectual (-4third.44 - 5.64) [6.97,37.83] its the as Vitamin B1 (mg/kg DW) 3.07 ± 0.13 2.94of ± 0.13 and 0.13 ± 0.17 -0.24,0.49owner. 0.474 (2.19 - 5.60) (2.39 - 3.44) is (2.39 - 3.36) (-0.66regulatory - 0.68) [0.37,6.35] a document any such its Vitamin B2 (mg/kg DW) 1.42 ± 0.046 1.42 ± 0.046 0.0015reproduction ± 0.050 document -0.099,0.10of 0.976 (1.34 - 1.91) (1.24 - 1.65) (1.16 - 1.61)owner (-0.30 - 0.45)this [0.91,2.30] under of this Vitamin B6 (mg/kg DW) 6.22 ± 0.23 rights 6.26the ± 0.23 -0.036 ± 0.18 -0.41,0.34 0.838 (5.08 - 7.47) documentand/or rights (5.28 - 6.99) to of (5.37 - fall6.80) use (-0.72 - of1.10) [3.12,9.30] Vitamin E (mg/kg DW) 6.77 ± 0.42 6.63 ± 0.42 0.14 ± 0.36the -0.64,0.91 0.714 (2.71 - 13.94) This may distribution,and (5.55 - 8.62) rights (2.72 - 9.02) owner (-2.35 - 3.83) [0,20.49] Antinutrient subject the violate Phytic Acid (% DW) 0.75be ± 0.050copy 0.73 ± 0.050 0.016 ± 0.027 -0.037,0.069 0.537 (0.50 - 0.94) (0.53 - 0.87) (0.56 - 0.88)of (-0.15 - 0.18) [0.21,1.22] and Secondary Metabolite may documentpublication, It exploitation Ferulic Acid (µg/g DW) 2131.38this ± 108.09 2148.05 ± 108.09 -16.67 ± 50.08 -116.98,83.65 0.74 (1412.68 - 2297.36) (1790.25 - 2525.31)any (1878.66 - 2669.85) (-330.17 - 264.79) [1136.69,2806.24] permission p-Coumaric Acid (µg/g DW) 194.25 ± 7.12 183.96prohibited ± 7.12 10.28 ± 7.08 -4.73,25.30 0.165 (99.30 - 285.75) (166.11 - 253.04) the (167.76 - 210.13) (-24.37 - 70.84) [0,378.57] commercial be 1 DW = dry weight; SE = standard error; CI = confidence interval. 2 With 95% confidence, interval Furthermore,contains 99%any of the values expressed in the population of commercial lines. Negative limits were set to zero. Consequently,without

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Table G-7. Literature and historical ranges of components of corn forage and grain

Tissue/ Literature ILSI Component1 Range2 Range3

Forage Proximates (% dw) Ash 2.43-9.64a; 2-6.6b 1.527 – 9.638 Carbohydrates 83.2-91.6b; 76.5-87.3a 76.4 – 92.1 Fat, total 0.35-3.62b; 1.42-4.57a 0.296 – 4.570 a b Moisture (% fw) 56.5-80.4 ;55.3-75.3 49.1 – 81.3 and Protein 4.98-11.56a and3.14 – 11.57regime. AG Fiber (% dw) Acid detergent fiber (ADF) 18.3-41.0b; 17.5-38.3a 16.13 – 47.39 Bayer property protectionpublishing Neutral detergent fiber (NDF) 26.4-54.5of b; 27.9-54.8a 20.29 – 63.71contents its parties.data therefore Minerals (% dw) and/oror affiliates. b Calcium property0.0969-0.3184 intellectualthird 0.0714 –may 0.5768 its b Phosphorous 0.1367-0.2914as 0.0936 – 0.3704 the of and is regulatory owner. a document Grain any such its reproductiondocument Proximates (% dw) owner this of d b Ash 1.1-3.9under; 0.89-6.28of this 0.616 – 6.282 and/orrights the b a Carbohydratesdocument to 77.4-87.2fall ; 82.2-88.1of rights 77.4 – 89.5 of d use b Fat, total 3.1-5.7 ; 2.48-4.81the 1.742 – 5.823 MoistureThis (% fw) may distribution,7-23andd; 8.18-26.2 b 6.1 – 40.5 rights owner Protein subject 6-12d; 9.7-16.1c 6.15 – 17.26 the violate be copy Fiber (% dw) of dand a,b Acid detergentmay fiberdocument (ADF)publication, 3.3-4.3 ; 2.46-11.34 1.82 – 11.34 It exploitation d b Neutral detergentthis fiber (NDF) 8.3-11.9 ; 7.58-15.91 5.59 – 22.64 Total dietary fiber (TDF)any 10.99-11.41h 8.82 – 35.31 permission prohibited Minerals the be Calcium (% dw)commercial 0.01-0.1d 0.00127 – 0.02084 d CopperFurthermore, (mg/kgany dw) 0.9-10 0.73 – 18.50 IronConsequently, (mg/kg dw)without 1-100d 10.42 – 49.07 Magnesium (% dw) 0.09-1d 0.0594 – 0.194 Manganese (mg/kg dw) 0.7-54d 1.69 – 14.30 Phosphorous (% dw) 0.26-0.75d 0.147 – 0.533 Potassium (% dw) 0.32-0.72d 0.181 – 0.603 Zinc (mg/kg dw) 12-30d 6.5 – 37.2

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Table G-7 (continued). Literature and historical ranges of components of corn forage and grain

Tissue/ Literature ILSI Component1 Range2 Range3

Grain Amino Acids (% dw) Alanine N/A 0.439 – 1.393 Arginine N/A 0.119 – 0.639 Aspartic acid N/A 0.335 – 1.208 Cystine N/A 0.125 – 0.514 and Glutamic acid N/A 0.965and – 3.536 regime. Glycine N/A AG 0.184 – 0.539 Histidine N/A 0.137 – 0.434 Isoleucine N/A 0.179 – 0.692 Bayer property protectionpublishing Leucine N/Aof 0.642 – 2.492contents its Lysine N/A parties.data 0.172 – 0.668 therefore Methionine N/A 0.124and/or – or0.468 affiliates. Phenylalanine propertyN/A intellectualthird 0.244 – 0.930may its Proline N/Aas 0.462 – 1.632 the of and Serine is N/A regulatory 0.235 – 0.769owner. a document Threonine any suchN/A 0.224 –its 0.666 reproductiondocument Tryptophan ownerN/A this 0.0271of – 0.215 Tyrosine N/Aunder of 0.103 – 0.642 rightsthe this Valine documentand/or N/A rights0.266 – 0.855 to of fall use of the Fatty AcidsThis may(% distribution,totaland fat) (% total fatty acid) rights owner 16:0 Palmitic subject 7-19e 7.94 – 20.71 16:1 Palmitoleic 1e the violate 0.095 – 0.447 be copy 18:0 Stearic 1-3ofe 1.02 – 3.40 eand 18:1 Oleicmay documentpublication, 20-46 17.4 – 40.2 It exploitation e 18:2 Linoleic this 35-70 36.2 – 66.5 18:3 Linolenic any 0.8-2e 0.57 – 2.25 permission e 20:0 Arachidic prohibited0.1-2 0.279 – 0.965 20:1 Eicosenoic the be - 0.170 – 1.917 22:0 Behenic commercial - 0.110 – 0.349 Furthermore,any VitaminsConsequently, (mg/kgwithout dw) Folic acid 0.3d 0.147 – 1.464 Niacin 9.3-70d 10.37 – 46.94 e Vitamin B1 3-8.6 1.26 – 40.00 e Vitamin B2 0.25-5.6 0.50 – 2.36 d e Vitamin B6 5.3 ; 9.6 3.68 – 11.32 Vitamin E 3-12.1e; 17-47d 1.5 – 68.7

Monsanto Company 06-CR-166U Page 254 of 262 CBI Deleted Version

Table G-7 (continued). Literature and historical ranges of components of corn forage and grain

Tissue/ Literature ILSI Component1 Range2 Range3

Grain Antinutrients (% dw) Phytic acid 0.48-1.12a 0.111 – 1.570 Raffinose 0.08-0.30e 0.020 – 0.320

regime.and Secondary Metabolites and AG (µg/g dw) Ferulic acid 113-1194f; 3000g 291.9 – 3885.8 f p-Coumaric acid 22-75 Bayer property53.4 protection – 576.2publishing of contents

1 its fw=fresh weight; dw=dry weight; Niacin =Vitamin B3; Vitaminparties. B1 =Thiamine;data Vitamintherefore B2 and/oror =Riboflavin; Vitamin B6 =Pyridoxine ; N/A = not available as percent dry wt. 2 a affiliates.b c Literature range references: property2002a. intellectual Sidhuthird et al ., 2000. Jugenheimer,may 1976. d e its g h Watson, 1987. Watson, 1982. Classen et al., as1990. Dowd and Vega, 1996. Choi et al., 1999. 3 the of and ILSI range is from ILSI Crop isComposition Database, 2006.regulatory owner. a document any such its 4 3 reproductiondocument Conversions: % dw x 10 = µg/g dw; mg/gowner dw x 10 = mg/kg thisdw; mg/100gof dw x 10 = mg/kg dw under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 255 of 262 CBI Deleted Version

G.6. References

AACC. 1998. Method 32.20. Approved Methods of the American Association of Cereal Chemists, 9th Ed. Albala-Hurtado S., M.T. Veciana-Nogues, M. Izquierdo-Pulido, and M.C. Vidal-Carou. 1997. Determination of free and total furfural compounds in infant milk formulas by high-performance liquid chromatography. J. Agric. Food Chem. 45:2128- 2133. AOAC. 2000. Official Methods of Analysis, 17th Ed. Association of Official Analytical Chemists, Gaithersburg, MD.

AOCS. 1997. Method Ce 1-62. Official Methods and Recommended Practicesregime. ofand the American Oil Chemists Society, 5th Ed. American Oiland Chemists Society, AG Champaign, IL. Bradstreet, R.B. 1965. The Kjeldahl Method for Organic Nitrogen. Academic Press, New Bayer property protectionpublishing York, NY. of contents Brobst, K.M. 1972. Gas-liquid chromatography of trimethylsilyl derivatives.its In Methods parties.data therefore in Carbohydrate Chemistry. Vol. 6. Academic Press, New York,and/or orNY. affiliates. Choi, I.H., J.H. Son, and K.H. Nahm.property 1999. intellectualDietarythird fiber fraction for grainsmay containing its high levels of water-soluble non-starchas polysaccharides. Japan Poultry Science. the of and 36:269-274. is regulatory owner. a documentits Classen, D., J.T. Arnason, J.A.any Serratos, such J.D.H. Lambert,reproduction C. Nozzolillo, and B.J.R. this documentof Philogene. 1990. Correlation ofowner phenolic acid content of maize to resistance to under of Sitophilus zeamais, therights maizethe weevil, in CIMMYT'sthis collections. J. Chem. Eco. documentand/or rights 16:301-315. to of fall use of the Cort, W.M.,This T.S. Vincente, E.H. Waysek,may distribution, andand B.D. Williams. 1983. Vitamin E content of rights owner feedstuffs determinedsubject by high-performance liquid chromatographic fluorescence. J. Agric. beFood Chem. 31:1330-1333. the violate copy of Dowd, P.F. and F.E. Vega. 1996. Enzymatic andoxidation products of allelochemicals as a basismay for resistancedocument publication,against insect s: Effects on the corn leafhopper Dalbulus It exploitation maidis. Natural Toxins. 4:85-91. this any George, C., W.P. Ridley, J.C. Obert, M.A. Nemeth, M.L. Breeze, and J.D. Astwood. permission 2004. Composition of grain prohibitedand forage from corn rootworm-protected corn event the MON 863 iscommercial equivalent to be that of conventional corn (Zea mays L.). J. Agric. Food Chem. 52:4149-4158. Furthermore,any Hagerman,Consequently, A.E. andwithout R.L. Nicholson. 1982. High-performance liquid chromatographic determination of hydroxycinnamic acids in maize mesocotyl. J. Agric. Food Chem. 30:1098-1102. ILSL-CCD, 2006. International Life Science Institute Crop Composition Database. Version 3.0. Available at: http://www.cropcomposition.org Jugenheimer, R.W. 1976. Corns for special purposes and uses. Pages 215-233 in Corn: Improvement, Seed Production and Uses. John Wiley and Sons, New York.

Monsanto Company 06-CR-166U Page 256 of 262 CBI Deleted Version

Kalthoff, I.M., and E.B. Sandell. 1948. Quantitative Inorganic Analysis. MacMillan, New York, NY. Lehrfeld, J. 1989. High-performance liquid chromatography analysis of phytic acid on a pH- stable, macroporous polymer column. Cereal Chem. 66:510-515. Lehrfeld, J. 1994. HPLC separation and quantitation of phytic acid and some inositol phosphates in foods: problem and solution. J. Agric. Food Chem. 42:2726-2731. Mason, B.S. and H.T. Slover. 1971. A gas chromatographic method for the determination of sugars in foods. J. Agric. Food Chem. 19:551-554. McMurray, C.H., W.J. Blanchflower, and D.A. Rice. 1980. Influence of extraction techniques on determination of α-tocopherol in animal feedstuffs. Journal of Association of Official Analytical Chemists. 63:1258-1261. and and regime. 2002a.AG Compositional analysis of grain from commercial YieldGard corn borer corn (MON 810) and conventional corn grown in U. S. in 2001. Monsanto Technical Report MSL-18125 (Study 02-01- Bayer property protectionpublishing 39-19). Monsanto Company, St. Louis,of MO. contents its Ridley, W.P., R.S. Sidhu, P.D. Pyla, M.A. Nemeth, M.L.parties. Breeze,data and J.D. thereforeAstwood. 2002b. Comparison of the nutritional profile of glyphosate-tolerantand/oror corn event affiliates. NK603 with that of conventionalproperty cornintellectual (Zeathird mays L.). J. Agric.may Food Chem. its 50:7235-7243. as the of and is regulatory owner. Sidhu, R.S., B.G. Hammond, R.L. Fuchs, J.-N. aMutz, L.R. documentHolden, B. George, and T. any such its Olson. 2000. Glyphosate-tolerant corn: Thereproduction compositiondocument andof feeding value of grain from glyphosate-tolerant cornowner is equi valent thisto that of conventional corn (Zea under of this mays L.). J Agric.and/or Foodrights Chem.the 48:2305-2312. document to fall of rights Speek, A. J., J. Schijver, and W.H.P.of Schreurs. use1985. Vitamin E composition of some the seed Thisoils as determinedmay by high-performancedistribution,and liquid chromatography with rights owner fluorometricsubject quantitation. J. Food Sci. 50:121-124. USDA. 1970. Forage and Fiber Analysis. Agriculturethe violate Handbook No. 379. United States be copy Department of Agriculture, Washington,of D.C. and may documentpublication, USDA. 1973.It Energy value of foods.exploitation Pages 2-11 in Agriculture Handbook No. 74. United States Departmentthis any of Agriculture, Washington, D.C. Watson, S.A. 1982. Corn: Amazingpermission Maize - General Properties. Vol. II: Part 1 Plant Products. CRC Press, Boca Raton,prohibited FL. the be Watson, S.A. 1987.commercial Structure and composition. Pages 53-82 in Corn: Chemistry and Furthermore,Technology.any Watson, S.A., and P.E. Ramstad, Eds.; American Association of CerealConsequently, Chemist.without St. Paul, MN

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Appendix H: USDA Notifications Approved for MON 89034

Field trials of MON 89034 were conducted in the U.S. since 2001. The protocols for these trials include field performance, agronomics, and generation of field materials and data necessary for this petition. In addition to the phenotypic assessment data provided for MON 89034, observational data on pest and disease stressors were collected from these product development trials. The majority of the final reports have been submitted to the USDA. However, some final reports, mainly from the 2005-2006 seasons, are still in preparation. A list of trials conducted under USDA notification and the status of the final reports for these trials are provided in Table H-1. and and regime. AG

Monsanto Company 06-CR-166U Page 258 of 262 CBI Deleted Version

Table H-1. USDA notifications approved for MON 89034 and status of trials conducted under these notifications.

USDA No. Effective Date (m/d/y) Release Site (State) Trial Status 2001 Field Trials 01-348-04n 1/13/2002 PR (2) Submitted to USDA 01-348-07n 1/11/2002 HI (6) Submitted to USDA

2002 Field Trials 02-009-12n 2/8/2002 IL (3) Submitted to USDA 02-009-15n 2/8/2002 IA (6) Submitted to USDA 02-017-12n 3/20/2002 AL, MS, OR, TN, WA Submitted to USDA and 02-242-10n 10/1/2002 PR (2) and Submittedregime. to USDA AG 2003 Field Trials 03-024-06n 2/23/2003 AL MS Submitted to USDA Bayer property protectionpublishing 03-034-25n 3/5/2003 of OR, TN (2) Submittedcontents to USDA 03-034-27n 3/5/2003 IL (2) Submittedits to USDA parties.data therefore 03-042-06n 3/13/2003 MO and/orSubmittedor to USDA 03-043-04n 3/17/2003 affiliates. IL (11) Submitted to USDA property intellectualthird may 03-043-05n 3/17/2003 its IA (11) Submitted to USDA 03-052-57n 3/23/2003the as TN Submitted to USDA of and owner. 03-161-05n is8/5/2003 HI (5) regulatory Submitted to USDA a document 03-219-04n 9/5/2003any such PR (2) itsSubmitted to USDA reproductiondocument 03-265-09n 11/21/2003 owner HI (6) this of Submitted to USDA under of 03-280-01n 11/6/2003rightsthe PR (3) this Submitted to USDA documentand/or rights to of fall use of 2004 Field Trials the 04-009-01n This 3/25/2004may distribution, HIand (5) Submitted to USDA rights owner 04-009-02n subject 3/25/2004 PR (4) Submitted to USDA 04-014-05n be 2/26/2004 GAthe violate Submitted to USDA 04-014-08n copy 2/13/2004 of TN (2) Submitted to USDA and 04-021-02n may documentpublication,2/20/2004 AL, MS Submitted to USDA It exploitation IA (2), IL (4), IN, KS, NE, 04-021-09n 2/20/2004 Submitted to USDA this any OH 04-021-11n 2/20/2004 AR Submitted to USDA permission 04-021-14n 2/20/2004prohibited TN Submitted to USDA the 04-023-09n commercial2/25/2004 be IA (12) Submitted to USDA 04-023-10n 2/25/2004 IA (8) Submitted to USDA 04-023-11nFurthermore, any 2/25/2004 WI (3) Submitted to USDA 04-028-10nConsequently, without 3/24/2004 MN (5) Submitted to USDA 04-028-23n 2/27/2004 TN Submitted to USDA 04-028-24n 2/27/2004 CO (3), NE (4) Submitted to USDA 04-028-25n 2/27/2004 IA (4) Submitted to USDA 04-028-26n 2/27/2004 OR (2), WA Submitted to USDA 04-028-27n 2/27/2004 IL (15) Submitted to USDA 04-028-28n 2/27/2004 KS (2) Submitted to USDA Table H-1 continues on next page.

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Table H-1 (continued). USDA notifications approved for MON 89034 and status of trials conducted under these notifications.

USDA No. Effective Date (m/d/y) Release Site (State) Trial Status 04-028-29n 2/27/2004 IN (4) Submitted to USDA 04-028-30n 2/27/2004 MI (2) Submitted to USDA 04-028-31n 2/27/2004 MO (2) Submitted to USDA 04-028-32n 2/27/2004 NE (4) Submitted to USDA 04-028-33n 2/27/2004 OH (2) Submitted to USDA 04-040-01n 4/2/2004 MD (2) Submitted to USDA 04-040-02n 3/12/2004 NE Submitted to USDA 04-040-03n 3/10/2004 IL (6) Submitted to USDA and 04-041-03n 3/11/2004 IL (10) and Submittedregime. to USDA 04-041-04n 3/11/2004 KS (2) AG Submitted to USDA 04-044-07n 3/14/2004 IA (6) Submitted to USDA 04-056-06n 3/26/2004 IA (2), IL (2), IN, MO (3) Submitted to USDA Bayer property protectionpublishing 04-079-02n 4/26/2004 of IN, MN, MO, WI Submittedcontents to USDA 04-085-15n 4/24/2004 NE (3) Submittedits to USDA parties.data therefore 04-090-03n 6/18/2004 HI (5) and/orSubmittedor to USDA 04-093-03n 5/2/2004 affiliates. IL Submitted to USDA property intellectualthird may 04-093-04n 5/2/2004 its IA Submitted to USDA 04-189-03n 9/27/2004the as HI (15) Submitted to USDA of and owner. 04-217-02n 9/20/2004is PR (2) regulatory Submitted to USDA a document 04-231-03n 9/27/2004any such PR (3) itsSubmitted to USDA reproductiondocument 04-260-01n 11/1/2004 owner HI this of Submitted to USDA under of 04-273-01n 10/18/2004rightsthe IA, IL (2), NE this Submitted to USDA 04-274-08n documentand/or10/28/2004 PR (2) rights Submitted to USDA to of fall use of 04-309-03n 1/10/2005 HI (9) the Submitted to USDA 04-309-04n This 1/10/2005may distribution, PRand (3) Submitted to USDA rights owner 04-337-02n subject 12/16/2004 MS Submitted to USDA 04-337-03n be 12/20/2004 ALthe violate Submitted to USDA 04-358-20n copy 1/25/2005 of GA (2) Submitted to USDA and 04-358-21n may documentpublication,1/24/2005 IA (14) Submitted to USDA 04-358-22nIt 1/24/2005exploitation IA (15) Submitted to USDA this 04-358-23n any 1/24/2005 IL (11) Submitted to USDA 04-358-24n 1/25/2005permission IN (3) Submitted to USDA 04-358-25n 1/24/2005prohibited KS (2) Submitted to USDA the 04-358-26n commercial2/7/2005 be MD Submitted to USDA 04-358-27n 1/26/2005 MI (3) Submitted to USDA 04-358-28nFurthermore, any 2/9/2005 MN (8) Submitted to USDA 04-358-29nConsequently, without 1/24/2005 MO (2) Submitted to USDA 04-358-30n 1/25/2005 NE (5) Submitted to USDA 04-358-31n 1/25/2005 OH (2) Submitted to USDA 04-358-32n 1/27/2005 TN (2) Submitted to USDA 04-358-33n 1/25/2005 WI (3) Submitted to USDA 04-362-09n 1/18/2005 TN (3) Submitted to USDA 04-362-11n 1/18/2005 AR Submitted to USDA Table H-1 continues on next page.

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Table H-1 (continued). USDA notifications approved for MON 89034 and status of trials conducted under these notifications.

USDA No. Effective Date (m/d/y) Release Site (State) Trial Status 2005 Field Trials IA, IL (3), IN, KS, NE, OH 05-025-04n 2/22/2005 In Progress (2) 05-026-19n 2/14/2005 IL (8), IN, WI (3) In Progress 05-028-02n 2/14/2005 CO (5), KS, NE (3) In Progress 05-028-11n 2/22/2005 IA (4), IL (9) In Progress 05-028-12n 2/22/2005 HI (4) In Progress 05-039-09n 3/10/2005 NE, OR, WA In Progressregime. and IA (2) IL (2), IN (3), MI, and 05-039-11n 3/28/2005 In Progress MN, MO (2),AG NE, NY, WI 05-040-15n 3/10/2005 LA Submitted to USDA IABayer (5), IL (2), IN (2), NE (4), 05-049-13n 3/24/2005 property protectionIn Progresspublishing ofTX (2) contents 05-055-03n 4/11/2005 IL (2), MN (5), MO (2) In Progressits parties.data therefore 05-056-10n 4/1/2005 MO and/orInor Progress 05-063-06n 4/1/2005 affiliates. IL (2) Submitted to USDA property intellectualthird may 05-104-03n 5/17/2005 its HI (10) In Progress as 05-129-01n 5/31/2005the of NEand (6) In Progress is regulatory owner. 05-131-01n 6/9/2005 LA a documentInits Progress 05-133-05n 7/20/2005any such HI (4), PR reproduction(2) In Progress this documentof 05-201-06n 9/26/2005 owner PR (3) In Progress under of 05-201-10n 9/6/2005rights the HI (5) this In Progress documentand/or rights 05-201-11n 9/26/2005to of fall PR (3) use of In Progress the This may distribution,and 2006 Field Trials rights owner 06-025-04n subject 3/30/2006 AL, AR, LA, MS (2), TN (2) In Progress 06-027-06n be 2/27/2006 GAthe (4) violate In Progress copy of 06-027-07n 3/6/2006 OHand (2) In Progress 06-027-08n may document3/6/2006publication, WI (3) In Progress It exploitation 06-027-09n this 3/7/2006 MN (9) In Progress 06-027-11n any 3/1/2006 MI (3) In Progress 06-027-12n 2/27/2006permission NE (4) In Progress prohibited 06-027-13n the2/27/2006 MO (2) In Progress 06-027-14n commercial2/27/2006 be TN (4) In Progress 06-027-15n 2/27/2006 KS (2) In Progress Furthermore,any 06-027-17nConsequently, without 4/5/2006 IA (5) In Progress 06-030-01n 2/28/2006 IN (4) In Progress 06-030-02n 2/28/2006 IL (13) In Progress 06-030-04n 3/7/2006 IA (12) In Progress 06-030-05n 3/7/2006 IA (13) In Progress 06-030-06n 2/28/2006 IL (12) In Progress 06-031-25n 2/28/2006 NE (2) In Progress 06-032-12n 4/5/2006 IA (12) In Progress Table H-1 continues on next page.

Monsanto Company 06-CR-166U Page 261 of 262 CBI Deleted Version

Table H-1 (continued). USDA notifications approved for MON 89034 and status of trials conducted under these notifications.

USDA No. Effective Date (m/d/y) Release Site (State) Trial Status 06-033-07n 5/18/2006 HI (7) In Progress 06-037-08n 5/18/2006 HI (5) In Progress 06-037-10n 5/26/2006 PR (3) In Progress 06-037-11n 3/16/2006 PR (3) In Progress 06-038-05n 4/6/2006 IA, IL (3), IN (2), TX (3) In Progress 06-046-06n 3/23/2006 CO (5), NE (2) In Progress 06-046-10n 3/16/2006 IL (4), MO (5) In Progress 06-046-11n 4/17/2006 IA (8), NE (6), SD In Progress and 06-052-11n 3/23/2006 MS andIn Progress regime. 06-060-09n 4/24/2006 IA (2) AG In Progress 06-060-10n 4/24/2006 IA (14) In Progress IA (3), IL (4), IN (2), KS, 06-073-01n 6/12/2006 Bayer In Progress MN property protectionpublishing of contents 06-073-02n 5/5/2006 IA (7), IL (5), NE, OH (2) In Progress its 06-081-02n 5/9/2006 IA parties.data In Progress therefore and/oror 06-082-05n 5/18/2006 HI (3), PR (2) In Progress affiliates. 06-089-11n 5/15/2006 property IA (5),intellectual IL, thirdNE In Progressmay its 06-090-04n 5/18/2006 HIas (6) In Progress the of and 06-090-05n 6/12/2006is PR (3) regulatory In Progressowner. a document 06-090-06n 5/18/2006any such HI (5) In itsProgress reproductiondocument 06-090-07n 5/26/2006 owner PR (3) this ofIn Progress 06-108-03n 5/26/2006 PR of In Progress rights under this 06-108-04n and/or5/26/2006 the HI, IN (2), PR In Progress document to of fall of rights 06-108-05n 5/18/2006 HI, PR (2)use In Progress the 06-108-06n This 5/26/2006 may distribution, HI, andIN In Progress rights owner 06-114-03n subject5/26/2006 HI, IL In Progress 06-118-02n 5/9/2006 IA In Progress be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Page 262 of 262 and and regime. AG

January 23, 2007

This addendum provides further information requested by USDA-APHIS in the Agency’s letter to Monsanto Company dated January 8, 2007, concerning Monsanto’s petition for the determination of non-regulated status for corn MON 89034. In the letter, USDA requested Monsanto Company to provide a short summary of a list of studies on the non- target organisms (NTOs) of MON 89034. In addition, the Agency requested Monsanto Company to provide an update on the trial status of USDA notifications approved for MON 89034. regime.and and AG The following provides a short summary for each NTO study with the Agency’s request highlighted in bold font and the full reference of the studies in italic. Bayer property protectionpublishing 1. Evaluation of Dietary Effects of Lyophilizedof Leaf Tissue from Corncontents MON 89034 its in a Chronic Exposure Study with Collembola (Folsomiaparties.data candida ). therefore and/oror affiliates. 2006. Evaluationproperty of Dietary intellectual Effectsthird of Lyophilized Leaf Tissuemay from Corn its MON 89034 in a Chronic Exposure Studyas with Collembola (Folsomia candida). the of and Monsanto Technical Reportis MSL-20169. regulatory owner. a document any such its reproductiondocument The objective of this study was toowner determine the thispotential effectof of chronic dietary exposure of lyophilized corn leaf tissueunder of MON of89034 on survival and reproduction of rightsthe this Collembola (documentFolsomiaand/or candida ). Collembola were exposedrights for 28-days to lyophilized to of fall use of leaf tissue, combined with Brewer’s yeast, from eitherthe MON 89034 or a conventional control cornThis with similar backmayground distribution, geneticsand at 50% (w/w) of diet, yielding dose levels rights owner of 80 µg Cry1A.105/gsubject diet and 70 µg Cry2Ab2/g diet for the MON 89034 treatments, which resulted in a safety factor (margin ofthe exposure violate or MOE) of 19X for Collembola to be copy both proteins based on the estimatedof maximum expected concentrations of the two 1 and proteinsmay in the topdocument 15publication, cm soil . Additionally, positive controls (treatments with It exploitation thiodicarb) werethis included to demonstrate the validity of the test system. No adverse effect on survival andany reproduction was observed in the treatment containing leaf tissue permission from MON 89034. Therefore,prohibited the NOEC (no-observed-effect-concentration) was determined to be ≥80 µg Cry1A.105/g diet and ≥70 µg Cry2Ab2/g diet. the be commercial 2.Furthermore, Evaluationany of Exposure to MON 89034 with the Cladoceran Daphnia magna: An AcuteConsequently, Static-renewalwithout Test with Corn Pollen.

2006. Evaluation of Exposure to MON 89034 with the Cladoceran Daphnia magna: An acute static-renewal test with corn pollen. Monsanto Study WL-2005-011.

1 Details on assessment of the maximum expected environmental concentrations and calculation of safety factor (MOE) are presented in Section VIII.A.3.

Monsanto Company 06-CR-166U Addendum Page 1 of 14 The objective of this study was to determine the potential for acute effects to the cladoceran, Daphnia magna, during a static-renewal test with pollen from MON 89034. The test procedure followed the methodology of U.S. EPA OPPTS Guideline Number 850.1010, which provides specific guidance for testing Daphnia magna. Initially, a limit test was performed at a concentration of 120 mg pollen/l from MON 89034, including a control group containing 120 mg pollen/l from a conventional corn line with a similar genetic background to MON 89034, and a diluent water control. At the end of the limit test, there was 17% mortality/immobility and no mortality in the control groups. Therefore, a dose-response test was conducted under static-renewal conditions at concentrations of 6.3, 13, 25, 50, 100 and 120 mg pollen/l. There were no mortalities, immobile daphnids or signs of toxicity noted in any control or test group during the exposure in the dose-response test. Based on the combined results of the limit test and regime.and the dose-response test using pollen from MON 89034, the NOECand was determined to be AG ≥100 mg pollen/l. Under a well accepted toxicity classification system for freshwater ecotoxicology, if no adverse effects are observed at a concentration of 100 mg/l, the 2 tested material is classified as practically non-toxicBayer (EPA,property 1985) protection. publishing of contents its 3. Evaluation of Potential Effects of Exposure toparties. Cry1A.105data Protein thereforein an Acute Study with the Earthworm in an Artificial Soil Substrate. and/oror affiliates. property intellectualthird may its as 2006. Evaluation of Potential Effects the of and of Exposure to Cry1A.105is Protein in an Acute Studyregulatory with the Earthwormowner. in an Artificial a document Soil Substrate. Monsanto Technicalany such Report MSL-20147. its reproductiondocument owner this of The objective of this study was to evaluateunder th e potentialof effects of acute exposure of the rightsthe this Cry1A.105 proteindocument and/oradministered to the earthworm, Eiseniarights fetida, during a 14-day to of fall use of exposure period when mixed in an artificial soil substrate.the A single concentration of 178 mg Cry1A.105This protein/kg soilmay dry weightdistribution,and was tested, which resulted in a safety factor rights owner (margin of exposure)subject of 41X based on the maximum expected environmental concentration for the protein in the top 15the cm ofviolate soil. Appropriate negative and positive be copy controls were also included in the study.of The results showed that there was no mortality and in the assaymay controldocument grouppublication, (soil only), control substance group (soil with 25 mM CAPS It exploitation buffer), and thethis Cry1A.105 protein group during the 14-day test. The positive control groups of 15 mg anyand 30 mg chloroacetamide/kg soil resulted in 48% and 100% permission mortalities, respectively, demonstratingprohibited the validity of the test system. A slight loss in average individual earthworm body weight from test initiation to test termination was the be noted in all test groups,commercial which was expected since the earthworms were not fed during the 14-dayFurthermore, test anyperiod. There was no significant difference (p>0.05) in body weight losses betweenConsequently, the Cry1A.105without protein treatment group and the control substance group. The study concluded that the NOEC for earthworms was ≥ 178 mg Cry1A.105 protein/kg dry soil.

4. Evaluation of the Dietary Effect(s) of a Cry1A.105 Protein on Honeybee Larvae (Apis mellifera L.). The report for this study states that honey bee larvae were dosed

2 EPA. 1985. Hazard evaluation division, standard evaluation procedure: acute toxicity for freshwater invertebrates. PB86-129269.

Monsanto Company 06-CR-166U Addendum Page 2 of 14 with at least a 10X safety factor. In the summary of this study for the petition, explain what the safety factor is based on (e.g., 10X expression in pollen).

2006. Evaluation of the Dietary Effect(s) of a Cry1A.105 Protein on Honeybee Larvae (Apis mellifera L.). Monsanto Study CA-2005-071.

The objective of this study was to evaluate potential dietary effects of Cry1A.105 protein when administered to honeybee larvae. The protein was tested at a concentration of 1100 µg Cry1A.105/ml using a 25 mM CAPS buffer, which resulted in a safety factor of approximately 125X based on the maximum Cry1A.105 protein expression level (8.8 µg/g fwt) in pollen from MON 89034. In addition, appropriate negative and positive controls were included in the study. The results revealed that the survival rate for the regime.and honeybee larvae in the negative controls was 92.5% for both theand 25 mM CAPS buffer and AG water treatments. The Cry1A.105 protein treatment yielded a 95% survival rate, comparable to the negative control groups. Behavioral observations at emergence indicated no adverse behavior or morphologicalBayer effects. property Based onprotection statisticalpublishing analyses and behavioral observations there were no significantof effects on the developmentcontents or survival its of honeybees treated with either the Cry1A.105 proteinparties. or datathe buffer/water controls.therefore The survival rates for the positive control treatments were 26.4% and/orand 5.0%or for 200 µg and affiliates. 2000 µg potassium arsenate/ml,property respectively, intellectual cthirdonfirming the validity mayof the test system. its In conclusion, the NOEC of Cry1A.105as protein on honeybee larvae was ≥1100 µg/ml. the of and is regulatory owner. a document 5. Evaluation of the Dietaryany Effect(s)such of a Cry1A.105 Protein itson Adult Honeybees reproductiondocument (Apis mellifera L.). In the summaryowner of this studythis for the petition,of explain what the under of safety factor is based on (e.g.,rights 10Xthe expression in pollen).this documentand/or rights to of fall use of 2006. Evaluation of the Dietary Effect(s)the of a Cry1A.105 Protein on Adult HoneybeesThis (Apis melliferamay L.). distribution, Monsantoand Study CA-2005-072. rights owner subject The objective of this study was to evaluatethe potential violate dietary effects of Cry1A.105 protein be copy on the adult honeybee during chronic feeding.of The protein was tested at a concentration and of 550 µgmay Cry1A.105/ml documentpublication, in a 30% sucrose solution, which resulted in a safety factor of It exploitation approximately this63X based on the maximum Cry1A.105 protein expression level (8.8 µg/g fwt) in pollen fromany MON 89034. In addition, appropriate negative controls (12.5 mM permission CAPS buffer in 30% sucrose, prohibitedand 30% sucrose in water) and a positive control (100 µg/ml potassium arsenate in 30% sucrose) were included in the study. Adult honeybees the be (0 to 5 days old)commercial were exposed to the test and control solutions continually for the test period.Furthermore, Theany number of dead bees was assessed on a daily basis. The study was terminatedConsequently, on daywithout 20 and all the bees in each cage were counted to determine the exact number of bees present in each cage. The potassium arsenate positive control produced 100% mortality by Day 2, confirming the validity of the test system. Based on statistical analyses and behavioral observations there were no significant effects on the development or survival of honeybees treated with either the Cry1A.105 protein or the negative controls. Thus, the NOEC of Cry1A.105 protein on adult honeybees was ≥550 µg/ml.

Monsanto Company 06-CR-166U Addendum Page 3 of 14 6. Honey bee dietary toxicity studies submitted with this petition utilized Cry1A.105 protein as the test material. Studies with Cry2Ab2 protein should also be submitted and summarized for the petition.

Studies (below) regarding the effects of Cry2Ab2 on honeybees have been previously submitted to USDA in Bollgard II cotton (MON 15985) submission (USDA Petition No. 00-342-01p). The summaries of the studies are provided below.

2000. Evaluation of dietary effect(s) of purified Bacillus thuringiensis Cry2Ab2 protein on honey bee larvae. Monsanto Technical Report MSL-16961.

The objective of this study was to evaluate potential dietary effects of Cry2Ab2 protein regime.and when administered to honey bee larvae. The protein was testedand at two concentrations of AG 1 and 100 µg Cry2Ab2/ml in 10 mM sodium carbonate buffer. The concentration of 100 µg/ml resulted in a safety factor of approximately 213X based on the maximum Cry2Ab2 protein expression level (0.47 µg/g fwt)Bayer in pollen property from MON protection 89034.publishing In addition, appropriate negative and positive controlsof were included in the study. Survivalcontents of honey its bee larvae was assessed on days 7 and 12 after treatment.parties. data The results showedtherefore that mean mortality at Day 12 was 17.5% and 18.75% for honey bee larvaeand/or exposedor to 1 and 100 µg affiliates. Cry2Ab2/ml solutions, respectively.property Mean intellectual mortalitythird at Day 12 in themay buffer (10 mM its sodium carbonate) and water control groupsas was 21.25% and 10.0%, respectively. No the of and significant difference wasis observed in mortalityregulatory between the Cry2Ab2owner. treatments and the a document buffer control treatment usingany thesuch Dunnett’s test. The mortality inits the positive control reproductiondocument treatments was 19.89% and 76.79%owner for 50 µg andthis 1000 µgof potassium arsenate/ml, respectively, confirming the validity underof the testof system. In conclusion, this study rightsthe this indicated thatdocument the NOECand/or of the Cry2Ab2 protein on honey rightsbee larvae was ≥100 µg/ml. to of fall use of the This 2000. Evaluationmay of thedistribution, dietaryand effect(s) of insect protection protein 2 on rights 3 owner adult honey beessubject (Apis mellifera L.) . Monsanto Technical Report MSL-16176. the violate be copy The objective of this study was to ofevalua te the toxicity of the Cry2Ab2 protein and administeredmay in thedocument dietpublication, to adult honey bees. Two levels of the Cry2Ab2 protein were It exploitation evaluated at 3.4this and 68 µg/ml in 5 mM sodium carbonate solution with 30% sucrose. The concentration ofany 68 µg/ml resulted in a safety factor of approximately 145X based on permission the maximum Cry2Ab2 proteinprohibited expression level (0.47 µg/g fwt) in pollen from MON 89034. In addition, three control groups were included: a buffer control (5 mM sodium the be carbonate buffercommercial with 30% sucrose), an assay control (30% sucrose in water), and a positiveFurthermore, controlany received 100 µg potassium arsenate/ml in 30% sucrose solution. The beesConsequently, were allowedwithout to feed ad libitum in cages inside a dark, environmentally controlled room. The number of dead bees in each cage was assessed on a daily basis. On Day 19, the study was terminated as the cumulative percentage of mortality in the assay control group (22.4%) exceeded the pre-determined criterion of 20%. At termination, the cumulative mortality of 3.4 and 68 µg Cry2Ab2/ml treatments was 26.1% and 19.0%, respectively. The buffer control group had a mortality of 21.7%. No significant

3 The insect protection protein 2 referred to in the title of the report is synonymous with the Cry2Ab2 protein.

Monsanto Company 06-CR-166U Addendum Page 4 of 14 difference was noted between the Cry2Ab2 protein treatments and the control treatment. No behavioral or morphological abnormalities were observed in bees exposed to the test or negative control substance treatments. The positive control group produced 100% mortality by Day 5 of the study, confirming the validity of the test system. In conclusion, this study indicated that the NOEC of the Cry2Ab2 protein on adult honey bees was ≥68 µg/ml.

7. Evaluation of Potential Dietary Effects of Cry1A.105 Protein on the Ladybird Beetle, Coleomegilla maculata (Oleoptera: Coccinellidae). In the summary of this study for the petition, explain what the safety factor is based on (e.g., 10X expression in pollen).

regime.and 2006. Evaluation of Potential Dietary Effectsand of Cry1A.105 Protein on AG the Ladybird Beetle, Coleomegilla maculata (Coleoptera: Coccinellidae). Monsanto Technical Report MSL-20150. Bayer property protectionpublishing The objective of this study was to examineof the potential for dietary contentseffects of the its Cry1A.105 protein on the mortality and developmentparties. of thedata ladybird beetle, thereforeColeomegilla maculata using an agar-based artificial diet. The Cry1A.105 prand/oroteinor was incorporated at affiliates. 240 µg/g of diet, which resultedproperty in a safetyintellectual factorthird of approximately may27X based on the its maximum Cry1A.105 protein expressionas level (8.8 µg/g fwt) in pollen from MON 89034. the of and In addition, appropriate isnega tive controls (25 regulatorymM CAPS buffer, purifiedowner. water) and a a document positive control (100 µg potassiumany such arsenate/g diet) were includedits in the study. The reproductiondocument results showed that there were no significantowner differencesthis for the meanof survival percentage of C. maculata among the Cry1A.105under protein treatmentof group (88.5%), buffer control rightsthe this group (87.5%),document andand/or the water control group (91.6%). rights The positive control group to of fall use of (potassium arsenate) only produced a survival rate of the2.08 %, confirming the validity of the test system.This Likewise, theremay were distribution, no andsign ificant differences for the mean percentage rights owner of C. maculata subjectlarvae that developed to adults among the Cry1A.105 protein treatment group (88.5%), buffer control group (85.4%),the andviolate the water control group (90.6%). None be copy of the insects in the potassium arsenateof positive control group developed to the adult and stage. mayIn addition, document therepublication, were no significant differences in the mean weight of C. It exploitation maculata adultsthis among the Cry1A.105 protein treatment group (10.8 mg) and the buffer control group (11.2 anymg). In conclusion, the NOEC of Cry1A.105 on ladybird beetle was permission ≥240 µg/g of diet. prohibited

the be 8. Evaluation ofcommercial Potential Dietary Effects of Cry2Ab2 Protein on the Ladybird Beetle,Furthermore, Coleomegillaany maculata (Coleoptera: Coccinellidae). Consequently,without 2006. Evaluation of Potential Dietary Effects of Cry2Ab2 Protein on the Ladybird Beetle, Coleomegilla maculata (Coleoptera: Coccinellidae). Monsanto Technical Report MSL-20151.

The objective of this study was to examine the potential for dietary effects of the Cry2Ab2 protein on the mortality and development of the ladybird beetle, Coleomegilla maculata using an agar-based artificial diet. The test substance was incorporated at 120

Monsanto Company 06-CR-166U Addendum Page 5 of 14 µg Cry2Ab2 protein/g of diet, which resulted in a safety factor of approximately 255X based on the maximum Cry2Ab2 protein expression level (0.47 µg/g fwt) in pollen from MON 89034. In addition, appropriate negative controls (50 mM CAPS buffer, purified water) and a positive control (100 µg potassium arsenate/g diet) were included in the study. The results showed that there were no significant differences for the mean survival percentage of C. maculata among the Cry2Ab2 protein treatment group (94.7%), buffer control group (88.8%), and the water control group (91.6%). The positive control group (potassium arsenate) only produced a survival rate of 2.08%, confirming the validity of the test system. Likewise, there were no significant differences for the mean percentage of C. maculata larvae that developed to adults among the Cry2Ab2 protein treatment group (92.6%), buffer control group (85.3%), and the water control group (90.6%). None of the insects in the potassium arsenate positive control group developed regime.and to the adult stage. In addition, there were no significant differeandnces in the mean weight of AG C. maculata adults among the Cry2Ab2 protein treatment group (10.3 mg) and the buffer control group (10.5 mg). In conclusion, the NOEC of Cry2Ab2 on ladybird beetle was ≥120 µg/g of diet. Bayer property protectionpublishing of contents its 9. Evaluation of Potential Dietary Effects of Cry1A.105parties.data Protein on Minutetherefore Pirate Bugs, Orius insidiosus (Hemiptera: Anthocoridae). In the summaryand/oror of this study for affiliates. the petition, explain what theproperty safety intellectualfactorthird is based on (e.g., 10Xmay expression in its pollen). as the of and is regulatory owner. a document 2006. Evaluationany such of Potentia l Dietary Effects of Cry1A.105its Protein on reproductiondocument Minute Pirate Bugs, Orius insidiosusowner (Hemiptera: Anthocoridae).this of Monsanto Technical Report MSL-20170. under of rightsthe this documentand/or rights to of fall use of The purpose of this study was to determine the potentialthe diet ary effects of the Cry1A.105 protein onThis mortality and developmmay distribution,ent ofand minute pirate bugs, Orius insidiosus. The rights owner Cry1A.105 proteinsubject was incorporated into a pollen-based diet for treatment of the test group. A buffer control group was fed thewith violatethe base pollen diet treated with 25 mM be copy CAPS buffer. An assay control group wasof fed with the base pollen diet only. In addition, and a positivemay control documentgrouppublication, was fed the base pollen diet treated with 15 µg potassium It exploitation arsenate/g of diet,this which demonstrated the validity of the test system. The study was first started with a test anyat the maximum dose level of 240 µg Cry1A.105 protein/g of diet. permission This treatment produced a meanprohibited survival rate of 47% for Orius insidiosus, while 88% survival rate was observed in the buffer control and assay control groups in this initial the be test. Based on commercialthe results of this maximum dose test, three dose-response tests were conductedFurthermore, withany Cry1A.105 exposure levels at 30, 60, 120, and 240 µg/g of diet. Each dose-responseConsequently, testwithout was performed independently at a different time using separate groups of Orius. For the three dose response replicate tests, mean survival of 55% was observed for the 240 µg Cry1A.105/g of diet treatments. In the buffer control and assay control groups, mean survival rates of 89% and 91%, respectively, were observed in the dose response tests. Based on the results of these dose response tests, the LC50 value was empirically determined to be >240 µg Cry1A.105/g of diet. The recorded endpoints of measurement (survival and development) of each dose-response replicate in the 30, 60, and 120 µg Cry1A.105/g of diet treated Orius was equal to or greater than those of the

Monsanto Company 06-CR-166U Addendum Page 6 of 14 buffer control and assay control groups. Therefore, the NOEC of Cry1A.105 to Orius was ≥120 µg/g of diet, which resulted in a safety factor of 14X based on the maximum Cry1A.105 protein expression level (8.8 µg/g fwt) in pollen from MON 89034.

10. Evaluation of Potential Dietary Effects of Cry2Ab2 Protein on Minute Pirate Bugs, Orius insidiosus (Hemiptera: Anthocoridae). In the summary of this study for the petition, explain what the safety factor is based on (e.g., 10X expression in pollen).

2006. Evaluation of Potential Dietary Effects of Cry2Ab2 Protein on Minute Pirate Bugs, Orius insidiosus (Hemiptera: Anthocoridae). Monsanto Technical Report MSL-20171. regime.and and AG The purpose of this study was to determine the potential dietary effects of the Cry2Ab2 protein on mortality and development of minute pirate bugs, Orius insidiosus. The Cry2Ab2 protein was incorporated into Bayera pollen-based property diet protectionat 100publishing µg/g of diet for treatment of the test group, which wasof equivalent to a safety factor ofcontents approximately its 213X based on the maximum Cry2Ab2 protein expressionparties.data level (0.47 µg/g thereforefwt) in MON 89034 pollen. A buffer control group was fed with the base pollenand/oror diet treated with 50 affiliates. mM CAPS buffer. An assay propertycontrol group intellectual wasthird fed with the base pollenmay diet only. In its addition, a positive control group wasas fed the base pollen diet treated with 15 µg the of and potassium arsenate/g of isdiet. The results demonstratedregulatory that the test substanceowner. treatment a document (100 µg Cry2Ab2/g of diet)any produced such a mean survival rate of 91%its for Orius insidiosus, reproductiondocument which was similar to the survival ratesowner of the bufferthis control groupof (81%) and the assay control group (88%). Statistical analysisunder detectedof no significant differences in the rightsthe this survival percentagedocument and/orbetween the Cry2Ab2 protein treatment,rights buffer control and assay to of fall use of control treatments. The percent survival for the Oriusthe exposed to the potassium arsenate positive Thiscontrol group was 40%,may confirming distribution,and the validity of the test system. The percent rights owner of nymphs that subjectdeveloped to adults for the Cry2Ab2 treatment, buffer control, and assay control treatments was 93%, 91%, and 95%,the reviolatespectively. In conclusion, the NOEC of be copy Cry2Ab2 to Orius insidiosus was ≥100 ofµg/g of diet. and may documentpublication, It exploitation 11. Evaluationthis of Potential Effects of Exposure to Cry1A.105 Protein in an Acute Study with the anyParasitic Wasp, Ichneumon promissorius (Hymenoptera: permission Ichneumonidae). In the summaryprohibited of this study for the petition, explain what the safety factor is based on (e.g., 10X expression in pollen). the be commercial Furthermore,any 2006. Evaluation of Potential Effects of ExposureConsequently, to withoutCry1A.105 Protein in an Acute Study with the Parasitic Wasp, Ichneumon promissorius (Hymenoptera: Ichneumonidae). Monsanto Technical Report MSL-20149.

The objective of this study was to evaluate the potential effects of acute exposure to the Cry1A.105 protein administered to the non-target parasitic wasp, Ichneumon promissorius. Parasitic wasps were exposed to the test substance Cry1A.105 protein at a concentration of 240 µg/ml in a 30% sucrose solution diet, which resulted in a safety factor of approximately 27X based on the maximum Cry1A.105 protein expression level

Monsanto Company 06-CR-166U Addendum Page 7 of 14 (8.8 µg/g fwt) in pollen from MON 89034. In addition, appropriate negative controls (25 mM CAPS buffer in 30% sucrose and 30% sucrose alone) and positive controls (100 and 400 µg/ml potassium arsenate in 30% sucrose) were included in the study. The results showed that at test termination on Day 21, the mortality in the assay control group (30% sucrose alone), control substance group (25 mM CAPS buffer in 30% sucrose), and the Cry1A.105 protein treatment group was 10%, 8%, and 7%, respectively. There was no significant difference (p>0.05) in mean mortality between the Cry1A.105 protein treatment group and the control substance group. All surviving wasps were normal in appearance and behavior. The mortality rates for the positive controls of 100 and 400 µg/ml potassium arsenate in 30% sucrose were 70% and 97%, respectively, confirming the validity of the test system. In conclusion, the NOEC of the Cry1A.105 protein for the parasitic wasp, Ichneumon promissorius, was ≥240 µg/ml. regime.and and AG 12. Evaluation of Potential Effects of Exposure to Cry2Ab2 Protein in an Acute Study with the Parasitic Wasp, Ichneumon promissorius (Hymenoptera: Ichneumonidae). In the summary of thisBayer study forproperty the petition, protectionpublishing explain what the safety factor is based on (e.g., 10X expressionof in pollen). contents its parties.data therefore 2006. Evaluationand/oror of Potential Effects affiliates. of Exposure to Cry2Ab2 Proteinproperty in an Acuteintellectual Studythird with the Parasiticmay Wasp, Ichneumon its promissorius (Hymenoptera: Ichneumonidaeas ). Monsanto Technical Report MSL-20148. the of and is regulatory owner. a document The objective of this study anywas tosuch evaluate the potential effects of itsacute exposure to the reproductiondocument Cry2Ab2 protein administered to theowner non-target parasiticthis wasp, Ichneumonof promissorius. Parasitic wasps were exposed to the testunder substance of Cry2Ab2 protein at a concentration of rightsthe this 100 µg/ml indocument a 30%and/or sucrose solution diet, which resultedrights in a safety factor of to of fall use of approximately 213X based on the maximum Cry2Ab2the protein expression level (0.47 µg/g fwt)This in pollen from MONmay 89034. distribution, Inand add ition, appropriate negative controls (50 mM rights owner CAPS buffer in subject30% sucrose and 30% sucrose alone) and positive controls (100 and 400 µg/ml potassium arsenate in 30% sucrose)the wereviolate included in the study. The results be copy showed that at test termination on Day of21, the mortality in the assay control group (30% and sucrose mayalone), controldocument substancepublication, group (50 mM CAPS buffer in 30% sucrose), and the It exploitation Cry2Ab2 proteinthis treatment group was 10%, 3%, and 3%, respectively. There was no significant differenceany (p>0.05) in mean mortality between the Cry2Ab2 protein treatment permission group and the control substanceprohibited group. All surviving wasps were normal in appearance and behavior. The mortality rates for the positive controls of 100 and 400 µg/ml the be potassium arsenatecommercial in 30% sucrose were 70% and 97%, respectively, confirming the validityFurthermore, of theany test system. In conclusion, the NOEC of the Cry2Ab2 protein for the parasiticConsequently, wasp, withoutIchneumon promissorius, was ≥100 µg/ml.

13. Evaluation of Potential Dietary Effects of MON 89034 with the Northern Bobwhite: an Eight-day Dietary Study with Corn Grain. An explanation of how this study can be used in a risk assessment for migratory birds should be included.

Monsanto Company 06-CR-166U Addendum Page 8 of 14 2006. Evaluation of Potential Dietary Effects of MON 89034 with the Northern Bobwhite: an Eight-day Dietary Study with Corn Grain. Monsanto Technical Report WL-2005-012.

The objective of this study was to examine the potential of effects from a dietary exposure to corn grain from MON 89034 to bobwhite quail (Colinus virginianus). Bobwhites are a commonly used surrogate species to develop data on dietary toxicity and are one of EPA’s preferred test species. The test procedure followed the methodology of U.S. EPA ecological effects test guideline OPPTS Guideline Number 850.2200, which provides specific guidance for testing bobwhite. Groups of 30 bobwhite quail, 10 days of age, were fed basal diets (with the corn component removed) mixed with either finely ground test, control, or reference corn grain at a limit dose of 50% by weight of the diet regime.and for eight days. The control substance was a corn grain from a andconvent ional corn line that AG has a similar genetic background to MON 89034, and the reference substances were three conventional corn varieties. The results showed that there was no mortality in the test group fed diet containing 50% MON 89034,Bayer and allproperty the birds protection in thepublishing test group were normal in appearance and behavior throughoutof the test. There was a singlecontents incidental its mortality in the control substance group and in two ofparties. the datare ference groups.therefore There were no statistically significant (p>0.05) treatment-related effects and/oron bodyor weight and feed affiliates. consumption among birds fedproperty MON 89034intellectual whenthird compared with themay control group. its Therefore, the 8-day dietary NOEC wasas determined to be ≥ 50% corn grain from MON the of and 89034. is regulatory owner. a document any such its reproductiondocument The results from the short-term studyowner with bobwhitethis demonstrate of that no significant risk to wild avian species is anticipated fromunder consumption of of grain from MON 89034. This rightsthe this study is considereddocument and/orto be acceptable for assessing short-termrights risk to wild migratory bird to of fall use of populations because: 1) the study followed accepted themethodology for assessing short- term riskThis to wild avian populatmayions distribution, and and2) juvenile birds were tested at a high dietary rights owner level of corn grainsubject from MON 89034. be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Addendum Page 9 of 14

Update on USDA Notification Trials

In the Agency’s letter, it was stated that “All field test reports for trials terminated six months or more ago should be submitted. Appendix H: USDA Notifications Approved for MON 89034 in the petition should be amended to indicate that all field test reports that are due have been submitted particularly those from 2005 that are outstanding”.

Table H-1 has been amended to include the most recent updated trial status as of January 23, 2007 (the Amended Table H-1 is attached, starting from the next page). All field test reports that are due have been submitted to the USDA. The USDA regulations specify that a field test report be submitted to the agency within 6 months after regime.and termination of the field test. We have noted the most recent guidanceand on the USDA web AG site, USDA-APHIS Biotechnology Regulatory Services User’s Guide, “Chapter 6, Notification” which states that APHIS considers the field test report to be due no later than 6 months after expiration of the notification.Bayer propertyMonsanto protectionfollowspublishing this most recent guidance on the due date of field test reports.of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Addendum Page 10 of 14 Amended Table H-1. USDA notifications approved for MON 89034 and status of trials conducted under these notifications.

USDA No. Effective Date (m/d/y) Release Site (State) Trial/Report Status 2001 Field Trials 01-348-04n 1/13/2002 PR (2) Submitted to USDA 01-348-07n 1/11/2002 HI (6) Submitted to USDA

2002 Field Trials 02-009-12n 2/8/2002 IL (3) Submitted to USDA 02-009-15n 2/8/2002 IA (6) Submitted to USDA 02-017-12n 3/20/2002 AL, MS, OR, TN, WA Submitted to USDA and 02-242-10n 10/1/2002 PR (2) and Submittedregime. to USDA AG 2003 Field Trials 03-024-06n 2/23/2003 AL MS Submitted to USDA Bayer property protectionpublishing 03-034-25n 3/5/2003 of OR, TN (2) Submittedcontents to USDA 03-034-27n 3/5/2003 IL (2) itsSubmitted to USDA parties.data therefore 03-042-06n 3/13/2003 MO and/oror Submitted to USDA 03-043-04n 3/17/2003 affiliates. IL (11) Submitted to USDA property intellectualthird may 03-043-05n 3/17/2003its IA (11) Submitted to USDA 03-052-57n the3/23/2003 as TN Submitted to USDA of and owner. 03-161-05n is 8/5/2003 HI (5)regulatory Submitted to USDA a document 03-219-04n any9/5/2003 such PR (2) its Submitted to USDA reproductiondocument 03-265-09n 11/21/2003owner HI (6) this of Submitted to USDA under of 03-280-01n rights11/6/2003the PR (3) this Submitted to USDA documentand/or rights to of fall use of 2004 Field Trials the 04-009-01nThis 3/25/2004may distribution, and HI (5) Submitted to USDA rights owner 04-009-02n subject 3/25/2004 PR (4) Submitted to USDA 04-014-05n be 2/26/2004 the GA violate Submitted to USDA 04-014-08n copy 2/13/2004 of TN (2) Submitted to USDA and 04-021-02nmay documentpublication,2/20/2004 AL, MS Submitted to USDA It exploitation IA (2), IL (4), IN, KS, NE, 04-021-09n 2/20/2004 Submitted to USDA this any OH 04-021-11n 2/20/2004 AR Submitted to USDA permission 04-021-14n 2/20/2004prohibited TN Submitted to USDA the 04-023-09n commercial2/25/2004 be IA (12) Submitted to USDA 04-023-10n 2/25/2004 IA (8) Submitted to USDA Furthermore,04-023-11n any 2/25/2004 WI (3) Submitted to USDA 04-028-10nConsequently, without 3/24/2004 MN (5) Submitted to USDA 04-028-23n 2/27/2004 TN Submitted to USDA 04-028-24n 2/27/2004 CO (3), NE (4) Submitted to USDA 04-028-25n 2/27/2004 IA (4) Submitted to USDA 04-028-26n 2/27/2004 OR (2), WA Submitted to USDA 04-028-27n 2/27/2004 IL (15) Submitted to USDA 04-028-28n 2/27/2004 KS (2) Submitted to USDA Amended Table H-1 continues on next page.

Monsanto Company 06-CR-166U Addendum Page 11 of 14 Amended Table H-1 (continued). USDA notifications approved for MON 89034 and status of trials conducted under these notifications.

USDA No. Effective Date (m/d/y) Release Site (State) Trial/Report Status 04-028-29n 2/27/2004 IN (4) Submitted to USDA 04-028-30n 2/27/2004 MI (2) Submitted to USDA 04-028-31n 2/27/2004 MO (2) Submitted to USDA 04-028-32n 2/27/2004 NE (4) Submitted to USDA 04-028-33n 2/27/2004 OH (2) Submitted to USDA 04-040-01n 4/2/2004 MD (2) Submitted to USDA 04-040-02n 3/12/2004 NE Submitted to USDA 04-040-03n 3/10/2004 IL (6) Submitted to USDA and 04-041-03n 3/11/2004 IL (10) and Submittedregime. to USDA 04-041-04n 3/11/2004 KS (2)AG Submitted to USDA 04-044-07n 3/14/2004 IA (6) Submitted to USDA 04-056-06n 3/26/2004 IA (2), IL (2), IN, MO (3) Submitted to USDA Bayer property protectionpublishing 04-079-02n 4/26/2004 of IN, MN, MO, WI Submittedcontents to USDA 04-085-15n 4/24/2004 NE (3) itsSubmitted to USDA parties.data therefore 04-090-03n 6/18/2004 HI (5) and/oror Submitted to USDA 04-093-03n 5/2/2004 affiliates. IL Submitted to USDA property intellectualthird may 04-093-04n 5/2/2004its IA Submitted to USDA 04-189-03n the9/27/2004 as HI (15) Submitted to USDA of and owner. 04-217-02n is 9/20/2004 PR (2)regulatory Submitted to USDA a document 04-231-03n any9/27/2004 such PR (3) its Submitted to USDA reproductiondocument 04-260-01n 11/1/2004owner HI this of Submitted to USDA under of 04-273-01n rights10/18/2004the IA, IL (2), NEthis Submitted to USDA 04-274-08n documentand/or 10/28/2004 PR (2) rights Submitted to USDA to of fall use of 04-309-03n 1/10/2005 HI (9) the Submitted to USDA 04-309-04nThis 1/10/2005may distribution, and PR (3) Submitted to USDA rights owner 04-337-02n subject 12/16/2004 MS Submitted to USDA 04-337-03n be 12/20/2004 the AL violate Submitted to USDA 04-358-20n copy 1/25/2005 of GA (2) Submitted to USDA and 04-358-21nmay documentpublication,1/24/2005 IA (14) Submitted to USDA 04-358-22nIt 1/24/2005exploitation IA (15) Submitted to USDA this 04-358-23n any 1/24/2005 IL (11) Submitted to USDA 04-358-24n permission1/25/2005 IN (3) Submitted to USDA 04-358-25n 1/24/2005prohibited KS (2) Submitted to USDA the 04-358-26n commercial be2/7/2005 MD Submitted to USDA 04-358-27n 1/26/2005 MI (3) Submitted to USDA Furthermore,04-358-28n any 2/9/2005 MN (8) Submitted to USDA 04-358-29nConsequently, without 1/24/2005 MO (2) Submitted to USDA 04-358-30n 1/25/2005 NE (5) Submitted to USDA 04-358-31n 1/25/2005 OH (2) Submitted to USDA 04-358-32n 1/27/2005 TN (2) Submitted to USDA 04-358-33n 1/25/2005 WI (3) Submitted to USDA 04-362-09n 1/18/2005 TN (3) Submitted to USDA 04-362-11n 1/18/2005 AR Submitted to USDA Amended Table H-1 continues on next page.

Monsanto Company 06-CR-166U Addendum Page 12 of 14 Amended Table H-1 (continued). USDA notifications approved for MON 89034 and status of trials conducted under these notifications.

USDA No. Effective Date (m/d/y) Release Site (State) Trial/Report Status 2005 Field Trials IA, IL (3), IN, KS, NE, OH 05-025-04n 2/22/2005 Submitted to USDA (2) 05-026-19n 2/14/2005 IL (8), IN, WI (3) Submitted to USDA 05-028-02n 2/14/2005 CO (5), KS, NE (3) Submitted to USDA 05-028-11n 2/22/2005 IA (4), IL (9) Submitted to USDA 05-028-12n 2/22/2005 HI (4) Submitted to USDA Submitted to USDA 05-039-09n 3/10/2005 NE, OR, WA regime.and IA (2) IL (2), IN (3), MI,and Submitted to USDA 05-039-11n 3/28/2005 MN, MOAG (2), NE, NY, WI 05-040-15n 3/10/2005 LA Submitted to USDA BayerIA (5), IL (2), IN (2), NE (4), Submitted to USDA 05-049-13n 3/24/2005 property protectionpublishing of TX (2) contents 05-055-03n 4/11/2005 IL (2), MN (5), MO (2) Submittedits to USDA parties.data therefore 05-056-10n 4/1/2005 MO and/oror Submitted to USDA 05-063-06n 4/1/2005 affiliates. IL (2) Submitted to USDA property intellectualthird may 05-104-03n 5/17/2005its HI (10) Submitted to USDA as 05-129-01n the5/31/2005 of andNE (6) Submitted to USDA is regulatory owner. 05-131-01n 6/9/2005 LAa documentits Submitted to USDA 05-133-05n 7/20/2005any such HI (4),reproduction PR (2) Submitted to USDA this documentof 05-201-06n 9/26/2005 owner PR (3) In progress* under of 05-201-10n rights9/6/2005the HI (5) this In Progress* documentand/or rights 05-201-11n to 9/26/2005of fall PRuse (3) of In Progress* the This may distribution,and 2006 Field Trials rights owner 06-025-04n subject 3/30/2006 AL, AR, LA, MS (2), TN (2) In Progress the violate 06-027-06n be copy 2/27/2006 GA (4) In Progress of In Progress 06-027-07n 3/6/2006 and OH (2) 06-027-08nmay documentpublication,3/6/2006 WI (3) In Progress It exploitation 06-027-09n this 3/7/2006 MN (9) In Progress 06-027-11n any 3/1/2006 MI (3) In Progress 06-027-12n 2/27/2006permission NE (4) In Progress prohibited In Progress 06-027-13n the 2/27/2006 MO (2) 06-027-14n commercial2/27/2006 be TN (4) In Progress 06-027-15n 2/27/2006 KS (2) In Progress Furthermore,any In Progress 06-027-17nConsequently, without 4/5/2006 IA (5) 06-030-01n 2/28/2006 IN (4) In Progress 06-030-02n 2/28/2006 IL (13) In Progress 06-030-04n 3/7/2006 IA (12) In Progress 06-030-05n 3/7/2006 IA (13) In Progress 06-030-06n 2/28/2006 IL (12) In Progress 06-031-25n 2/28/2006 NE (2) In Progress 06-032-12n 4/5/2006 IA (12) In Progress Amended Table H-1 continues on next page.

Monsanto Company 06-CR-166U Addendum Page 13 of 14 Amended Table H-1 (continued). USDA notifications approved for MON 89034 and status of trials conducted under these notifications.

USDA No. Effective Date (m/d/y) Release Site (State) Trial/Report Status 06-033-07n 5/18/2006 HI (7) In Progress 06-037-08n 5/18/2006 HI (5) In Progress 06-037-10n 5/26/2006 PR (3) In Progress 06-037-11n 3/16/2006 PR (3) In Progress 06-038-05n 4/6/2006 IA, IL (3), IN (2), TX (3) In Progress 06-046-06n 3/23/2006 CO (5), NE (2) In Progress 06-046-10n 3/16/2006 IL (4), MO (5) In Progress 06-046-11n 4/17/2006 IA (8), NE (6), SD In Progress and 06-052-11n 3/23/2006 MS and In Progressregime. 06-060-09n 4/24/2006 IA (2) AG In Progress 06-060-10n 4/24/2006 IA (14) In Progress IA (3), IL (4), IN (2), KS, In Progress 06-073-01n 6/12/2006 Bayer MN property protectionpublishing of contents 06-073-02n 5/5/2006 IA (7), IL (5), NE, OH (2) In Progress its 06-081-02n 5/9/2006 IA parties.data In Progresstherefore and/oror 06-082-05n 5/18/2006 HI (3), PR (2) In Progress affiliates. 06-089-11n 5/15/2006property intellectual IA (5),third IL, NE Inmay Progress its 06-090-04n 5/18/2006 as HI (6) In Progress the of and 06-090-05n is6/12/2006 PR (3)regulatory Inowner. Progress a document 06-090-06n 5/18/2006any such HI (5) itsIn Progress reproductiondocument 06-090-07n 5/26/2006 owner PR (3) this of In Progress 06-108-03n 5/26/2006 PR of In Progress rights under this 06-108-04n and/or5/26/2006 the HI, IN (2), PR In Progress document to of fall of rights 06-108-05n 5/18/2006 HI, PRuse (2) In Progress the 06-108-06nThis 5/26/2006may distribution,and HI, IN In Progress rights owner In Progress 06-114-03n subject 5/26/2006 HI, IL 06-118-02n 5/9/2006 IA In Progress be the violate ______copy of * It is noted that as of January 23, 2007, when andthis addendum is made, there are three notifications for may documentpublication, 2005It field trials of which the exploitationreports have not been submitted. Among the three notifications, no plantings occurredthis under any 05-201-06n, and a field test report will be submitted to that effect. The two field trials under notifications 05-210-10n and 05-201-11n are still in progress. These two notifications permission involve field trials in continuous plantingprohibited situations in Hawaii and Puerto Rico. We plan to submit these reports prior to the due datethe as defined by 6 months after notification expiration (as shown in the table below). commercial be USDA No. Trial/Report Notification Notification 6 months 6 months Furthermore,any Status effective date Expiration after after Consequently,without termination notification dateδ expiration 05-201-06n In progress 9/26/2005 9/26/2006 Not planted 3/26/2007 05-201-10n In progress 9/6/2005 9/6/2006 2/28/2007 3/6/2007 05-201-11n In progress 9/26/2005 9/26/2006 3/11/2007 3/26/2007 δ Termination date refers to the destruction of the last plants in the trials.

Monsanto Company 06-CR-166U Addendum Page 14 of 14 and and regime. AG

Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without “major Federal action” to include actions with effects that may be major and which are potentially subject to Federal control and responsibility.3 Once it is clear that a proposed action triggers the requirement of NEPA and is not categorically excluded, the federal agency must decide what NEPA document to prepare. The CEQ regulations direct agencies to determine whether, under the agency’s own supplemental procedures, the proposal is one which “normally requires an environmental impact statement.” If so, an environmental impact statement (EIS) is prepared. If not, the agency prepares an environmental assessment (EA).4

We believe an EA would satisfy the NEPA requirement for the determination of non- regulated status for MON 89034 because the action is “characterized by its limited scope (particular sites, species, or activities) and potential effect (impacting relatively few regime.and environmental values or systems). Individuals and systems that mayand be affected can be AG identified. Methodologies, strategies, and techniques employed to deal with the issues at hand are seldom new or untested. Alternative means of dealing with those issues are well established. Mitigation measures are generallyBayer available property and have protection beenpublishing successfully employed.”5 of contents its parties.data therefore Our understanding is that an EA leads to one of two determinations;and/or the orfirst possibility is affiliates. that the proposed action will not resultproperty in si gnificantintellectualthird impacts to the humanmay environment, its in which case a “Finding of No Signifiascant Impact” (FONSI) is prepared which the of and documents the agency’s rationaleis for its decision.regulatory The second possibilityowner. is that the a document proposed action will result in anysignificant such impacts to the human environment,its in which reproductiondocument case the agency proceeds to prepare owneran EIS to informthis its decision-makingof on the proposed action. under of rightsthe this documentand/or rights to of fall use of Monsanto believes the Petition and the 38 volumes of thethe attachment that Monsanto submitted toThis APHIS on October 25,may 2006, distribution, theand addendum submitted on January 23, 2007, rights owner and the addendum subjectsubmitted herein collectively provide substantial data, information, and evidence that MON 89034 will not havethe anyviolate significant impact on the human be copy environment, i.e., a finding of no significantof impact or FONSI. and may documentpublication, It exploitation Our conclusion thatthis there are no significant environmental impacts resulting from the deregulation of MON any89034 is based on the comprehensive assessments which have permission taken into consideration both the “context”prohibited and “intensity” that deregulation of MON 89034 may have on the human environment. The term “context” refers to the setting the be within which the proposedcommercial action takes place. We have considered the potential impacts of MONFurthermore, 89034 anyon the human environment in the following context: Consequently,without (i) The affected crop: the biology and genetics of corn, pollination and outcrossing characteristics and gene flow potential, and assessment of weediness and plant pest potential (Petition sections II, VIII.B, and VIII.C).

3 40 C.F.R. § 1508.18. 4 40 C.F.R. § 1501.4. 5 7 C.F.R. § 372.5

Letter to USDA, May 18, 2007 Page 2 of 8 Monsanto Company file No. 06-CR-166U (ii) The target insect pests, non-target organisms, and threatened and endangered species: (Petition sections VIII.A.2 to VIII.A.5; addendum submitted on January 23, 2007; points No. 1 and 2 of the current addendum). (iii) Affected agricultural practices: (Petition sections VIII.D.2, VIII.E, and VIII.F). (iv) Affected regions and locality: farms in all states where corn is grown (Petition section VIII.D.1). (v) Affected interests: the interests of U.S. farmers (field corn growers, specialty corn growers, organic corn growers, and farmers specializing in other major crops such as soybeans and cotton), poultry and dairy farmers who use corn as animal feeds, and grain handlers and traders (Petition section I.C; points No. 4 and 5 of the current addendum). (vi) Land use and agricultural production: U.S. cropland and uses for major crops; regime.and supply and demand of corn grain for utilizations as food,and feed, production of AG ethanol for fuel, and for export (point No. 5 of the current addendum; Petition section I.C). Bayer property protectionpublishing With respect to the intensity element, CEQof regulations provide ten factors tocontents guide the 6 7 its analysis. The term “intensity” refers to the severity of parties.the impact.data We have thereforetaken into consideration the potential direct, indirect, and cumulative impactsand/or of theor deregulation of affiliates. MON 89034 on the human environment.property intellectualthird may its as the of and (1) Impacts that may be beneficialis or adverse withregulatory respect to the use of MONowner. 89034 in a document the U.S. any such its reproductiondocument owner this of a. MON 89034 is expected to protectunder corn yieldsof against damage caused by rightsthe this lepidopterandocument insectand/or pests, thus increasing cropland productivrights ity and increasing to of fall use of farmers’ income (point No. 5 of the current addendum).the b. MONThis 89034 is expected tomay reduce distribution, chemicaland pesticide use in corn and reduce the rights owner environmentalsubject impact of the conventional agricultural production of corn (point No. 5 of the current addendum). the violate be copy c. MON 89034 is expected to reduceof mycotoxin levels commonly found in and conventionalmay corndocument grain.publication, Lower levels of mycotoxin in the grain are beneficial to It exploitation human andthis animal health (Petition section I.C.; point No. 5 of the current addendum). any permission d. MON 89034 is expected toprohibited provide an effective insect resistance management (IRM) tool and increase the durability of Bt corn technology (Petition sections I.C. the be and VIII.F; pointscommercial No. 1 and 6 of the current addendum). e.Furthermore, MON 89034any is not likely to have any impact on the weediness potential of corn andConsequently, raises nowithout concerns of ourcrossing with wild relatives of corn in the U.S.; and unintended cross pollination between corn varieties targeted for different market segments is manageable under the well-established agricultural practices and marketing standards in the U.S. (Petition sections II, VIII.B and VIII.C; point No. 4 of the current addendum).

6 40 C.F.R. § 1508.27. 7 40 C.F.R. § 1508.27(b).

Letter to USDA, May 18, 2007 Page 3 of 8 Monsanto Company file No. 06-CR-166U f. MON 89034 is not expected to have any adverse impact on current agricultural practices (Petition section VIII.D; point No. 5 of the current addendum). g. MON 89034 is not likely to pose any risk to non-target organisms, and endangered and threatened species (Petition sections VIII.A.2 to VIII.A.5; addendum submitted January 23, 2007; points No. 1 and 2 of the current addendum). MON 89034 contains two insecticidal proteins, therefore, this product is also subject to regulatory review and oversight by EPA. Monsanto has filed applications to the EPA for the registration of the plant-incorporated protectants Cry1A.105 and Cry2Ab2 proteins, and the genetic material (vector PV-ZMIR245) necessary for their production. EPA registration processes include extensive assessments regarding potential effects of MON 89034 on non-target organisms, and threatened and endangered species. A copy of Monsanto’s regime.and submission to EPA was provided to APHIS as an attachmentand (38 volumes in total) AG to the Petition on October 25, 2006. It is our understanding that APHIS consults with EPA and confirms EPA’s regulatory responsibility with respect to assessment of potential effects of MONBayer 89034 onproperty non-target protection organismspublishing including threatened and endangered species. of contents its parties.data therefore (2) The degree to which the proposed action affects public healthand/or or safety.or affiliates. property intellectualthird may its a. MON 89034 has no potential adverseas effect on human and animal health. The the of and food and feed derivedis from MON 89034 areregulatory at least as safe and nutritiousowner. as those a document derived from corn varietiesany currentlysuch in commerce. MON 89034its is expected to reproductiondocument reduce mycotoxin levels in cornowner grain and therebythis provideof added benefit to human and animal health comparedunder to c onventionalof this corn grain (Petition section and/orrights the I.C.; pointdocument No. 5 of the current falladdendum ). Underof therights “Coordinated Framework to of 8 use for the Regulation of Biotechnology” , the responsibilitiesthe of regulatory oversight of biotechnology-derivedThis maycrops distribution,fall andon three lead federal agencies, including rights owner USDA, EPA,subject and FDA. Monsanto has submitted a premarket notification entitled “Food and Feed Safety and Nutritionalthe A ssessmentviolate of the Lepidopteran-Protected be copy Corn MON 89034” to the FDA. ofThe key conclusions of that assessment have and beenmay included indocument thepublication, Petit ion section VI.D. It is our understanding that APHIS It exploitation consults withthis FDA and confirms FDA’s regulatory responsibility over the food and feed safety anyassessment on MON 89034. FDA is responsible for all the food permission and feed safety issues exceptprohibited the safety of the two insecticidal proteins which falls into the jurisdiction of EPA. the be b. Monsanto hascommercial filed applications to the EPA for the registration of the plant- Furthermore,incorporatedany protectants Cry1A.105 and Cry2Ab2 proteins, and the genetic materialConsequently, (vectorwithout PV-ZMIR245) necessary for their production. In July 2006, EPA established a temporary exemption from the requirement of a tolerance for Cry1A.105 protein and the genetic material necessary for its production in all corn commodities (40 CFR §174.453). Likewise, temporary exemption from the requirement of a tolerance for Cry2Ab2 protein and the genetic material necessary for its production in corn was also granted by EPA in July 2006 (40 CFR

8 Federal Register. 1986. Coordinated framework for regulation of biotechnology; announcement of policy. 1986 June 26; 51(123):23302-50.

Letter to USDA, May 18, 2007 Page 4 of 8 Monsanto Company file No. 06-CR-166U §174.454). Monsanto is petitioning EPA to amend 40 CFR §174.453 and 40 CFR §174.454 by removing the time limitation for the exemption from the requirement of a tolerance for the Cry1A.105 and Cry2Ab2 proteins. It is expected that the EPA review will conclude with the registration of the MON 89034 and the issuance of tolerance exemption for the two proteins relative to the food and feed safety.

(3) Unique characteristics of the geographic area such as proximity to historic or cultural resources, park lands, prime farmlands, wetlands, wild and scenic rivers, or ecologically critical areas.

Like any other corn variety, MON 89034 will be grown in existing cropland regime.and (point No. 5 of the current addendum). As such, the plantingand of MON 89034 is AG not likely to affect historic or cultural resources, park lands, wetlands, wild and scenic rivers, or ecologically critical areas in any way different from the cultivation of current corn varieties. BayerMON 89034property is substantially protectionpublishing equivalent to conventional corn except for the lepidopterof an protection trait (Petitioncontents section its VII). MON 89034 has no plant pest risk comparedparties. to conventionaldata corntherefore (Petition section VIII.B). MON 89034 poses no significant risk to and/orendangeredor butterflies affiliates. and moths in the order Lepidopteraproperty (Petitionintellectualthird section VIII.A.4). may its as the of and (4) The degree to which theis effects on the qualityregulatory of the human environmentowner. are likely a document to be highly controversial.any such its reproductiondocument owner this of The term “controversial” as used inunder the NEPA ofcontext, this based on CEQ guidelines, and/orrights the refers notdocument to the amount of publicfall opposition, butof to whererights there is a substantial to of use 9 dispute as to the size, nature, or effect of the majorthe federal action. Although thereThis is general oppositionmay fromdistribution, andcerta in groups or individuals to plant rights owner biotechnologysubject in general, there is no substantial dispute as to the size, nature, or effect of MON 89034 corn on the humanthe violateenvironment based on scientific data be copy and evidence. of and may documentpublication, It exploitation (5) The degree tothis which the possible effects on the human environment are highly uncertain or involveany uniq ue or unknown risks. permission prohibited MON 89034 is a lepidopteran insect protected Bt corn product. Similar Bt corn the be products (suchcommercial as events MON 810, line 1507, and Bt11) have completed the Furthermore,regulatoryany processes by EPA, FDA, and USDA, and have been successfully cultivatedConsequently, bywithout farmers in the U.S. and other countries in large areas for nearly a decade. In 2006, 40% of corn acreage in the U.S. was planted with corn varieties possessing Bt traits, including lepidopteran protection or coleopteran protection, or the combined Bt trait products. The cultivation and consumption records and experiences of Bt corn have shown, with a great degree of certainty, significant beneficial impacts and no adverse impacts on human and animal health and the environment. Similar conclusions of no significant adverse impacts on human

9 Hanley v. Kleindienst, 471 F.2d 823 (2d Cir. 1972), cert. denied, 412 U.S. 908 (1973).

Letter to USDA, May 18, 2007 Page 5 of 8 Monsanto Company file No. 06-CR-166U environment have been reached for MON 89034 [Petition sections VI, VII, and V.III; points No. 5 of the current addendum; also refer to the above points (1) and (2)].

(6) The degree to which the action may establish a precedent for future actions with significant effects or represents a decision in principle about a future consideration.

The review, approval and deregulation of biotechnology-derived crops have been conducted on case-by-case basis. There have been 71 deregulations by USDA- APHIS from 1992 to April 2007.10 There is no indication that APHIS or any other federal agencies intend to use deregulation of MON 89034 as a precedent for future biotechnology-derived crop deregulations or registrations. regime.and and AG (7) Whether the action is related to other actions with individually insignificant but cumulatively significant impacts. Significance exists if it is reasonable to anticipate a cumulatively significant impact on theBayer environment. property Significance protectionpublishing cannot be avoided by terming an action temporof ary or by breaking it down contentsinto small its component parts. parties.data therefore and/oror affiliates. "Cumulative impact" is theproperty impact onintellectual thethird environment which resultsmay from the its incremental impact of the actionas when added to other past, present, and the of and reasonably foreseeableis future actions regardlessregulatory of what agency (Federalowner. or non- a document Federal) or person undertakesany such such other actions. Cumulative itsimpacts can result reproductiondocument from individually minor but collectivelyowner significantthis actions oftaking place over a period of time.11 The attached addendumunder provides,of as indicated below, additional rightsthe this assessmentsdocument regardingand/or potential cumulative impacts ofrights MON 89034, taking into to of fall use of considerations other biotechnology-derived crops thederegulated and cultivated in the pastThis and present, and thosmaye likelydistribution, toand be cultivated in the future (points No. 1, 2, rights owner 3, and 4 ofsubject the current addendum). These assessments concluded that MON 89034 is unlikely to pose a significantthe violateimpact on the human environment be copy considering the past, present, and oflikely future deregulations and approvals of and biotechnology-derivedmay documentpublication, crops. It exploitation this (8) The degree to whichany the action may adversely affect districts, sites, highways, permission structures, or objects listed inprohibited or eligib le for listing in the National Register of Historic Places or may cause loss or destruction of significant scientific, cultural, or the be historical resources.commercial Furthermore,any LikeConsequently, any otherwithout corn variety, MON 89034 will be grown on existing cropland (point No. 5 of the current addendum). As such, it is not likely to affect districts, sites, highways, structures, or objects listed in or eligible for listing in the National Register of Historic Places or may cause loss or destruction of significant scientific, cultural, or historical resources.

10 Petitions of Nonregulated Status Granted or Pending by APHIS. http://www.aphis.usda.gov/brs/not_reg.html 11 40 C.F.R. § 1508.7

Letter to USDA, May 18, 2007 Page 6 of 8 Monsanto Company file No. 06-CR-166U

(9) The degree to which the action may adversely affect an endangered or threatened species or its habitat that has been determined to be critical under the Endangered Species Act of 1973.

The potential risks to non-target organisms, and threatened and endangered species by the introduction of MON 89034 has been assessed by Monsanto and the assessment has been submitted to EPA for review. A summary of the assessment have been included in the Petition section VIII.A. It was concluded that MON 89034 poses no significant risk to these organisms. This conclusion is consistent with other approved Bt corn products. Substantial number of studies conducted over the past decade by industry and the academic community on regime.and registered and deregulated insect-protected crops that produceand a variety of Cry AG proteins have demonstrated that these crops have no adverse effects on biodiversity, tested populations of natural enemies, and other ecologically important non-target arthropods. ImportanBayertly, whenproperty expressed protection inpublishing Bt crops, even sensitive non-target lepidopteran andof coleopteran species have been showncontents to be its exposed to very low levels of the Cry proteins. Theparties. exposuredata levels weretherefore too low to pose a significant risk to populations of the sensitiveand/or non-targetor species affiliates. including threatened and endangeredproperty speciintellectualesthird (Petition sections VIII.A3,may VIII.A.4, its and VIII.A5). as the of and is regulatory owner. a document (10) Whether the action threatensany such a violation of Federal, State, itsor local law or reproductiondocument requirements imposed for the protectionowner of the environment.this of under of rightsthe this There is nodocument indicationand/or that this action would violate Federarightsl, State, or local law or to of fall use of requirements imposed for the protection of thethe environment. Concurrent jurisdictionsThis exist among femayderal distribution, agencies,and namely, USDA, EPA, and FDA under rights owner the “Coordinatedsubject Framework for the Regulation of Biotechnology” regarding the regulation of biotechnology-derived crops.the violate As mentioned above, Monsanto has be copy submitted registration or premarketof consul tation documents to EPA and FDA, in and additionmay to the Petitiondocumentpublication, to USDA-APHIS (point No. 6 of the current addendum). It exploitation Furthermore,this a number of states have statutes or regulations on the cultivation and environmental releaseany of biotechnology-derived insect protected crops, such as Bt permission corn. Monsanto will seek registrationsprohibited and approvals from those states in which it plans to market MON 89034 prior to the commercialization. Deregulation of the be MON 89034commercial by USDA does not serve as a substitute for the approval or Furthermore,registrationany requirement of other federal or state agencies but constitutes an essentialConsequently, elementwithout towards overall regulatory compliance for this product.

In conclusion, the Petition, the prior submitted addendum, and the addendum attached herein, together, provide substantial evidence that MON 89034 is not likely to pose any significant impact on the human environment. Monsanto requests a determination from APHIS that MON 89034 and any progeny derived from crosses between MON 89034 and other commercial corn varieties be granted non-regulated status.

Letter to USDA, May 18, 2007 Page 7 of 8 Monsanto Company file No. 06-CR-166U and and regime. AG

May 18, 2007

Table of Contents

Summary...... 2 1. Assessment of Potential Cumulative Impact of MON 89034 with other Bt Crops on Insect Pest Resistance Development and Evolution ...... 5 regime.and 2. Assessment of Potential Cumulative Impact of Deregulationand of MON 89034 on Bt AG Protein Accumulation in Soil Environment...... 6 3. Assessment of Potential Cumulative Impact of MON 89034 in Combined Trait Products Resulted from Conventional BreedBayering withproperty other Biotechnology-Derivedprotectionpublishing Products...... of contents .. 9 its 4. Assessment of Potential Impact of Deregulation ofparties. MONdata 89034 on Biodiversity,therefore Preservation of Corn Germplasm Purity, and Specialty Cornand/or Production...... or 10 affiliates. 5. Assessment of the Potentialproperty Impact of Deregulationintellectualthird of MON 89034 mayon Land Use and its Agricultural Production ...... as 14 the of and 6. Mitigation and Remediationis Measures beforeregulatory and after Registration owner.and Deregulation a document of MON 89034 in the U.S...... any such its 18 reproductiondocument References...... owner this of 20 under of rightsthe this Table 1. Certifieddocument organicand/or and total U.S. acreage of selectedrights crops (USDA-ERS, 2007).. to of fall use of ...... the 13 Table 2. ThisU.S. corn acreages, production,may distribution, vaandlue, supply, demand, and export shipment rights owner (USDA-NASS,subject 2007)...... 15 Table 3. Average annual U.S. corn utilizationthe duringviolate 2002-2006...... 16 be copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 1 of 26 Addendum to Section VIII (Environmental Assessment and Impact on Agronomic Practices) of USDA Petition No. 06-298-01p (i.e., Monsanto Company File No. 06- CR-166U) for Determination of Non-regulated Status for MON 89034

Summary

This addendum provides additional information and assessments to section VIII of Monsanto Company (Monsanto) File No. 06-CR-166U for the “Determination of Non- regulated Status for MON 89034” (Petition). Specifically, the addendum addresses the following topics: (1) potential cumulative impact of MON 89034 with other Bt crops on insect pest resistance development and evolution; (2) potential cumulative impactand of and regime. deregulation of MON 89034 on Bt protein accumulationAG in the soil environment; (3) potential cumulative impact of MON 89034 in combined trait products resulted from conventional breeding with other biotechnology-derived corn products; (4) potential impact of deregulation of MON 89034 onBayer biodi versity,property preservation protection publishingof corn germplasm purity, and specialty corn production; (5)of potential impact of deregulation contentsof MON 89034 its on land use and agricultural production; and (6) mitigationparties.data and remediationtherefore measures and/or before and after registration and deregulation of MON 89034 in the U.S.or affiliates. property intellectualthird may its The introduction of MON 89034 in theas U.S. is not expected to result in an increase in the the of and risk of resistance developmentis and evolution inregulatory the target pests to the owner.Cry proteins. This a document conclusion takes into considerationany such the potential cumulative impactits from Bt crops reproductiondocument approved in the past, present, and likelyowner to be introducedthis in the future.of MON 89034 will under of be introduced with an insectrights resistancethe management this(IRM) plan under the regulatory documentand/or rights oversight of EPA. Theto effectiveness of fall of this useplan willof be monitored through surveys of target pest susceptibility, and the implementation of thethe plan will be tracked through a ComplianceThis Assurance Programmay (CAP) distribution, thatand is co mparable to the CAP used for current Bt rights owner corn products. subject Adoption of MON 89034 should reduce the risk of the resistance evolution in lepidopteran pests towards Crythe proteinsviolate because MON 89034 will provide be copy two Bt proteins (Cry1A.105 and Cry2Ab2)of that are independently active against the and lepidopteranmay pests documentof corn.publication, Also, by introducing distinctive properties in insecticidal It exploitation modes of action,this the selection pressure on Cry1Ab and Cry1F, the active ingredients of the existing lepidopteranany protected corn products, will be reduced. permission prohibited There is no significant risk of any potential cumulative adverse impact from Cry1A.105 the be and Cry2Ab2 proteinscommercial derived from MON 89034 to the environment and soil-dwelling organisms.Furthermore, any Numerous studies conducted in both laboratories and fields demonstrated littleConsequently, or no cumulativwithout e impact on the soil microflora from the use of biotechnology- derived crops expressing Cry proteins. In the past decade, a number of Bt corn and Bt cotton products have been approved and commercialized in the U.S. and around the world. The active insecticidal Cry proteins in these crops include Cry1Ab, Cry1Ac, Cry1F, Cry2Ab2, Cry3Bb1, Cry34Ab1, Cry35Ab1, and Cry3A proteins. Many laboratory soil degradation studies have been conducted with these Cry proteins. In most studies, a rapid initial dissipation of the Cry proteins was observed, sometimes followed by a slower decline of the remaining soil-bound trace levels of the Cry proteins. In

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 2 of 26 addition, a number of field monitoring studies have been conducted to assess the potential accumulation of Cry proteins following several years of sustained Bt corn or Bt cotton cultivation. The results showed no persistence or accumulation of Cry proteins in the fields. Given the structural and functional similarities of the Cry1A.105 and Cry2Ab2 with those Cry proteins, and the rapid degradation of both of these proteins in studies conducted and submitted in the Petition section VIII.A.5, the risk of any potential cumulative adverse impact from Cry1A.105 and Cry2Ab2 proteins to the environment and soil-dwelling organisms is negligible.

Crop varieties produced by conventional plant breeding have a demonstrated long history of safety. For biotechnology-derived corn products, once single events are assessed and determined to be safe for human and animal consumption and safe to the environment, regime.and then combining the unrelated single events through conventionaland breeding should not AG pose any new characteristics which would change the safety assessment conclusions. The safety assessments conducted for the single event products are sufficient to cover any event combinations that may occur for Bayerunrelated traits.property Breeders protection publishinghave put in place standard testing and assessment proceduresof to examine and confirm the contentsequivalence of its the combined trait products to the single event productsparties.data in phenotypes,therefore agronomic characteristics, and the efficacy of the traits. In the case of and/orcombiningor closely related affiliates. traits, e.g., two or more Bt proteins,property any potentialintellectualthird cumulativ e effects willmay be assessed on its case-by-case basis. So far, no cumulativeas effects have been documented for all registered, the of and combined trait productsis of Bt corn. MON 89034regulatory may be combinedowner. with the existing a document approved and deregulated anycorn suchevents including rootworm protectionits trait, herbicide reproductiondocument tolerance trait, or high lysine corn ownertrait by conventionalthis breeding.of The combined trait products are not expected to interact at underthe molecular of or protein levels based on their well rightsthe this characterized documentmodesand/or of action. The assessments on anyrights existing corn products in to of fall use of commerce and on MON 89034 apply to the combined thetrait products. No new cumulative impacts Thisshould be expected bymay these distribution,combinations.and rights owner subject There is no evidence to suggest that the introductionthe violate of MON 89034 corn will adversely be copy impact biodiversity, genetic purity of cornof germplasm, or specialty corn production even and consideringmay the potentialdocumentpublication, cumulative effect of multiple biotechnology-derived traits. It exploitation There has beenthis a long history of preserving, protecting and enhancing corn germplasm. Measures of preservationany of corn germplasm purity including isolation, detasseling and permission the use of cytoplasmic male sterilityprohibited have been well established and have a track record of effectiveness for many years. The adoption and large scale cultivation of the be biotechnology-derivedcommercial corn in the past decade did not have any negative impact on biodiversity,Furthermore,any genetic purity of corn, or specialty corn production including organic corn in the U.S.Consequently, without

Introduction of MON 89034 will not have any adverse impact on agricultural land use, instead, it will help to protect corn yields and sustain agricultural land productivity. The introduction and rapid adoption of biotechnology-derived corn products in the past decade had no significant impact on the cropland use in the U.S. The total crop area in the U.S. has remained relatively steady for decades, as was the case with field corn planting acreage. From 1996 to 2006, the total annual field corn acres fluctuated around

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 3 of 26 80 million acres, while in the same time span the adoption rate for biotechnology-derived corn products increased from none to 61%. Farmers rotate crops and adjust planting acreages of different crops based on the market demand and commodity prices. U.S. and global corn market demand remains high for food, feed, ethanol production, and other industrial uses. Planted corn acres are projected to significantly increase in 2007 compared to previous years due to the increased need of corn for ethanol production. This increase is not related to biotechnology-derived corn. Corn products that maximize the yield potential may reduce the overall acres needed for such new uses. Adoption of biotechnology-derived corn has been demonstrated to protect corn yields, increase agricultural land output, and alleviate pressure on the reduction of acreages of other large crops (such as soybeans, wheat, and cotton), which translates into sustained benefits to the U.S. farmers and consumers. regime.and and AG Monsanto employs a rigorous product compliance and stewardship program which considers many factors. Consistent with Monsanto’s commitments, MON 89034 will not be launched without regulatory approvalsBayer in the U.S.property and key protectioncorn importpublishing countries to ensure global compliance with local regulationsof and the flow of internationalcontents trade. We its commit to the leading industry standards on seedparties. qualitydata and controltherefore to prevent adventitious presence of unapproved traits. Monsanto’s stewardshipand/oror policy is the shared affiliates. responsibility of Monsanto, ourproperty licensees intellectualand ourthird customers to insure thatmay its products are its used properly. We are committed toas legal and ethical obligations to ensure that our the of and products and technologiesis are safe and environmentallyregulatory responsible,owner. and do not pose a document undue risks to human healthany or thesuch environmen t during any stage itsof their life cycle. In reproductiondocument the U.S., under the Coordinated Frameworowner k for thethis Regulation of of Biotechnology, the responsibility for regulatory oversight underof biotechnology-derived of crops falls on three lead rightsthe this federal agenciesdocument – and/orEPA, FDA, and USDA. Prior to therights commercialization of MON to of fall use of 89034, Monsanto will seek approvals and clearances fromthe those three agencies and any necessaryThis approvals at the statemay level distribution, as well.and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 4 of 26

1. Assessment of Potential Cumulative Impact of MON 89034 with other Bt Crops on Insect Pest Resistance Development and Evolution

The first Bt corn product expressing the Cry1Ab protein was approved for commercial use in the U.S. in 1995 (Federal Register, 1995). Since then, additional Cry1Ab expressing biotechnology-derived corn, along with Cry1F, Cry3Bb1, Cry34Ab1, Cry35Ab1, and Cry3A expressing corn, and Cry1F, Cry1Ac and Cry2Ab2 expressing cotton have been approved and cultivated in the U.S. and other countries (EPA, 2007; FDA, 2007; USDA, 2007a; James, 2006). and and regime. Currently, Bt corn products are grown on overAG 40 million acres in the U.S. and on substantial areas in Canada, Argentina, Spain and the Philippines, and on smaller areas in a number of other countries (James, 2006). In many parts of the northern U.S. Corn Belt, Bt corn adoption exceeds 50% of the corn Bayeracreage. Theproperty consistently protection highpublishing adoption of Bt corn potentially exerts selection for resistanceof to these Cry proteins by thecontents targeted pests, its such as European corn borer, southwestern corn borer,parties. corndata earworm, and corntherefore rootworm. and/or or affiliates. As a mitigation measure to reduceproperty the likelihoointellectualthirdd of the development ofmay resistant insects, its Insect Resistance Management (IRM)as plans have been developed and implemented for the of and all Bt corn and cotton productsis in all countries inregulatory which they have beenowner. cultivated. These a document IRM plans and their underlyingany such strategies received extensive expertits review and are reproductiondocument required conditions of the EPA registrationowner of these productsthis (EPA,of 2001a, b; 2005a, b, c, under of d, e, f, g; 2006). Also therights effectivenessthe of these plansthis has been monitored annually documentand/or rights through surveys of targetto pest of susceptibility fall (Farinouse etof al., 2004; ABSTC, 2006; Siegfried et al., 2006). No instances of insect resistance developmentthe to any of the Bt corn or Bt cotton eventsThis have been confirmedmay distribution, in anyand of the target pests anywhere in the world rights owner (Tabashnik et al.,subject 2003; Farino et al., 2004; ABSTC, 2006). Even when highly sensitive methods have been used to search for Bt resistthe antviolate alleles, no evidence of an increase in Bt be copy resistant allele frequency has been observedof during the eleven years of commercial Bt and corn usemay (Bourguet document et al.,publication, 2003; Stodola et al., 2006). Therefore, the risk of resistance It exploitation evolving to thethis currently commercial Bt corn and Bt cotton products has been managed effectively through anythe current agricultural practices and IRM programs. This in planta permission Bt protein expression approach prohibitedcontrasts with the use of traditional Bt microbial pesticide spray applications where no IRM plans have been implemented and field resistance has the be been observed, mostcommercial notably in the diamondback moth (Tabashnik et al., 1997). Furthermore,any As withConsequently, all previouslywithout commercialized Bt corn products, MON 89034 will be introduced with an effective IRM plan. Monsanto has drafted such a plan, which is being reviewed by the EPA ( 2006). EPA will mandate an IRM plan as part of the EPA registration conditions for MON 89034. The components of this plan are based on the best available science and employ comparable approaches to the plans which have been successfully implemented for the current biotechnology-derived Bt corn products. The effectiveness of this plan will be monitored through annual surveys of target pest susceptibility, and the implementation of the plan will be tracked through a Compliance Assurance Program

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 5 of 26 (CAP) that is comparable to the CAP used for current Bt corn products. The CAP will employ a combination of educational programs and surveys of growers’ compliance. The results of the susceptibility surveys and the CAP will be reported to EPA for review on a regular basis. Therefore, the introduction of MON 89034 in the U.S. is not expected to result in an increase in the overall risk of resistance development and evolution in the target pests to the Cry proteins.

To the contrary, the adoption of MON 89034 should reduce the overall risk of the resistance evolution in lepidopteran pests towards Cry proteins in a number of important ways. First, the introduction of MON 89034 into the marketplace will provide two Bt proteins that are independently active against the lepidopteran pests of corn. By introducing new and distinctive properties in insecticidal modes of action, the selection regime.and pressure on Cry1Ab and Cry1F, the active ingredients ofand the current lepidopteran AG protection corn, will be reduced, decreasing the risk of resistance development in European corn borer, southwestern corn borer, and corn earworm. Second, MON 89034 contains a combination of two Bt proteinsBayer – Cry1A.105 property and Cry2Ab2 protectionpublishing - each of which is highly effective against these corn ofpest s and which differ significantlycontents in key its characteristics in their insecticidal modes of action parties.such datathat this producttherefore exhibits two independent and effective modes of action. and/oror affiliates. property intellectualthird may its Crops producing two or more insecticidalas proteins are sometime called pyramided the of and products (Roush, 1998) isand have been shown regulatoryto have a lower associatedowner. risk of insect a document resistance than products contaiany ningsuch a single Bt protein for a similarits spectrum of pests. reproductiondocument Mathematical modeling (Caprio, 1998;owner Rous h, 1998)this and experimentalof studies in model systems (Zhao et al., 2003) have shownunder that insectof resistance evolves many times more rightsthe this slowly to productsdocument and/orwith two highly effective Bt proteinsrights than products with a single to of fall use of highly effective Bt protein. MON 89034 will bethe introduced by Monsanto as a replacementThis for the Cry1Ab expressingmay distribution, MONand 810 (YieldGard® corn borer) and also may rights owner displace other lepidopteransubject protection, single Bt protein corn products in the marketplace. The net effect will be to convert much ofthe the violate corn-growing area in the U.S. and other be copy countries from single Bt protein productsof to MON 89034 and combined trait products, and with a maysignificant associateddocumentpublication, reduction in the risk of resistance evolution in the target It exploitation pests. this any permission prohibited 2. Assessment of Potential Cumulative Impact of Deregulation of MON 89034 on Bt the Protein Accumulationcommercial in Soil be Environment Furthermore,any LongConsequently, before biotechnology-derivewithout d Bt crops were developed, the agriculture industry had been using whole Bt microbes and spores as microbial pesticides on crops for insect control, a pest management practice still in application in organic crop production and forestry today. Bacillus thuringiensis is a Gram-positive, soil dwelling bacterium. It also occurs naturally in the caterpillars of some moths and butterflies as well as on the surface of plants (Madigan and Martinko, 2005). Upon sporulation, Bt produces a variety of

® YieldGard is a registered trademark of Monsanto Technology, LLC.

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 6 of 26 crystal (Cry) proteins which display specific activities against insect species of the orders Lepidoptera (moths and butterflies), Diptera (flies and mosquitoes) and Coleoptera (beetles).

Spray applications with microbial pesticide formulations consisting of whole Bt cells and spores have been used in organic agriculture in the U.S. for over four decades (Betz et al.; 2002). No significant environmental impact has been reported with this practice despite findings of detectable concentrations of Cry proteins long after spraying (Smith and Barry, 1998). The soil degradation of Cry proteins from microbial Bt sprays was initially 14 documented by radioisotope labeling and monitoring of the liberated CO2 (West, 1984). The half-life of the sprayed Bt pesticides was reported to be less than six days in soil. Later studies of aerial Bt-spray applications indicated that Bt spores persisted for two regime.and years in soil and one year in leaf samples (Smith and Barry, 1998).and Field studies in Italy AG indicated that Bt spray residues were detectable by insect bioassay for 28 months after spraying, while the bacterial spores (Btk) were detected for 88 months. However, the insecticidal activity decreased markedly betweenBayer 14 andproperty 28 months protection (Vettopublishingri et al., 2003). of contents its In contrast to Bt spray applications with microbial pesticideparties.data form ulations, biotechnology-therefore derived plants have built-in protection through the expression ofand/or oneor or more specific Cry affiliates. proteins. This approach offersproperty some environmentalintellectualthird advantages overmay the Bt microbial its pesticide formulations. First, Bt cropsas are very selective and specific with respect to the the of and active insecticidal ingredients,is normally one, two,regulatory or three Cry proteinsowner. compared to a a document unknown number of Cry anyproteins such from the Bt cells and sporesits in the microbial reproductiondocument formulations. Second, Bt crops areowner readily degradable,this exposingof the Cry proteins to microbial soil degradation. As a consequeunder nce,of the Cry proteins expressed in the rightsthe this biotechnology-deriveddocumentand/or crops have a lower tendency for accumulationrights in the environment. to of fall use of A recent study compared the concentration of Cry1Abthe protein from a biotechnology- derived ThisBt corn and from Dipelmay® Btdistribution, sprayand formulations in soil and surface water. The rights owner report concludedsubject that Cry1Ab from the biotechnology-derived Bt corn degraded faster in soil and water than the Cry1Ab proteinthe containedviolate in the Dipel® microbial spray be copy formulations (Douville et al., 2005). of and may documentpublication, It exploitation In the past decade,this a number of biotechnology-derived Bt corn and Bt cotton products have been approvedany and commercialized in the U.S. and around the world. The active permission insecticidal Cry proteins in theseprohibited crops include Cry1Ab, Cry1Ac, Cry1F, Cry2Ab2, Cry3Bb1, Cry34Ab1, Cry35Ab1, and Cry3A proteins (EPA, 2007). In 2006, there were the be 22 countries growingcommercial biotechnology-derived crops, and there were 19 million hectares (or 47Furthermore, million acres)any planted to Bt crops including corn and cotton (James, 2006). In the U.S.,Consequently, 40% of cornwithout acreage and 57% of the cotton acreage were planted with varieties possessing Bt insect protection traits in 2006 (USDA-NASS, 2006). Many laboratory soil degradation studies have been conducted with various Cry proteins. In most studies, a rapid initial dissipation of the Cry proteins was observed, sometimes followed by a slower decline of the remaining soil-bound trace levels of the Cry proteins (Palm et al., 1996; Sims and Holden, 1996; Sims and Ream, 1997; Herman et al., 2001; Herman et al.,

® DiPel is a registered trademark of Abbott Laboratories.

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 7 of 26 2002a, b; 2002a, b; Stotzky, 2004; Hopkins and Gregorich, 2005; 2006).

In addition to the laboratory studies, a number of field monitoring studies have been conducted to assess the potential accumulation of Cry proteins following several years of sustained Bt corn or Bt cotton production. These studies have shown no persistence or accumulation of Cry proteins in fields where corn expressing Cry1Ab protein (Dubelman et al., 2005) or Cry3Bb1 protein (Ahmad et al., 2005), and cotton expressing Cry1Ac protein (Head et al., 2002) were grown continuously for several years. The results of these long-term field studies suggest that there were no significant accumulation of Bt proteins from continuous cultivation of Bt crops.

regime.and Published studies show little or no cumulative impact on the soiland microflora from the use AG of biotechnology-derived crops expressing Cry proteins. For example, a recent field study over a 3-year cropping cycle concluded that growing MON 863, which expresses the Cry3Bb1 protein, showed no adverse impactBayer on theproperty soil ecology protection (Devarepublishing et al., 2007). Another study conducted in two fieldsof in Germany showed that corn-rhizospherecontents its microbial communities exposed to the Cry1Ab proteinparties. fromdata MON 810 weretherefore less affected by Cry1Ab protein than by other environmental factors, such asand/or the orage of the plants and affiliates. field heterogeneities (Baumgarteproperty and Teebe, intellectual 2005).third A separate study mayto assess the effect its of Bt-corn on soil microbial communityas structure concluded that Cry proteins had only the of and minor effects on soil microbialis community prregulatoryofiles when comparedowner. to their non-Bt a document isolines (Blackwood and Buyer,any such 2004). A season-long field studyits conducted with the reproductiondocument Cry3A protein expressed in biotechnology-deowner rived thispotato showedof no adverse effects towards soil-dwelling microorganisms under(Donega n ofet al., 1996). In a study conducted in rightsthe this Kansas duringdocument the 2000and/or and 2001 growing seasons, the numbersrights of soil mites, collembola to of fall use of and nematodes observed in plots planted with Cry3Bb1-producingthe corn were similar to those observedThis in plots plantedmay with distribution,non-Btand corn (Al-Deeb et al., 2003). Other published rights owner reports showed subjectthat Cry proteins had no microbiocidal or microbiostatic activity in vitro against selected bacteria, fungi, and algaethe (Koskellaviolate and Stotzky, 2002), and had no be copy apparent effect on earthworms, nematodes,of protozoa, bacteria and fungi (Saxena and and Stotzky,may 2001). documentpublication, It exploitation this MON 89034, the productany of this petition, expresses two Cry proteins – Cry1A.105 and permission Cry2Ab2. MON 89034 is intendedprohibited to replace MON 810 corn and to compete with other existing biotechnology-derived, lepidopteran protection corn products. Given the the be structural and functionalcommercial sim ilarities of Cry1A.105 and Cry2Ab2 with other Cry proteins, andFurthermore, the lackany of soil accumulation of other Cry proteins derived from large scale cultivationConsequently, of Btwithout corn and Bt cotton in the past decade, and the rapid degradation of both of these proteins in studies conducted and submitted in the Petition section VIII.A.5, the risk of any potential cumulative adverse impact from Cry1A.105 and Cry2Ab2 proteins to the environment and soil-dwelling organisms is negligible.

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 8 of 26

3. Assessment of Potential Cumulative Impact of MON 89034 in Combined Trait Products Resulted from Conventional Breeding with other Biotechnology-Derived Products

Different biotechnology-derived corn products have been combined through conventional breeding practices to produce combined trait corn products that meet the broader needs of corn growers. For example, insect protection traits have been combined through conventional breeding with herbicide tolerance traits, and lepidopteran insect protection traits have been combined through conventional breeding with coleopteran insect protection traits to produce corn products possessing a combination of traits in anda single and regime. plant. AG

In the past one and a half decades, over 20 biotechnology-derived corn products have completed the regulatory process in the U.S.Bayer under theproperty regulatory protection overspublishingight of EPA, FDA, and USDA (EPA, 2007; FDA, 2007;of USDA, 2007a). Three majorcontents types of its biotechnology-derived corn traits have been developedparties. anddata widely adoptedtherefore by the U.S. and/or corn growers, who increasingly choose to purchase and plant combinedor trait products. affiliates. These three major types of theproperty biotechnology-derived intellectualthird corn traits includemay (1) lepidopteran its pest protection corn, (2) coleopteran pestas protection corn, and (3) herbicide tolerant corn the of and including the glyphosateis tolerance trait and theregulatory glufosinate tolerance owner.trait. In addition, a document other corn trait such as highany lysine such corn was also developed and approvedits in the U.S. reproductiondocument owner this of under of Different combined trait productsrightsthe have been produced fromthis the approved and deregulated documentand/or rights single event corn productsto throughof fall conventiona usel breeding,of and have been cultivated by corn farmers in the U.S. A survey by the National Agriculturalthe Statistics Service showed that that This61% of total U.S. cornmay acreage distribution, inand 2006 was planted with biotechnology-derived rights owner varieties (USDA-NASS,subject 2006). The percentages of insect protected corn, herbicide tolerant corn, and combined trait corn prtheoducts violate possessing both insect protection and be copy herbicide tolerance traits were 25%, 21%of and 15%, respectively. It should be noted that and in this surveymay the insectdocumentpublication, protected corn includes both single event and combined trait Bt It exploitation corn products. this any permission Biotechnology-derived, combinedprohibited trait or combined event products produced by conventional breeding from the approved and deregulated single event products should the be be subject to thecommercial same regulatory oversight that is applied to conventional crop varieties thatFurthermore, are producedany by conventional plant breeding. The safety assessments conducted for the singleConsequently, eventwithout products are sufficient to cover any trait combinations that may occur for unrelated traits. Crop varieties produced by conventional plant breeding have a demonstrated long history of safety. For biotechnology-derived products, once single events are assessed and determined to be safe for human and animal consumption and safe to the environment, then combining the unrelated single events through conventional breeding should not pose any new characteristics which would change the safety assessment conclusions. Any assessments conducted for the single trait products can be used as a basis for any potential trait combinations based on the type of genes, the

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 9 of 26 localization of the proteins or other active ingredients, and the modes of action of the traits. Breeders have put in place standard testing and assessment procedures to further examine and confirm the equivalence of the combined trait products to the single event products in phenotypes, agronomic characteristics, and the efficacy of the traits. In the case of combining closely related traits, e.g., two or more Bt proteins, any potential cumulative effects will be assessed on case-by-case basis. No cumulative effects have been reported for all registered combined trait products of Bt corn or Bt cotton (EPA, 2007).

MON 89034 may be combined with the existing approved and deregulated corn events including rootworm protection, herbicide tolerance traits, or high lysine corn trait by conventional breeding. The combined trait products are not expected to interact at the regime.and molecular or protein levels based on their well characterizedand mode s of action. The AG assessments on any existing corn products in commerce and on MON 89034 apply to the potential combined trait products. No new cumulative impacts should be expected by these combinations. Bayer property protectionpublishing of contents its parties.data therefore 4. Assessment of Potential Impact of Deregulation of MONand/or 89034or on Biodiversity, affiliates. Preservation of Corn Germplasmproperty Purity, intellectual andthird Specialty Corn Productionmay its as the of and The Petition section IIis has discussed, in greatregulatory detail, the outcrossingowner. potential of a document contemporary domestic cornany varie suchties and their wild relatives. Sectionits VII presented reproductiondocument data on the phenotypic, agronomic, ownerecological interactionsthis and compositionalof assessment under of of MON 89034. Section rightsVIII.Bthe and VIII.C examinedthis the potential of weediness and documentand/or rights gene flow of MON 89034to corofn. Thesefall analysesuse conclude of d that (1) MON 89034 exhibits no characteristics that would cause increased weedinessthe and is not likely to become invasiveThis or form self-sustainingmay populations distribution,and outside of agricultural fields; (2) unconfined rights owner cultivation shouldsubject not lead to increased weediness of sexually compatible relatives (of which there are none in the U.S.); and (3)the use violateof MON 89034 in breeding for improved be copy corn hybrids will not reduce or limit theof genetic diversity available to corn breeders in the and future. mayIn addition, document the publication,assessment in section VIII also concluded that it is unlikely that It exploitation MON 89034 wouldthis have long-term direct or indirect effects on non-target organisms common to agriculturalany production areas or on threatened and endangered species permission recognized by the U.S. Fish andprohibited Wildlife Service and the National Marine Fishery Service. Based on these analyses, there is negligible potential for adverse effects on the be biodiversity fromcommercial MON 89034 compared to conventional and previously approved insect protectionFurthermore, cornany already under cultivation. Consequently,without Since the development of hybrid corn systems in the 1930’s, the corn industry has created and adopted systems to maintain and preserve the purity of inbred corn lines, hybrids and corn populations developed for various uses including food, feed, processing, and specialty uses such as popcorn, sweet corn, high oil corn, white corn, blue corn, high protein corn (Smith et al., 2004; Sprague and Dudley, 1988). These systems have been developed and implemented over several decades to maintain and meet industry standards on genetic purity and seed quality. Biotechnology-derived corn traits have

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 10 of 26 been incorporated into these systems while achieving accepted purity standards for the corn industry and taking into consideration the cumulative effects of biotechnology- derived corn products.

To maintain the genetic purity of inbred parents, hybrids and corn populations, production activities for each type are isolated from one another and from commercial grain production (Smith et al. 2004; Sprague and Dudley, 1988). Isolation is achieved through various means but may include physical separation to prevent cross pollination, temporal isolation by planting at different times to stagger pollination times of different materials, detasseling, and the use of cytoplasmic male sterility. Additionally, production guidelines and operating procedures are used to ensure genetic purity and quality throughout the entire production process from planting and growth of the crop, through regime.and harvesting, transport, conditioning, packaging, storage and sale.and With these operating AG procedures being widely used in the corn industry, the industry has been able to introduce new varieties including biotechnology-derived corn into the marketplace while still maintaining the desired levels of purity andBayer quality ofproperty individual protection varieties.publishing Thus, over the last 70 years, systems have been developeof d to maintain the purity andcontents quality of the its diverse corn types needed to meet the needs and desiresparties. ofdata corn farmers andtherefore consumers. These systems have allowed the introduction of biotechnology-derivedand/oror traits into the affiliates. desired inbred lines while at propertythe same intellectualtime allowingthird for the maintenancemay of desired its conventional inbred lines. Therefore, asno significant single or cumulative impact on the the of and production of corn seedsis for various desired usesregulatory has been observed. Theowner. introduction of a document MON 89034 is not expectedany to havesuch any effect on the well establishedits effective processes reproductiondocument already in place to insure genetic isolation.owner this of under of rightsthe this There has beendocument a longand/or history of preserving, protecting andrights enhancing corn germplasm. to of fall use of The introduction of biotechnology-derived corn has hadthe little impact on genetic diversity and germplasmThis resources of maycorn, evendistribution, andcons idering the potential cumulative effect of rights owner multiple biotechnology-derivedsubject traits being introduced to corn. Plant breeders, institutions, government and non-governmentthe agenciviolatees take great strides to maintain corn be copy germplasm resources. A full review ofof this topic is presented in Chapter 1.3 of the book and Corn: Origin,may History,document publication,Technology and Production (Smith et al., 2004). As noted in the It exploitation text, corn geneticthis diversity is maintained through public and private plant breeding efforts, seed conservation inany gene banks, germplasm collections maintained by the International permission Maize and Wheat Improvement prohibitedCenter (CIM MYT) and other governmental agencies, on- farm conservation and enhancement activities in Latin America, and conservation of corn the be wild relatives incommercial Mexico and Central America. These efforts maintain and preserve corn geneticFurthermore, diversityany necessary for improving the base genetics of corn, separate from or in combinationConsequently, withwithout the breeding practices used to introduce biotechnology-derived traits into new corn varieties. Thus, the genetic diversity of corn will continue to be maintained.

The introduction and large scale cultivation of biotechnology-derived corn in the past decade did not have any negative impact on organic corn production or other specialty corn production in the U.S. It is well recognized that some corn seed companies and farmers specialize in small specialty use corn, such as organic corn, sweet corn and

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 11 of 26 popcorn. Organic farmers may prefer to use spray applications of Bt microbial pesticides over their crops for insect control instead of using the biotechnology-derived, built-in insect protection Bt traits. These seed manufacturers and growers have been using the same established processes and methods (e.g., isolation, detasseling, and use of cytoplasm male sterility) as described above to ensure the genetic purity of their corn products. Organic producers, like the producers of other specialty corn products including popcorn, use process based standards to assure that products produced meet the specifications set by the industry or the purchasers of the products.

Organic farming is a small sector of the U.S. agriculture industry with only about 0.5% of all U.S. cropland and 0.5% of all U.S. pasture being certified organic in 2005 (USDA- ERS, 2007). Only a small percentage of the top U.S. field crops (0.16% of corn, 0.17% regime.and of soybeans, and 0.48% wheat) were grown under certifiedand organic farming systems AG (Table 1). Although small, organic production has been one of the fastest growing segments of U.S. agriculture for the past decade. The U.S. had less than one million acres of certified organic farmland when CongressBayer passedproperty the Organicprotectionpublishing Foods Production Act of 1990. By the time USDA implemenof ted national organic standardscontents in 2002, its certified organic farmland had doubled, and doubledparties. againdata between 2002therefore and 2005 (USDA-ERS, 2007). For example, from 1995 to 2005 the organicand/or coorrn acreage increased affiliates. by 300% in the U.S. (Table 1property), while duringintellectual thirdthe same time frame themay biotechnology- its derived corn acreage increased from asnone to 52% of total U.S. corn acreage in 2005 the of and (USDA-NASS, 2006). is These national statisticsregulatory do not suggest thatowner. the increase in a document organic corn production andany the such increase in biotechnology-derivedits corn production are reproductiondocument correlated, but they do indicate thatowner the a doption of thisbiotechnology-derived of corn did not adversely affect organic corn production.under Acco rdingly,of there is no reason to expect that rightsthe this Monsanto’s seconddocument and/orgeneration lepidopteran protection cornrights product, MON 89034, will to of fall use of have any negative impact on the production of organic thecorn or other specialty corn. This may distribution,and rights owner In conclusion, measuressubject of preservation of corn germplasm purity including isolation, detasseling and the use of cytoplasmic malethe ster violateility have been well established and have be copy a track record of effectiveness over manyof years. The adoption and large scale cultivation and of biotechnology-derivedmay documentpublication, corn in the past decade did not have any negative impact on It exploitation biodiversity, geneticthis purity of corn, or the production of specialty corn including organic corn in the U.S. Thereany is no evidence to suggest that the introduction of MON 89034 permission corn will adversely impact biodiversity,prohibited geneti c purity of corn germplasm, or small scale specialty use corn production even considering the potential cumulative effect of multiple the be biotechnology-derivedcommercial traits. It is also concluded based on past experience and standards thatFurthermore, the introductionany of MON 89034 will not have an impact on the production of specialtyConsequently, corn products,without including organic corn.

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 12 of 26 Table 1. Certified organic and total U.S. acreage of selected crops (USDA-ERS, 2007).

Certified U.S. Cropland Total certified organic (acres) organic to 2005 (acres) Item total (%)

1995 1997 2000 2001 2002 2003 2004 2005 regime.and and Total cropland: 638,500 850,173 1,218,905 1,302,392 1,299,632 1,451,601AG 1,452,353 1,723,271 340,650,083 0.51% Grains--

Corn 32,650 42,703 77,912 93,551 96,270 Bayer105,574 property99,111 protection130,672publishing 81,759,000 0.16% of contents Wheat 96,100 125,687 181,262 194,640 217,611 234,221 214,244 277,487its 57,229,000 0.48% parties. data therefore Oats and/oror 13,250 29,748 29,771 33,254 53,459 46,074 42,616 46,465 4,246,000 1.09% affiliates. may Barley property intellectualthird 17,150 29,829 41,904 31,478 its34,031 30,265 26,629 39,271 3,875,000 1.01% the as Sorghum of and owner. -- 3,075 1,602 is938 3,043 4,152regulatory 4,453 6,042 6,454,000 0.09% a document any such its Rice reproductiondocument 8,400 11,043 26,870 29,022 22,381owner 20,152 this 22,173 of 26,428 3,384,000 0.78%

Spelt under of this 12,350 1,704 12,606 and/or 7,639rights the 6,939 9,719 6,203 8,169 -- -- document of rights Millet to of fall 18,550 12,285 15,103 23,366 17,575 use26,935 21,036 14,175 565,000 2.51% the Buckwheat This may distribution,and 13,250 7,616 10,599 rights14,311 8,388 owner8,086 7,960 6,364 -- -- subject Rye 2,900 4,365 be7,488 7,056 9,644 the 11,616violate 10,289 8,597 1,433,000 0.60% Beans-- copy of and Soybeans may documentpublication, 47,200 82,143It 136,071 174,467exploitation 126,540 122,403 114,239 122,217 72,142,000 0.17%

Dry beans this -- 4,641 14,010any 15,080 2,430 9,836 7,642 10,561 1,659,300 0.64%

Dry peas & lentils permission 5,900 5,187 10,144 9,362 prohibited7,476 16,188 15,893 17,757 571,000 3.11% Oilseeds-- the be commercial Flax 5,850 Furthermore,8,053 25,076 20,672 20,484 14,940 35,104 30,843 983,000 3.14% any Sunflowers 14,200 Consequently,10,894 19,342without 15,295 7,624 7,121 9,742 6,087 2,709,000 0.22%

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 13 of 26 5. Assessment of the Potential Impact of Deregulation of MON 89034 on Land Use and Agricultural Production

The introduction and rapid adoption of biotechnology-derived corn products in the past decade had no significant impact on cropland use in the U.S. The total crop area in the U.S. remained relatively steady for decades, as was the case with field corn acreage. From 1996 to 2006, the total annual field corn acres fluctuated around 80 million acres (Table 2), while at the same time frame the adoption rate for biotechnology-derived corn products increased from none to 61% (USDA-NASS, 2006). Farmers rotate crops and adjust planting acreages of different crops based on the market demand and commodity prices. U.S. and global corn market demand remains high for food, feed, andethanol and regime. production, and other industrial uses. Adoption AGof biotechnology-deri ved corn has been proven to protect corn yields, increase agricultural land productivity, and alleviate pressure on the reduction of acreages of other large crops (such as soybeans, wheat, and cotton), which translates into sustained benefitsBayer to farmersproperty and protectionconsumers.publishing Introduction of MON 89034 will not have any adverseof impact on the agricultural landcontents use, instead it its will help to protect corn yields and sustain agriculturalparties. landdata productivity. therefore and/or or affiliates. According to the national census,property the tota intellectuall harvestedthird cropland areas frommay 1974 to 2002 its varied annually from 295 to 326 millionas acres. Corn is the largest crop grown in the U.S. the of and in terms of acreage plantedis and net value. Inregulatory 2006, corn was plantedowner. on 78.3 million a document acres with a total productionany ofsuch 10.5 billion bushels of grain valuedits at $ reproductiondocument (Table 2), with an additional 6.5 millionowner acres of cornthis crops harvestedof for silage use. under of The acreages of other majorrights cropsthe (in million acres) in 2006this were 75.5 for soybeans, 57.3 documentand/or rights for wheat, 15.2 for cotton,to of3.5 forfall barley, use2.8 forof rice, 2.0 for sunflower, 1.4 for sugarbeets, 1.1 for potatoes, and 1.0 for canola (USDA-NASS,the 2007). This may distribution,and rights owner Corn grain and itssubject processed products have long been a very important component of the U.S. agricultural production essential to thethe na tion’sviolate economy. Because of its high starch be copy content, corn grain is used as a valuableof energy source in animal feed for domestic and livestock,may such as cattle,documentpublication, pigs and poultry. Corn is also extensively processed to generate It exploitation a wide range ofthis food and industrial products including corn syrup, glucose, dextrose, starch, cereals, andany ethanol. In addition, as the largest corn producer in the world, the permission U.S. also exports corn grain toprohibited a number of countries. As corn demand and supply fluctuate year over year, the average annual data of the most recent five years (2002-2006) the be were used to illustratecommercial the contemporary utilization profile of U.S. corn. The average annualFurthermore, total anycorn use from 2002-2006 was 10,683 million bushels, among which, feed use is highest,Consequently, takingwithout 55.5%, followed by export shipment (18.2%), ethanol for fuel (13.6%), and food (12.6%) (Table 3).

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 14 of 26 Table 2. U.S. corn acreages, production, value, supply, demand, and export shipment (USDA-NASS, 2007).

Endingand stock Production Supply Total Use Total regime. Planting Area Yield Price Value ($ and inventory Export (million Year (million (million (million (million acres) (bushels/acre) ($/bushel) billion) AG (million bushels) bushels) bushels) bushels) bushels)

Bayer property protectionpublishing 1987 66.20 7,131.30 119.80 of 12,016.40 7,757.32 contents4,259.08 1,716.43 its 1988 67.72 4,928.68 84.60 parties.9,190.55data 7,260.12 therefore1,930.43 2,028.45 1989 72.32 7,531.95 116.30 9,464.28 and/or8,119.83or 1,344.45 2,367.30 1990 74.17 7,934.03 118.50 affiliates. 9,281.90 7,760.66 1,521.24 1,726.63 property intellectualthird may 1991 75.96 7,474.77 108.60 its 9,015.65 7,915.34 1,100.31 1,583.92 as 1992 79.31 9,476.70 131.50 the of and 10,584.10 8,471.12 2,112.98 1,663.28 is regulatory owner. 1993 73.24 6,337.73 100.70 a 8,471.53document 7,621.38 850.15 1,328.32 any such its 1994 78.92 10,050.52 138.60 reproduction10,910.22 document 9,352.38 1,557.84 2,177.48 owner this of 1995 71.48 7,400.05 113.50 8,974.38 8,548.44 425.94 2,227.82 under of 1996 79.23 9,232.56 127.10 rightsthe 9,671.76this 8,788.60 883.16 1,797.37 documentand/or of rights 1997 79.54 9,206.83 126.70 to of fall use 10,098.80 8,791.00 1,307.80 1,504.43 1998 80.17 9,758.69 134.40 11,085.29the 9,298.32 1,786.97 1,984.19 This may distribution,and 1999 77.39 9,430.61 133.80 rights owner 11,232.33 9,514.78 1,717.55 1,936.57 2000 79.55 9,915.05 136.90subject 11,639.42 9,740.32 1,899.10 1,941.35 the 2001 75.70 9,502.58 be 138.20 violate11,411.83 9,815.40 1,596.43 1,904.77 copy of 2002 78.89 8,966.79 129.30 and 10,577.66 9,490.99 1,086.67 1,587.89 2003 78.60 10,089.22 may 142.20document publication, 11,189.97 10,231.88 958.09 1,899.82 It exploitation 2004 80.93 11,807.09 this 160.40 12,776.01 10,662.04 2,113.97 1,818.06 2005 81.78 11,114.08 148.00any 13,237.00 11,269.84 1,967.16 2,147.34 permission 2006 78.33 10,534.87 149.10 prohibited 12,512.03 11,760.00 752.03 2,250.00 the commercial be Furthermore,any Consequently,without

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 15 of 26

Table 3. Average annual U.S. corn utilization during 2002-2006.

Mean (million Standard deviation bushels) (million bushels) % to total use Feed and residual 5,926.5 224.4 55.5% Food 1,348.6 15.6 12.6% Ethanol 1,447.8 403.9 13.6% Export 1,940.6 236.5 18.2% Seed 20.3 0.4 0.2% Total use 10,683.8 791.0 100.0%

and Corn is emerging as an important renewable resource thatand ca n be processedregime. and fermented to produce ethanol as an alternative fuelAG and gasoline additive. In the recent years, significantly increased oil prices and the government regulations on cleaner air and alternative fuel (e.g. Energy Policy Act of 2005) have dramatically driven the demand for Bayer property protectionpublishing ethanol production from corn grain. Manyof new ethanol plants are being builtcontents in the Corn Belt. Current USDA projections indicated that U.S. corn acreage willits increase, while parties.data therefore other major crops such as soybeans and wheat will either decreaseand/or oror remain flat. As a result of the strong near-term economicaffiliates. outlook for corn, a sharp increase in corn property intellectualthird may planting is expected to occur for 2007.its Corn acreage is expected to increase from 78.8 to as 90.4 million acres from 2006the to of2007, representingand a 15% increase in a single year. Corn is regulatory owner. production is projected to stay strong througha 2016 (USDA,document 2007b).its This increase in projected acres of corn is independentany such of the biotechnology-derreproduction ived traits that have been this documentof commercialized in corn. The biotechnology-deriveowner d corn traits that have been introduced under of or with the introduction rightsof MONthe 89034 in the future,this combined with advances in documentand/or of rights conventional breeding methodto of arefall intended useto maximize the yield protection of corn. Therefore, these traits will hopefully help to meet thethe growing demands for corn and This may distribution,and reduce the number of increasedrights acres that mayowner need to be planted. subject be the violate Higher corn yieldscopy and sustained productivityof of U.S. cropland are also important to maintain the status for the U.S. as a keyand supplier of corn grain to the world. As the may documentpublication, world’sIt largest corn grain producer,exploitation the U.S. not only supplies the domestic market but also exports largethis quantitiesany of corn to other countries. In the past 20 years, U.S. corn export fluctuated year over year, but overall remained flat (Table 2). The average of permission annual corn export in 1987-2006prohibited was 1,879.6 million bushels and the average for the the most recent fivecommercial years (2002- be2006) was 1,940.6 million bushels (Table 3). The lead importing countries and regions of U.S. corn include Japan, Mexico, Korea, and Taiwan. USDAFurthermore, projectsany U.S. corn export will remain flat or slightly decrease over the next five yearsConsequently, due to thewithout strong domestic demand for corn.

The introduction and adoption of biotechnology-derived insect protection corn products in the past decade had no adverse impact on cropland use but protected corn yields and increased agricultural land output. Corn plants are subject to damage by both insect pests and weeds. Insect pests of corn such as corn borers and corn rootworms can be controlled by chemical pesticides or plant incorporated protectants such as Bt corn. No adverse effects to human and animal health and the environment have been documented

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 16 of 26 for the adoption and cultivation of biotechnology-derived corn in the past decade. In contrast, there have been well-documented economical and environmental benefits from the adoption of Bt corn, herbicide tolerance corn, and their combined trait products. The use of these biotechnology-derived corn in the U.S. increased corn yields, reduced mycotoxin levels in corn grain, enhanced simplicity and flexibility of insect pest control and weed management, reduced chemical insecticide and herbicide use, increased no-till corn acreage which resulted in less soil erosion and less runoff of pesticides and water (Koziel, 1993; Martin and Hyde, 2001; Carpenter and Gianessi, 2001; Gianessi et al., 2002; Shelton et al., 2002; Fawcett and Towery, 2002; Hyde et al., 2003; Carpenter et al., 2004; Sankula and Blumenthal, 2004; Sankula et al., 2005; Sankula, 2006; Wu, 2006; NCGA, 2007).

regime.and Numerous studies demonstrated that lepidopteran protectionand corn reduces mycotoxin AG levels in corn grain, resulting in healthier food and feed. Lepidopteran pests such as ECB, CEW and FAW cause substantial damage to stalks, ears, and leaves of developing corn plants. Some of these pests, notably CEWBayer and FAW,property cause damageprotection publishingto corn ears which provide entrance of mycotoxin-producingof fungi (Miller, 2001). Corncontents ears that are its protected from feeding damage caused by CEW andparties. FAWdata would have fewertherefore ports of entry for invasion by fungi, especially those that produce mycotoxins,and/oror increasing the affiliates. safety of grain for food andproperty feed use. intellectual Resultsthird of field studies mayhave consistently its demonstrated that Bt corn had significantlyas lower incidence and severity of Fusarium ear the of and rot and Aspergillus kernelis rot and produced cornregulatory grains with lower levelsowner. of fumonisins a document and aflatoxins (Munkvold anyet al.,such 1999; Bakan et al., 2002; Hammondits et al., 2003; reproductiondocument Windham et al., 1999). The resultsowner from field trials thisconducted beof tween 1997 and 2001, in Argentina, France, Italy, Turkey, andunder USA indicatedof that the overall fumonisin levels rightsthe this in Bt corn weredocument reducedand/or by 47% to 97% compared with rightsnon-Bt corn (Hammond et al., to of fall use of 2003). Windham et al. (1999) reported that when plantsthe were infested with southwestern corn borers,This a Bt corn hybrid mayprovided distribution, moreand than 75% reduction in aflatoxin compared rights owner with its non-Bt subjectcounterpart. Most of these prior studies included MON 810 as the Bt corn. Preliminary data for MON 89034 demonsthe violatetrates that MON 89034 is subject to even be copy less insect damage than MON 810, andof this lower level of insect damage tends to and translatemay into even documentlowerpublication, mycotoxin contamination in the corn grain. The data show It exploitation there is a statisticallythis significant reduction in aflatoxin levels in MON 89034 compared to MON 810 or non-Btany control corn. permission prohibited Monsanto’s first generation, biotechnology-derived, lepidopteran protection corn product the be MON 810 (YieldGardcommercial® corn borer) has been shown to protect corn yields and reduce chemical insecticide use. In 2005 about 34 U.S. states planted 27.9 million acres of corn Furthermore,any ® ® possessingConsequently, YieldGardwithout corn borer trait either as a single trait product (YieldGard corn corer) or combined trait products (YieldGard® Plus and YieldGard® Triple), resulting in an increased production of 109.3 million bushels or 6.12 billion pounds of corn valued at $ (Sankula, 2006). Net returns were estimated to be $ . In addition, the amount of pesticide required for pest control is estimated to decrease by 4.85 million pounds per year when using corn-borer protected traits. In percentage terms, MON 810 and combined trait corn containing MON 810 planted in 2005 improved the return on

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 17 of 26 investments that translates to 24% increase in yield, 27% decrease in pesticide use, and increased monetary gain of 26% compared to 2004 (Sankula, 2006).

MON 89034 is Monsanto’s second generation lepidopteran protection corn with dual effective dose that expands the pest spectrum beyond that of MON 810. MON 89034 is intended to replace MON 810, and is expected to be at least equally or even more economically advantageous depending on the level and type of pest infestation that occurs locally. There is no reason to expect that deregulation of MON 89034 will have any adverse impact on cropland use in the U.S., instead, like the existing biotechnology- derived lepidopteran protection corn, MON 89034 will protect corn yields, increase agricultural land productivity, and decrease mycotoxin levels in corn grain, which will translate into direct benefits for farmers and consumers. regime.and and AG 6. Mitigation and Remediation Measures before and after Registration and Deregulation of MON 89034 in the U.S. Bayer property protectionpublishing of contents its Under the Coordinated Framework for Regulation parties.of Biotechnology,data the thereforeresponsibility and/or for regulatory oversight of biotechnology-derived crops falls onor three lead federal affiliates. agencies: EPA, FDA, and USDAproperty (Federal intellectual Register,third 1986). Deregulationmay of MON 89034 its from USDA constitutes only one componentas of the overall regulatory oversight and the of and review of this product. is MON 89034 cannot regulatorybe released and marketedowner. until all three a document agencies have completed theirany review such and assessments under their respecits tive jurisdictions. reproductiondocument owner this of under of Food and feed from biotechnology-derivedrightsthe crops arethis subject to regulatory review by documentand/or rights FDA under the Federal Food,to of Drug, fall and Cosmeticuse Actof [21 U.S.C. 346 a(d)]. Since 1992, FDA has used a voluntary consultation process to workthe together with biotechnology- derived Thisproduct developers tomay identify distribution, andand resolve any issues regarding the safety and rights owner nutritional contentsubject of food and feed from biotechnology-derived crops that would necessitate legal action by the agency if thethe productsviolate were introduced into commerce. be copy Although this process is not legally binding,of to date, all biotechnology-derived crops and marketedmay in the U.S.document havepublication, completed the consultation process before commercialization. It exploitation Using the currentthis process, Monsanto has initiated and will complete a consultation with FDA prior to commercialany distribution of MON 89034 (refer to Petition section I.E.2). permission prohibited Monsanto has provided FDA a comprehensive data package which included: (1) the the be intended technicalcommercial effect of the modification of the corn plants; (2) a molecular characterizationFurthermore,any of the modification, including the identities, sources, and functions of the introducedConsequently, geneticwithout material; (3) information on the expressed protein products encoded by the introduced genes; (4) information on assessment of potential allergenicity of the introduced proteins; (5) information on assessment of potential toxicity of the introduced proteins; and (6) information on the compositional and nutritional characteristics of the food and feed derived from MON 89034. At the conclusion of the consultation process, FDA has the ability to notify the submitter if there are unresolved questions regarding the safety of the food under review, thereby effectively halting introduction of the food into commerce. As an ultimate mitigation measure, FDA has the authority to initiate a variety

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 18 of 26 of actions against food or feed in commerce if the agency has reason to believe that the food or feed is unsafe.

As presented in the Petition section I.E.1, because MON 89034 contains two insecticidal proteins, it is also subject to regulatory review and oversight by EPA under the Federal Insecticide, Fungicide and Rodenticide Act [7 U.S.C. §136(u)] and the Federal Food, Drug, and Cosmetic Act [21 U.S.C. 346 a(d)]. Monsanto has filed applications with EPA for the registration of the plant-incorporated protectants Cry1A.105 and Cry2Ab2 proteins, and the genetic material (vector PV-ZMIR245) necessary for their production. Monsanto has also submitted data to enable EPA to conduct a food safety review of those proteins in support of exemptions from the requirement of a tolerance. Sale or distribution of those insecticidal proteins or their presence in food or feed without the regime.and requisite EPA approvals would be a violation of federal law. and AG

Since 1995, EPA has reviewed the safety and environmental effects and approved the use of 23 biotechnology-derived Bt protein/cropBayer combinations property (E PA,protection 2007).publishing Over the past decade, many of these Bt crops, in particular,of Bt corn and Bt cotton have contentsbeen cultivated its in the U.S. over substantial acres. No adverse effectsparties.data on humans, animals,therefore or the environment have been documented from the use of these cropsand/or (Petitionor section VIII). affiliates. EPA has implemented a comprehensiveproperty intellectual datathird review and assessmentmay process on the its biotechnology-derived Bt crops and otheras plant-incorporated protectants or PIPs (EPA, the of and 2001b). The review includes,is but is not limitedregulatory to, the examinationowner. of data and a document information on (1) the sourceany ofsuch the genes; (2) the expression itsof the PIPs; (3) the reproductiondocument modifications to the introduced traitowner as compar ed to thatthis trait in naofture; (4) the biology of the recipient crops; (5) environmental fateunder of the PIPs;of (6) potential effects on non-target rightsthe this organisms, threateneddocumentand/or and endangered species; and (7) potentialrights toxicity and allergenicity to of fall use of of the PIPs on humans and animals. the This may distribution,and rights owner As an importantsubject mitigation and remediation measure, EPA has placed time restrictions on many of the registered PIPs and requiresthe re valuationviolate and re-registration prior to the be copy expiration date for those PIPs if the technologyof providers decide to continue to market and those productsmay (EPA,document 2007).publication, One concern with the use of Bt crops is the potential It exploitation development andthis evolution of resistance from the insect pests as has been observed with chemical insecticidesany in the past. As a preventative and remedial measure, EPA has permission implemented conditional registrationsprohibited with mandatory requirements for Bt crop developers to implement Insect Resistance Management (IRM) plans. As discussed the be earlier in this commercialaddendum, the IRM programs include refuge requirement, grower agreements,Furthermore,any IRM education, and Compliance and Assurance Program, and insect resistanceConsequently, monitoringwithout [refer to reference EPA (2005e) for an example of the details of these requirements]. Annual reporting to EPA is required with respect to these requirements and the sales of the product. It is expected that EPA will implement similar measures and requirements to MON 89034 and the combined trait products containing MON 89034.

Monsanto employs a rigorous product stewardship program that demonstrates respect for our customers, their markets and the environment. Our market stewardship program

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 19 of 26 considers many factors to ensure global integration and increased transparency. In keeping with past practice, we will not launch MON 89034 without regulatory approvals from key corn import countries to ensure global compliance and the flow of international trade. We commit to the industry practices on seed quality and control to prevent adventitious presence of unapproved traits. Before commercializing MON 89034, a detection method will be made available to grain producers, processors and buyers. Our stewardship policy is the shared responsibility of Monsanto, our licensees and our customers to insure that our products are used properly. We are committed to legal and ethical obligations to ensure that our products and technologies are safe and environmentally responsible, and do not pose undue risks to human health or the environment during any stage of their life cycle. As such, Monsanto has policies in place to monitor our procedures as we research, develop, design, manufacture, market and regime.and discontinue products through the product life cycle. and AG

References Bayer property protectionpublishing of contents its 1. ABSTC. 2006. Monitoring the Susceptibilityparties. ofdata Corn Lepidopteratherefore Pests to and/or Cry1Ab and Cry1F Proteins: 2005 Monitoring Results. Reportor submitted to EPA affiliates. by the IRM Technicalproperty Subcommittee intellectualthird of the Agriculturmayal Biotechnology its Stewardship Technical Committeeas (ABSTC). EPA MRID # 46874901. the of and 2. Ahmad, A., G.E.is Wilde, and K.Y. Zregulatoryhu. 2005. Detectabilityowner. of coleopteran- a document specific Cry3Bb1 proteinany such in soil and its effect on nontargetits surface and below- reproductiondocument ground arthropods. Environ. ownerEntomol. 34:385-394.this of under of 3. Al-Deeb, M.A., G.E.rights Wilde,the J.M. Blair, and T.C.this Todd. 2003. Effect of Bt corn documentand/or rights for corn rootwormto controlof fall on nontargetuse soilof microarthropods and nematodes. Environ. Entomol. 32:859-865. the 4. Bakan,This B., D. Melcion,may D. distribution,Richard-and Molard, and B. Cahagnier. 2002 Fungal rights owner growth subjectand Fusarium mycotoxin content in isogenic traditional maize and genetically modified maize grownthe in Franceviolate and Spain. J. Agric. Food Chem. be copy 50:728-731. of and 5. Baumgartemay S.document andpublication, C.C. Tebbe. 2005. Field studies on the environmental fate of It exploitation the Cry1Abthis Bt toxin produced by transgenic maize (MON 810) and its effect on bacterial communitiesany in the maize rhizosphere. Mol. Ecol. 14:2539-2551. permission 6. Betz, F.S., B.G. Hammond,prohibited and R.L. Fuchs. 2000. Safety and advantages of Bacillus thuringiensis protected plants to control insect pests. Regul. Toxicol. the be Pharmacol.commercial 32:156-173. 7.Furthermore, Blackwood,any C.B., and J.S. Buyer. 2004. Soil microbial communities associated Consequently,with Bt withoutand non-Bt corn in three soils. J. Environ. Quality. 33:832-836. 8. Bourguet, D., J. Chaufaux, M. Seguin, C. Buisson, J.L. Hinton, T.J. Stodola, P. Porter, G. Cronhohn, L.L. Buschman, and D.A. Andow. 2003. Frequency of alleles conferring resistance to Bt maize in French and US Corn Belt populations of the European corn borer, Ostrinia nubilalis. Theor. Appl. Genet. 106:1225- 1233. 9. Caprio, M. 1998. Evaluating resistance management strategies for multiple toxins in the presence of external refuges. J. Econ. Entomol. 91:1021-1031.

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 20 of 26 10. Carpenter, J.E., and L.P. Gianessi. 2001. Agricultural biotechnology: Updated benefits estimates. In Report published by the National Center for Food and Agricultural Policy, Washington, D.C. 11. Carpenter, J.E., S. Sankula, C.E. Silvers, and L.P. Gianessi. 2004. Insecticidal Bacillus thuringiensis plants versus chemical insecticides. ACS Symposium Series 866 - Agricultural Biotechnology Chapter 3:37-51. 12. Devare, M., L.M. Londono-R and J.E. Thies. 2007. Neither transgenic Bt maize (MON863) nor tefluthrin insecticide adversely affect soil microbial activity or biomass: A 3-year field analysis. Soil Biol. Biochem. in-press (accepted 8 March 2007). 13. Donegan, K.K., D.L. Schaller, J.K. Stone, L.M. Ganio, G. Reed, P.B. Hamm, and R.J. Seidler. 1996. Microbial populations, fungal species diversity and plant regime.and pathogen levels in field plots of potato plants andexpressing the Bacillus AG thuringiensis var. tenebrionis endotoxin. Trans. Res. 5:25-35. 14. Douville, M., F. Gagne, L. Masson, J. McKay, and C. Blaise. 2005. Tracking the source of Bacillus thuringiensis Cry1AbBayer endotoxin property in the protection environment.publishing Biochem. Systematics Ecol. 33:219-232. of contents its 15. Dubelman, S., B.R. Ayden, B.M. Bader, C.R.parties. Brown,data C. Jiang, andtherefore D. Vlachos. 2005. Cry1Ab protein does not persist in soil after 3 yearsand/or orof sustained Bt corn affiliates. use. Environ. Entomol. property34:915-921. intellectual third may its 16. as the of and 2002a. Aerobicis soil degradationregulatory of the Bacillus thuringiensisowner. Cry2Ab2 a document protein derived fromany cotton such leaf tissue. Monsanto Technicalits Report MSL-16892, reproductiondocument St. Louis, MO. owner this of 17. under of rightsthe this document2002b.and/or Aerobic soil degradation of the Bacillusrights thuringiensis Cry3Bb1 to of fall use of variant protein produced by corn rootworm theprotected corn event MON 863. MonsantoThis Technical Reportmay MSL-17102,distribution,and St. Louis, MO. rights owner 18. EPA. 2001a.subject Bacillus thuringiensis subspecies Cry1F protein and the genetic material necessary for its productionthe (plasmid violate insert PHI 8999) in corn Fact Sheet. be copy http://www.epa.gov/oppbppd1/biopesticidesof /ingredients/factsheets/factsheet_0064 and 81.htmmay . documentpublication, It exploitation 19. EPA. 2001b.this Biopesticides Registration Action Document - Bacillus thuringiensis Plant-Incorporatedany Protectants. permission http://www.epa.gov/oppbppd1/biopeprohibited sticides/pips/bt_brad.htm. 20. EPA. 2005a. Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the the be genetic materialcommercial necessary for their production (plasmid insert PHP 17662) in Furthermore,Eventany DAS-59122-7 corn (006490) Fact Sheet. Consequently,http://www.epa.gov/oppbppd1/biopesticideswithout /ingredients/factsheets/factsheet_0064 90.htm. 21. EPA. 2005b. Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their production (plasmid insert PHP 17662) in Event DAS-59122-7 corn & Bacillus thuringiensis Cry1F protein and the genetic material necessary for its production (plasmid insert PHI8999) in Event TC1507 corn (006490, 006481) Fact Sheet. http://www.epa.gov/oppbppd1/biopesticides/ingredients/factsheets/factsheet_0064

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 21 of 26 81-006490.htm. 22. EPA. 2005c. Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary for its production (vector ZMIR13L) in event MON 863 corn (006484) Biopesticide Registration Action Document (BRAD). http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006484. htm. 23. EPA. 2005d. Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary for its production (vector ZMIR13L) in event MON 863 corn & Bacillus thuringiensis Cry1Ab delta-endotoxin and the genetic material necessary for its production in corn (006430, 006484) Fact Sheet. http://www.epa.gov/oppbppd1/biopesticides/ingredients/factsheets/factsheet_0064 30-006484.htm. regime.and 24. EPA. 2005e. Bacillus thuringiensis Cry3Bb1 protein andand the genetic material AG necessary for its production (Vector ZMIR39) in Event MON 88017 corn (OECD Unique Identifier: MON-88Ø17-3) (006502, 006430) Fact Sheet. http://www.epa.gov/oppbppd1/biopesticidesBayer /ingredients/factsheets/factsheet_0064property protectionpublishing 30-006502.htm of contents its 25. EPA. 2005f. Bacillus thuringiensis var. aizawaiparties. straindata PS811 Cry1Ftherefore insecticidal crystal protein and the genetic material necessary forand/or its orproduction (plasmid affiliates. insert PHP12537) inproperty Event intellectualDAS-06275-8third corn (006491)may Fact Sheet. its http://www.epa.gov/oppbppd1/biopesticidesas /ingredients/factsheets/factsheet_0064 the of and 91.htm. is regulatory owner. a document 26. EPA. 2005g. Biopesticideany such Regi stration Action Document. Bacillusits thuringiensis reproductiondocument Cry1F corn. owner this of http://www.epa.gov/oppbppd1/biopesticidunder es/ingredients/tech_docs/brad_006481.of rightsthe this pdf. documentand/or rights to of fall use of 27. EPA. 2006. Modified Cry3A protein and the geneticthe material necessary for its productionThis (via elementsmay of pZM26)distribution,and in event MIR604 corn SYN-IR604-8 rights owner (006509)subject Fact Sheet. http://www.epa.gov/oppbppd1/biopesticidesthe violate/ingredients/factsheets/factsheet_0065 be copy 09.htm. of and 28. EPA.may 2007.document Currentpublication, & Previously Registered Section 3 PIP Registrations. It exploitation http://www.epa.gov/oppbppd1/biopesthis ticides/pips/pip_list.htm 29. Farinos, G.P.,any M. Poza, P. Hernandez-Crespo, F. Ortego, and P. Castanera, 2004. permission Resistance monitoring prohibitedof field populations of the corn borers Sesamia nonagrioides and Ostrinia nubilalis after 5 years of Bt maize cultivation in Spain. the be Entomol.commercial Exp. Applic. 110:23-30. 30.Furthermore, Fawcett,any R., and D. Towery. 2002. Conservation tillage and plant biotechnology: Consequently,How newwithout technologies can improve the environment by reducing the need to plow. Report of Conservation Technology Information Center, Purdue University, West Lafayette, IN. 31. FDA. 2007. List of Completed Consultations on Bioengineered Foods. http://vm.cfsan.fda.gov/~lrd/biocon.html. 32. Federal Register. 1986. Coordinated framework for regulation of biotechnology; announcement of policy. June 26, 1986, 51(123):23302-50.

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 22 of 26 33. Federal Register. 1995. Availability of determination of Nonregulated status for genetically engineered corn. Federal Register, Vol. 60, No. 119. June 21, 1995. 34. Gianessi, L.P., C.S. Silvers, S. Sankula, and J.E. Carpenter. 2002. Plant biotechnology: current and potential impact for improving pest management in US agriculture, an analysis of 40 case studies. www.ncfap.org/whatwedo/biotech- us.php 35. Hammond, B., K. Campbell, C. Pilcher, A. Robinson, D. Melcion, B. Cahagnier, J. Richard, J. Segueira, J. Cea, F. Tatli, R. Grogna, A. Pietri, G. Piva, and L. Rice. 2003. Reduction of fumonisin mycotoxins in Bt corn. Poster presented at the 42nd Annual Meeting of Society of Toxicology, Salt Lake City, UT. 36. 2006. Insect resistance management plan for second generation lepidopteran-protected corn, MON 89034. Monsanto Technical Report 06-RA- regime.and 39-06. and AG 37. Head, G., J.B. Surber, J.A. Watson, J.W. Martin, and J.J. Duan. 2002. No detection of Cry1Ac protein in soil after multiple years of transgenic Bt cotton (Bollgard) use. Environ. Entomol. Bayer31:30-36. property protectionpublishing 38. Herman, R.A., J.D. Wolt, and W.R.of Halliday. 2002a. Rapid degradationcontents of Cry1F its insecticidal crystal protein in soil. J. Agric. Foodparties. Chem.data 50:7076-7078.therefore 39. Herman, R.A., P.N. Scherer, and J.D. Wolt. 2002b. Rapidand/or degradationor of a binary, affiliates. PS149B1, δ-endotoxin propertyof Bacillus intellectual thuringiensisthird in soil, and a novelmay mathematical its model for fitting curve-linear deascay. Environ. Entomol. 31:208-214. the of and 40. Herman, R.A., S.L.is Evans, D.M. Shanaharegulatoryn, C.A. Mihaliak, G.A.owner. Bormett, D.L. a document Young, and J. Buehrer.any 2001.such Rapid degradation of Cry1F delta-endotoxinits in soil. reproductiondocument Environ. Entomol. 30:642-644.owner this of 41. Hopkins, D.W., and E.G. Gregorich.under 2005. of Decomposition of residues and loss of rightsthe this the δ-endotoxindocumentand/or from transgenic (Bt) corn (Zea maysrights L.) in soil. Can. J. Soil Sci. to of fall use of 85:19-26. the 42. Hyde,This J., M.A. Martin,may P.V. distribution, Preckel,and L.L. Buschman, C.R. Edwards, P.E. rights owner Sloderbeck,subject and R.A. Higgins. 2003. The value of Bt corn in southwest Kansas: A Monte Carlo simulation approach.the J. violateAgric. Resource Econ. 28:15-33. be copy 43. James, C. 2006. Global statusof of commercialized biotech/GM crops: 2006. and ISAAAmay Briefdocument No.publication, 35. It exploitation 44. Koskella,this J., and G. Stotzky. 2002. Larvicidal toxins from Bacillus thuringiensis subspp. kurstaki,any morrisoni (strain tenebrionis), and israelensis have no permission microbicidal or microbiostaticprohibited activity against selected bacteria, fungi, and algae in vitro. Can. J. Microbiol. 48:262-267. the be 45. Koziel, M.G.,commercial G.L. Beland, C. Bowman, N.B. Carozzi, R. Crenshaw, L. Crossland, Furthermore,J. Dawson,any N. Desai, M. Hill, S. Kadwell, K. Lewis, D. Maddox, K. McPherson, Consequently,M.R. Meghji,without E. Merlink, R. Rhodes, G.W. Warren, M. Wright, and S.V. Evola. 1993. Field performace of an elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Biotechnol. 11:194-200. 46. Madigan, M., and J. Martinko (eds) 2005. Brock Biology of Microorganisms, 11th ed., Prentice Hall. 47. Martin, M.A., and J. Hyde. 2001. Economic considerations for the adoption of transgenic crops: The case of Bt corn. J. Nematol. 33:173-177.

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 23 of 26 48. Miller, J.D. 2001. Factors that affect the occurrence of fumonisins. Environ. Health Persp. 109 (suppl 2):321-324. 49. 2006. Aerobic soil degradation of the purified Cry2Ab2 and Cry1A.105 proteins. Monsanto Technical Report MSL-20174, St. Louis, MO. 50. Munkvold, G.P., R.L. Hellmich, and L.G. Rice. 1999 Comparison of fumonisin concentrations in kernels of transgenic Bt maize hybrids and non-transgenic hybrids. Plant Dis. 83:130-138. 51. NCGA. 2007. No-till Corn on the Rise, NCGA Notes (3-26-07). http://www.ncga.com/news/notd/2007/march/032607.asp 52. Palm, C.J., D.L. Schaller, K.K. Donegan, and R.J. Seidler. 1996. Persistence in regime.and soil of transgenic plant produced Bacillus thuringiensis andkurstaki δ-endotoxin. Can. AG J. Microbiol. 42:1258-1262. 53. Roush, R.T. 1998. Two toxin strategies for management of insecticidal transgenic crops: can pyramiding Bayersucceed whereproperty pesticide protection mixturespublishing have not? Phil. Trans. R. Soc. Lond. 353:1777-1786.of contents its 54. Sankula, S. 2006. Quantification of theparties. impactsdata on US agriculturetherefore of biotechnology-derived crops planted in 2005. Nationaland/or Centeror for Food and affiliates. Agriculture Policy. propertyhttp://www.ncfap.org/what intellectualthird wedo/pdf/2005biotechimpacts-may its finalversion.pdf as the of and 55. Sankula, S., andis E. Blumenthal. 2004.regulatory Impacts on USowner. agriculture of a document biotechnology-derivedany crops such planted in 2003: an update ofits eleven case studies. reproductiondocument www.ncfap.org/whatwedo/biotech-us.phpowner this of 56. Sankula, S., G. Marmon, and E.under Blum enthal.of 2005. Biotechnology-derived crops rightsthe this planteddocument in 2004and/or – impacts on US agriculture. www.ncfap.org/whatwedo/biotech-rights to of fall use of us.php the 57. Saxena,This D. and G. Stotzky.may 2001.distribution,and Bacillus thuringiensis (Bt) toxin released from rights owner root exudatessubject and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, andthe fungi violate in soil. Soil Biol. Biochem. 33:1225- be copy 1230. of and 58. Shelton,may A.M.,document J.Z.publication, Zhao, and R.T. Roush. 2002. Economic, ecological, food It exploitation safety, andthis social consequences of the deployment of Bt transgenic plants. Annu. Rev. Entomol.any 47:845-81. permission 59. Siegfried, B.D., T. Spencer,prohibited A. Crespo, E. Pereira, and P. Marcon, 2006. Ten years of Bt resistance monitoring in the European corn borer: what we know, the be what we commercialdon't know, and what we can do better. Pp. 168-171 in Proceedings of Furthermore,The any9th International Symposium on the Biosafety of Genetically Modified Consequently,Organisms,without Jeju Island, Korea, September 24-29, 2006. 60. Sims, S., and L.R. Holden. 1996. Insect bioassay for determining soil degradation of Bacillus thuringiensis subsp. kurstaki Cry1A(b) protein in corn tissue. Environ. Entomol. 25:659-664. 61. Sims, S.R., and J.E. Ream. 1997. Soil inactivation of the Bacillus thuringiensis subsp. kurstaki CryIIA insecticidal protein within transgenic cotton tissue: laboratory microcosm and field studies. J. Agric. Food Chem. 45:1502-1505.

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 24 of 26 62. Smith, C.W., J. Betran, and E.C.A. Runge. (eds) 2004 Corn: Origin, History, Technology, and Production. John Wiley and Sons, Inc. 949pp. 63. Smith, R.A., and J.W. Barry. 1998. Environmental persistence of Bacillus thuringiensis spores following aerial application. J. Invertebrate Pathol. 71:263- 267. 64. Sprague, G.F., and J. W. Dudley. (eds) 1988. Corn and Corn Improvement, 3rd Edition. Agronomy Society of America. 986pp. 65. Stodola, T.J., D.A. Andow, A.R. Hyden, J.L. Hinton, J.J. Roark, L.L. Buschman, P. Porter, and G.B. Cronholm. 2006. Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in southern United States Corn Belt population of European corn borer (Lepidoptera: Crambidae). J. Econ. Entomol. 99: 502-507. 66. Stotzky, G. 2004. Persistence and biological activity in soil of the insecticidal regime.and proteins from Bacillus thuringiensis, especially from transgenicand plants. Plant Soil AG 266:77-89. 67. Tabashnik, B.E., Y. Carriere, T.J. Dennehy, S. Morin, M.S. Sisterson, R.T. Roush, A.M. Shelton, and J.Z. Zhao. 2003.Bayer Ins ectproperty resistance protection to transgenicpublishing Bt crops: Lessons from the laboratory and offield. J. Econ. Entomol. 96:1031-1038.contents its 68. Tabashnik, B.E., Y.B. Liu, T. Malvar, D.G. parties.Heckel,data L. Masson, V.therefore Ballester, F. Granero, J.L. Mensua, and J. Ferre. 1997. Global variationand/oror in the genetic and affiliates. biochemical basis of diamondbackproperty intellectual moththird resistance to Bacillus thuringiensismay . Proc. its Natl. Acad. Sci. USA 94:12780-12785.as the of and 69. USDA. 2007a. Petitionsis of Nonregulatedregulatory Status Granted or Pendingowner. by APHIS. a document http://www.aphis.usda.gov/brs/not_reg.htmlany such its reproductiondocument 70. USDA. 2007b. Agricultural ownerProjections to 2016.this Office of the Chief Economist, World Agricultural Outlook Board,under Unitedof States Department of Agriculture. rightsthe this Prepareddocument byand/or the Interagency Agricultural/Projectionsrights Committee. Long-Term to of fall use of Projections Report OCE-2007-1. the 71. USDA-ERS.This 2007. Organicmay production.distribution,and http://www.ers.usda.gov/Data/Organic/ rights owner 72. USDA-NASS.subject 2006. National Agricultural Statistics Service, United States Department of Agriculture. Acreagethe Report,violate Cr Pr2-5 (6-06) a. June 30, 2006. be copy http://usda.mannlib.cornell.edu/usdaof /nass/Acre/2000s/2006/Acre-06-30-2006.pdf and 73. USDA-NASS.may document 2007.publication, Online database (www.nass.usda.gov) at the United States It exploitation Departmentthis of Agriculture, National Agricultural Statistics Service (USDA- NASS). any permission 74. Vettori, C., D. Paffetti, D.prohibited Saxena, G. Stotzky, and R. Giannini. 2003. Persistence of toxins and cells of Bacillus thuringiensis subsp. kurstaki introduced in sprays the be to Sardiniacommercial soils. Soil Biol. Biochem. 35:1635-1642. 75.Furthermore, West,any A. W. 1984. Fate of the insecticidal, proteinaceous parasporal crystal of Consequently,Bacilluswithout thuringiensis in soil. Soil Biol. Biochem. 16:357-360. 76. Windham, G.L., W.P. Williams, and F.M. Davis. 1999 Effects of the southwestern corn borer on Aspergillus flavus kernel infection and aflatoxin accumulation in maize hybrids. Plant Dis. 83:535-540. 77. Wu, F. 2006. Mycotoxin reduction in Bt corn: potential economic, health, and regulatory impacts. Trans. Res. 15:277-289.

Monsanto Company 06-CR-166U Addendum to Section VIII, Page 25 of 26 78. Zhao, J.Z., J. Cao, Y.X. Li, H.L. Collins, R.T. Roush, E.D. Earle, and A.M. Shelton. 2003. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nat. Biotechnol. 21:1493-1497.

and and regime. AG

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