Food and Feed Safety and Nutritional Assessment of MON 88017 Corn

Conclusion Based on Data and Information Evaluated According to FDA’s Policy on Foods from New Plant Varieties

March 30, 2004

and FDA BNF 97 and regime. AG

Monsanto # 04-CR-109F Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. propertyPrepared intellectual by:third 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 Contributorsfall 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 Submitted by: Furthermore,any Consequently,without

Monsanto Company 800 North Lindbergh Blvd. St. Louis, MO 63167

Monsanto Company # 04-CR-109F Page 1 of 223 PART I:

TABLE OF CONTENTS Page PART I:…………………………………………………………………………… 2 Table of Contents…………………………………………………………………. 2 List of Tables……………………………………………………………………... 6 List of Figures…………………………………………………………………….. 8 Certification………………………………………………………………………. 10 Release of Information……………………………………………………………. 11 Abbreviations and Definitions……………………………………………………. 12 Narrative Summary……………………………………………………………….. 16

and PART II: Synopsis of consultation summary……………………………………..and regime. 20 Section 1. Name and address of the submitter…………………………….……...AG 20 Section 2. The subject of this summary and the plant species from which they Bayer property protectionpublishing were derived…………………………………………………….……..of contents 20 its Section 3. Distinctive designations given to the subjectparties. ofdata this summary……….therefore 20 and/oror Section 4. Identity and sources of the geneticaffiliates. material introduced into property intellectualthird may MON 88017…………………………………………………………...its 20 as the of and Section 5. The intended technicalis effect of MONregulatory 88017……………………….. owner. 21 a document any such its Section 6. The application and uses of MON 88017…………………………….reproductiondocument 22 owner this of Section 7. The applications for which MONunder 88017 of is not suitable……….…….. 24 rightsthe this documentand/or rights to of fall use of PART III: Status of submissions to other regulatory agencies…………………...the 25 This may distribution,and Section 1. Status of submissionrights to USDA-APHIS……………………………….. owner 25 subject Section 2. Status of submissionbe to EPA…………………………………………..the violate 25 copy of Section 3. Status of submissions to foreign governments………………………...and 26 may documentpublication, It exploitation PART IV: Development thisof MONany 88017………………………………………… 27 permission Section 1. History and biology of corn…………………………………………...prohibited 27 the 1.1. Scientific namecommercial and taxonom be ic classification of corn……………….. 27 1.2. Growth and reproductive characterstics of corn…………………….. 29 Furthermore,any 1.3. HistoryConsequently, of cornwithout development………………………………………… 29 Section 2. Characterization of the vector used in transformation………………... 31 2.1. The cp4 epsps gene and CP4 EPSPS protein………………………... 31 2.1.a. The Arabidopsis thaliana EPSPS transit peptide (CTP2)……….. 32 2.1.b. cp4 epsps regulatory sequences…………………………………. 32 2.2. The cry3Bb1 gene and Cry3Bb1 protein……………………………. 32 2.3. T-DNA borders……………………………………………….……... 33 2.4. Genetic elements outside of the T-DNA border…………………….. 33 Section 3. Characterization of the introduced genetic material………………….. 40 3.1. Molecular analysis – introduced DNA…..…………………………... 40 3.1.a. Insert and copy number………………………………………….. 44 3.1.b. cp4 epsps cassette intactness…………………………………….. 46

Monsanto Company # 04-CR-109F Page 2 of 223 Table of contents (cont.) Page PART IV. Section 3. (cont.) 3.1.c. p-ract + ract1 intron probe……………………………………….. 46 3.1.d. ctp2 + cp4 epsps probe…………………………………………... 48 3.1.e. NOS 3’ probe……………………………………………………. 48 3.1.f. cry3Bb1 cassette intactness……………………………………… 51 3.1.g. p-e35S probe…………………………………………………….. 51 3.1.h. wtCAB leader + ract1 intron probe……………………….…….. 51 3.1.i. cry3Bb1 probe…………………………………………….…….. 52 3.1.j. tahsp17 3’ probe…………………………………………………. 52 3.2. Analysis for backbone fragments……………………………………. 57 3.3. Insert stability in multiple breeding generations……………………. 59 3.4. Organization of the insert in MON 88017…………………………... 62 3.5. Inheritance of corn rootworm protection and glyphosate toleranceand and regime. traits in MON 88017……………………………….………………..AG 65 3.6. Summary of the molecular characterization of MON 88017………... 67 Section 4. Other data and information about developmentBayer property of MONprotectionpublishing 88017……... 67 of contents its parties.data therefore PART V: Presence of genes that encode resistance to antibiotics…………….….and/oror 68 affiliates. property intellectualthird may its as PART VI: Characterization of thethe prot ofeins introducedand into MON 88017…….…. 69 is regulatory owner. a document Section 1. Identity and function ofany the suchCP4 EPSPS and Cry3Bb1its proteins reproductiondocument present in MON 88017………………………………………………...owner this of 69 under of 1.1. Characterization of therights CP4the EPSPS protein producedthis in documentand/or of rights MON 88017………………………………………………….……...to of fall use 69 the This may distribution,and 1.1.a. Western blot analysisrights to confirmowner the identity of the CP4 EPSPS protein…………………………………………………………...subject 70 be the violate 1.1.b. Molecular weightcopy and purityof of the CP4 EPSPS protein………... 70 and 1.1.c. MALDI-TOFmay document MSpublication, tryptic map mass analysis……………………. 73 It exploitation 1.1.d. N-terminalthis sequence any analysis……………………………………. 73 1.1.e. Functional activity………………………………………………..permission 75 prohibited 1.1.f. Glycosylation analysis……………………………………………the be 75 1.1.g. Conclusionscommercial for characterization of the CP4 EPSPS protein Furthermore,any producedConsequently,without in MON 88017……………………………………….. 77 1.2. Characterization of the Cry3Bb1 protein produced in MON 88017… 77 1.2.a. Western blot analysis to confirm the identity of the Cry3Bb1 protein…………………………………………………………... 78 1.2.b. Molecular weight and purity of the Cry3Bb1 protein……….…… 79 1.2.c. MALDI-TOF MS tryptic map mass analysis…………………….. 82 1.2.d. Functional activity of the Cry3Bb1 protein……………………… 83 1.2.e. Glycosylation analysis of the Cry3Bb1 protein………………….. 83 1.2.f. Conclusions of the characterization of the Cry3Bb1 protein produced in MON 88017…………………………………………. 83 Section 2. Levels of the CP4 EPSPS and Cry3Bb1 proteins in MON 88017……. 86

Monsanto Company # 04-CR-109F Page 3 of 223 Table of contents (cont.)

Page Section 3. Estimate of dietary exposure………………………………………….. 91 3.1. Estimated dietary exposure to the CP4 EPSPS protein……………… 91 3.2. Estimated dietary exposure to the Cry3Bb1 protein………………… 92 3.3. Margins of exposure…………………………………………………. 92 Section 4. Assessment of the potential for allergenicity of the CP4 EPSPS and Cry3Bb1 proteins produced in MON 88017………………………….. 93 4.1. Assessment of the potential for allergenicity of the CP4 EPSPS protein………………………………………………………………. 95 4.1.a. Source of the CP4 EPSPS protein………………………………... 95 4.1.b. Proportion of the total protein – CP4 EPSPS……………………. 95 4.1.c. Bioinformatics analyses of sequence similarity of the CP4 EPSPS protein produced in MON 88017 to allergens and toxins…………and 96 and regime. 4.1.d. Stability of the CP4 EPSPS protein AGin simulated digestive fluids.. 96 4.2. Assessment of the potential for allergenicity of the Cry3Bb1 protein……………………………………………………………….Bayer property protectionpublishing 100 of contents 4.2.a. Source of the Cry3Bb1 protein……………………………….…..its 100 parties.data therefore 4.2.b. Proportion of the total protein – Cry3Bb1 ……………………….and/oror 100 affiliates. 4.2.c. Bioinformatics analysesproperty of sequence intellectualthird similarity of themay Cry3Bb1 its as protein produced inthe MON of 88017and to allergens and toxins………… 101 is regulatory owner. a document 4.2.d. Stability of the Cry3Bb1any such prot ein in simulated digestiveits fluids.. 101 reproductiondocument 4.3. Discussion and conclusions………………………………………….owner this of 106 under of Section 5. Safety assessment of therights CP4the EPSPS and Cry3Bb1this proteins………… 106 documentand/or of rights 5.1. Safety assessment ofto theof CP4 fall EPSPS use protein……………………….. 106 the This may distribution,and 5.1.a. Structural similarityrights of the CP4 EPSPSowner protein to known proteins and toxins………………………………………………………….subject 106 be the violate 5.1.b. Acute oral toxicitycopy study withof the CP4 EPSPS protein………….. 107 and 5.1.c. Safetymay of the documentdonorpublication, organism: Agrobacterium sp. strain CP4……. 107 It exploitation 5.1.d. Similaritythis of anyCP4 EPSPS to EPSPSs derived from food sources with a long history ofpermission safe consumption………………………….. 108 prohibited 5.1.e. Presence of CP4the EPSPS be protein in food and feed crops…………. 108 5.2. Safety assessmentcommercial of the Cry3Bb1 protein………………………….. 109 Furthermore,any 5.2.a. StructuralConsequently, withoutsimilarity of the Cry3Bb1 protein to known proteins and toxins…………………………………………………………. 109 5.2.b. Acute oral toxicity study with the Cry3Bb1 protein……………... 109 5.2.c. Safety of the donor organism: Bacillus thuringiensis……………. 111 5.2.d. Human and animal safety of B.t. Cry proteins…………………… 112 5.3. Conclusions………………………………………………………….. 112

Monsanto Company # 04-CR-109F Page 4 of 223 Table of contents (cont.)

Page PART VII: Food/feed safety and nutritional assessment of MON 88017 corn….. 117 Section 1. Corn as the comparable feed and food………………………………... 117 Section 2. Historical uses of corn………………………………………………… 117 2.1. History and utilization of corn……………………………………….. 117 2.2. Corn as food source………………………………………………….. 117 2.3. Corn as feed source…………………………………………………... 118 Section 3. Comparison of the composition and characteristics of MON 88017 to conventional corn……………………………………………………... 118 3.1. Levels of significant nutrients, anti-nutrients, and secondary metabolites……………………………………………………………. 118 3.2. Levels of naturally occurring toxicants and anti-nutrients…………... 134 3.3. Any intended changes to the composition of food and feed………….and 134 and regime. Section 4. Other information relevant to the safety AGand nutritional assessment of MON 88017…………………………………………………………... 134 Section 5. Substantial equivalence of MON 88017Bayer to conventionalproperty protectionpublishing corn………... 134 of contents Conclusions………………………………………………………………………..its 135 parties.data therefore REFERENCES…………………………………………………………………….and/oror 137 affiliates. APPENDIX I. Materials and methodsproperty used intellectualfor thethird molecular analysismay of its as MON 88017….…………………………………………………..the of and 149 is regulatory owner. a document APPENDIX II. Materials and methodsany such used for the characterizationits of the reproductiondocument introduced proteins in MONowner 88017……………………………... this of 153 under of APPENDIX III. Materials and methodsrightsthe used for analysisthis of expression of the documentand/or of rights introduced proteins………………………………………………to of fall use 167 the This may distribution,and APPENDIX IV. Materials and rightsmethods used forowner compositional analysis of MON 88017……………………………………………………..subject 173 be the violate Supplemental Summary: copy of and Fattymay and aminodocumentpublication, acid composition of MON 88017 and It exploitation conventionalthis any corn calculated as percent of dry weight, percent of total fat, and percentpermission of total protein…………………………….. 207 prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company # 04-CR-109F Page 5 of 223 List of Tables Page Table 1. Summary of genetic elements in vector PV-ZMIR39……………… 35 Table 2. Comparison of expected and observed segregation frequencies for progeny of MON 88017 plants……………………………………... 66 Table 3. N-terminal sequence of the CP4 EPSPS protein produced in MON 88017………………………………………………………… 74 Table 4. Levels of the Cry3Bb1 and CP4 EPSPS proteins in tissues of MON 88017………………………………………………………… 87 Table 5. Levels of the CP4 EPSPS protein in overseason tissues of MON 88017 ………………………………………………………... 88 Table 6. Levels of the Cry3Bb1 protein in overseason tissues of MON 88017………………………………………………………… 89 Table 7. Limits of detection and quantitation for the ELISA of the Cry3Bb1and and regime. and CP4 EPSPS proteins ……………………………………………AG 90 Table 8. Margins of exposure for dietary consumption of CP4 EPSPS and Cry3Bb1 proteins in MON 88017 whenBayer used property as food protection or animalpublishing of contents feed…………………………………………………………………..its 93 parties.data therefore Table 9. Specific activity of E. coli-produced CP4 EPSPS proteinand/oror after affiliates. property intellectualthird may digestion in simulated gastric fluid………………………………….its 100 as Table 10. Comparison of the deducedthe ofamino acidand sequence of native is regulatory owner. a document CP4 EPSPS to that of otherany EPSPSs………………………………... such its 108 reproductiondocument Table 11. Acute oral NOELs for various B.t.owner and plant-producedthis of Cry3 under of rightsthe this proteins………………………………………………………………documentand/or rights 111 to of fall use of Table 12. Comparison of proximates, fiber, and mineralthe content in forage This may distribution,and from MON 88017 andrights conventional cornowner for combined field sites… 121 Table 13. Comparison of thesubject amino acid content in grain from MON 88017 be the violate and conventional corncopy for combinedof field sites………………….…. 122 and may documentpublication, Table 14. ComparisonIt of the fatty acexploitationid content in grain from MON 88017 and conventionalthis corn any for combined field sites………………….…. 124 Table 15. Comparison of the mineralpermission content in grain from MON 88017 and prohibited conventional corn for combinedthe field sites……………………….. 125 commercial be Table 16. Comparison of the proximates and fiber content in grain from Furthermore,any MON 88017Consequently, andwithout conventional corn for combined field sites………. 126 Table 17. Comparison of the vitamin content in grain from MON 88017 and conventional corn for combined field sites…………………………. 127 Table 18. Comparison of the secondary metabolites and anti-nutrients content in grain from MON 88017 and conventional corn for combined field sites………………………………………………… 128 Table 19. Summary of statistically significant differences in composition of MON 88017 and conventional corn………………..………………. 129 Table 20. Literature and historical ranges of components of corn forage and grain………………………………………………………………… 131

Monsanto Company # 04-CR-109F Page 6 of 223 List of Tables (cont.)

Page APPENDIX IV Table 1. Comparison of the proximates, fiber, and mineral content in forage collected at the IA field site from MON 88017 and conventional 183 corn …………………………………………………………………. Table 2. Comparison of the proximates, fiber, and mineral content in forage collected at the IL field site from MON 88017 and conventional corn………………………………………………………………….. 184 Table 3. Comparison of the proximates, fiber, and mineral content in forage collected at the NE field site from MON 88017 and conventional corn………………………………………………………………….. 185 Table 4. Comparison of the amino acid content in grain collected at the IA field site from MON 88017 and conventional corn…………………and 186 and regime. Table 5. Comparison of the amino acid content in grainAG collected at the IL field site from MON 88017 and conventional corn………………… 188 Table 6. Comparison of the amino acid contentBayer in grainproperty collected protection atpublishing the NE of contents field site from MON 88017 and conventional corn…………………its 190 parties.data therefore Table 7. Comparison of the fatty acid content in grain collectedand/or ator the IA affiliates. field site from MON 88017 propertyand conventional intellectualthird corn…………………may 192 its as Table 8. Comparison of the fatty acidthe of content andin grain collected at the IL is regulatory owner. a document field site from MON 88017any andsuch conventional corn…………………its 193 reproductiondocument Table 9. Comparison of the fatty acid contentowner in grainthis collected of at the NE under of field site from MON 88017rights andthe conventional corn…………………this 194 documentand/or of rights Table 10. Comparison of the mineralto of conten fall t in grainuse collected at the IA field the This may distribution,and site from MON 88017rights and conventionalowner corn………………… 195 Table 11. Comparison of thesubject mineral content in grain collected at the IL field be the violate site from MON 88017copy and conventionalof corn……………………… 196 and Table 12. Comparisonmay of thedocument mineralpublication, content in grain collected at the NE It exploitation field site from MONthis any 88017 and conventional corn………………… 197 Table 13. Comparison of the proximatespermission and fiber content in grain collected prohibited at the IA field site fromthe M beON 88017 and conventional corn………. 198 Table 14. Comparison of thecommercial proximates and fiber content in grain collected Furthermore,any at the ILConsequently, field sitewithout from MON 88017 and conventional corn………. 199 Table 15. Comparison of the proximates and fiber content in grain collected at the NE field site from MON 88017 and conventional corn……… 200 Table 16. Comparison of the vitamin content in grain collected at the IA field site from MON 88017 and conventional corn……………………… 201 Table 17. Comparison of the vitamin content in grain collected at the IL field site from MON 88017 and conventional corn……………………… 202 Table 18. Comparison of the vitamin content in grain collected at the NE field site from MON 88017 and conventional corn………………… 203 Table 19. Comparison of the secondary metabolites and anti-nutrients content in grain collected at the IA field site from MON 88017 and conventional corn…………………………………………………… 204

Monsanto Company # 04-CR-109F Page 7 of 223 List of Tables (cont.)

Page Table 20. Comparison of the secondary metabolites and anti-nutrients content in grain collected at the IL field site from MON 88017 and conventional corn……………………………………………………………………… 205 Table 21. Comparison of the secondary metabolites and anti-nutrients content in grain collected at the NE field site from MON 88017 and conventional corn……………………………………………………………………… 206 Table 22. Fatty acid content in grain from MON 88017 and LH198xLH59 corn calculated as percent of dry weight…………….……………………….. 208 Table 23. Fatty acid content in grain from MON 88017 and LH198xLH59 corn calculated as percent of total fat………………………………………… 211 Table 24. Amino acid content in grain from MON 88017 and LH198xLH59 corn calculated as percent of dry weight…………….………………………..and 214 and regime. Table 25. Amino acid content in grain from MON 88017AG and LH198xLH59 corn calculated as percent of total protein……………….…………………… 219 Bayer property protectionpublishing of contents its parties.data therefore List of Figures and/oror affiliates. property intellectualthird may its Page as Figure 1. Plasmid map of vector PV-ZMIR39…………………………………the of and 34 is regulatory owner. a document Figure 2. Deduced amino acid sequenceany such of the CP4 EPSPS proteinits produced in reproductiondocument MON 88017…………………………………………………………..owner this of 38 under of rightsthe this Figure 3. Deduced aminodocument acidand/or sequence of the Cry3Bb1 proteinrights produced in to of fall use of MON 88017…………………………………………………………the 39 This may distribution,and Figure 4a. Schematic representationrights of the probesowner 1- 6 generated from the vector PV-ZMIR39 forsubject Southern blot analyses……………………………… 41 be the violate Figure 4b. Schematic representationcopy of the ofprobes 7 - 12 generated from the and may documentpublication, vector PV-ZMIR39It for Southernexploitation blot analyses……………………… 42 Figure 5. Schematic representationthis any of the insert present in MON 88017……….. 43 Figure 6. Southern blot analysis ofpermission MON 88017: insert and copy number……... 45 prohibited Figure 7. Southern blot analysisthe of MON 88017: p-ract + ract1 intron probe… 47 commercial be Figure 8. Southern blot analysis of MON 88017: ctp2 + cp4 epsps probe……... 49 Furthermore,any Figure 9. SouthernConsequently, blot analyswithout is of MON 88017: NOS 3’ probe……………….. 50 Figure 10. Southern blot analysis of MON 88017: p-e35S probe………………. 53 Figure 11. Southern blot analysis of MON 88017: wtCAB leader + ract1 intron probe………………………………………………………………….. 54 Figure 12. Southern blot analysis of MON 88017: cry3Bb1 probe……………… 55 Figure 13. Southern blot analysis of MON 88017: tahsp17 3’ probe……………. 56 Figure 14. Southern blot analysis of MON 88017: backbone analysis…………... 58 Figure 15. Breeding tree of MON 88017………………………………………… 60 Figure 16. Stability of integrated DNA in MON 88017…………………………. 61

Monsanto Company # 04-CR-109F Page 8 of 223 List of Figures (cont.)

Page Figure 17. Overlapping PCR analyses across the insert in MON 88017: Part I…. 63 Figure 18. Overlapping PCR analyses across the insert in MON 88017: Part II… 64 Figure 19. Western blot analysis demonstrating equivalence of CP4 EPSPS proteins produced in MON 88017 and E. coli………………………... 71 Figure 20. Molecular weight and purity of the plant-produced CP4 EPSPS protein demonstrated by SDS-PAGE analysis………………………... 72 Figure 21. MALDI-TOF MS coverage map of the CP4 EPSPS protein sequence produced in MON 88017………………………………………….….. 73 Figure 22. Glycosylation analysis of the CP4 EPSPS protein produced in MON 88017…………………………………………………………... 76 Figure 23. Polypeptides derived from the full-length Cry3Bb1 protein…………. 78 Figure 24. Western blot analysis demonstrating equivalence of the plant- andand E. and regime. coli-produced Cry3Bb1 protein……………………………………….AG 80 Figure 25. Molecular weight and purity of the Cry3Bb1 protein demonstrated by SDS-PAGE analysis…………………………………………………...Bayer property protectionpublishing 81 of contents Figure 26. MALDI-TOF MS coverage map of the Cry3Bb1 proteinits sequence parties.data therefore produced in MON 88017……………………………………………...and/oror 82 affiliates. Figure 27. Glycosylation analysis of propertythe Cry3Bb1 intellectualthird protein producedmay in its as MON 88017…………………………………………………………...the of and 85 is regulatory owner. a document Figure 28. Digestion of CP4 EPSPSany protein such in simulated gastric itsfluid reproductiondocument demonstrated by SDS-PAGE………………………………………….owner this of 98 under of Figure 29. Digestion of CP4 EPSPSrights proteinthe in simulatedthis gastric fluid documentand/or of rights demonstrated by westernto of blot fall analysis………………………………... use 99 the This may distribution,and Figure 30. Digestion of the Cry3Bb1rights protein in simulatedowner gastric fluid demonstrated bysubject SDS-PAGE analysis ……………………………….. 103 be the violate Figure 31. Digestion of thecopy Cry3Bb1 proteinof in simulated gastric fluid and demonstratedmay by westerndocumentpublication, blot analysis………………………………... 104 It exploitation Figure 32. Digestion of thethis Cry3Bb1any protein in simulated intestinal fluid demonstrated by westernpermission blot analysis………………………………... 105 prohibited Figure 33. Safety assessment ofthe new be varieties: the donor……………………….. 115 Figure 34. Safety assessmentcommercial of new varieties: proteins introduced from donor… 116 Furthermore,any Figure 35. SafetyConsequently, assessmentwithout of new varieties: the host plant…………………… 136

Monsanto Company # 04-CR-109F Page 9 of 223 CERTIFICATION

Monsanto Company is submitting this food and feed safety and nutritional assessment in compliance with the FDA’s 1992 policy statement regarding foods derived from new plant varieties (57 FR 22984). At the agency’s request, and where appropriate, this submission also complies with the recommendations contained in the proposed rule for Premarket Biotechnology Notice (PBN) Concerning Bioengineered Foods (66 FR 4706).

Specifically, as recommended in the proposed 21 CFR §192.25(a), the undersigned attests to the following:

1. It is the view of Monsanto Company (hereafter referred to as Monsanto) that: (i) MON 88017 is as safe and nutritious as other commercially available corn; and (ii) and the intended uses of the food and feed derived from MONand 88017 areregime. in compliance with all applicable requirements of the Federal Food,AG Drug and Cosmetic Act. 2. Monsanto will make available to FDA, upon request, relevant data or other Bayer property protectionpublishing information not included in this submissiofon, either during the coursecontents of FDA’s its evaluation of the submission, or for cause. parties.data therefore 3. Monsanto will make relevant data or other information notand/or includedor in this affiliates. property intellectualthird may submission available to FDA either: its (i) by allowing FDA to review and copy these as the of and data or information at Monsanto’sis offices in St.regulatory Louis, MO, duringowner. customary a document business hours; or (ii) by sendingany a suchcopy of these data or informationits to FDA. reproductiondocument of 4. Monsanto makes no claim of confidentialityowner regardingthis either the existence of this under of rightsthe this submission, or any ofdocument the and/ordata or other information containedrights herein. However, to of fall use of Monsanto reserves the right to make a claim of confidentialitythe regarding any relevant This may distribution,and data or other information notrights included in thisowner submission, but requested by FDA, subject either in the course ofbe its review of this submission,the violate or for cause. Any such claim of copy of confidentiality will be made at the time suchand data or information is provided, along may documentpublication, with an explanationIt for the basisexploitation of the claim. 5. To the best of Monsanto’sthis any knowledge, this submission is representative and balanced, permission including information, unfavorableprohibited as well as favorable, pertinent to the evaluation of the the safety, nutritional, commercialor other regulator be y issues that may be associated with MON 88017. Furthermore,any Consequently,without

Signature:

Date:

Director, North American Biotechnology Regulatory Affairs Monsanto Company 800 North Lindbergh Blvd. St. Louis, MO 63167

Monsanto Company # 04-CR-109F Page 10 of 223 Release of Information

Monsanto is submitting the information in this assessment for review by the FDA as part of the regulatory process. By submitting this information, Monsanto does not authorize its release to any third party. In the event the FDA receives a Freedom of Information Act request, pursuant to 5 U.S.C., § 552, and 21 CFR Part 20, covering all or some of this information, Monsanto expects that, in advance of the release of the document(s), FDA 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 understands that a 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 authorize the release, publication or other distribution of this information (including website posting) without Monsanto's prior notice and consent. 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

Monsanto Company # 04-CR-109F Page 11 of 223 Abbreviations1 and Definitions

~ approximately aa amino acid aad bacterial promoter and coding sequence for an aminoglycoside- modifying enzyme, 3’(9)-O-nucleotidyltransferase from the transposon Tn7 ADF acid detergent fiber ALLERGEN3 allergen and gliadin protein sequence database, compiled by Monsanto Company ALLPEPTIDES Protein sequence database comprised of GenPept, PIR and SwissProt, as curated by Monsanto Company avg average bp base pair B. subtilis Bacillis subtilis and and regime. BSA bovine serum albumin AG B.t. Bacillus thuringiensis CaMV cauliflower mosaic virusBayer property protectionpublishing of contents CFR Code of Federal Regulations its parties.data therefore CI confidence interval and/oror affiliates. CMS cytoplasmic maleproperty sterile intellectual third may its as CPB Colorado potatothe of beetle and is regulatory owner. a document CRW corn rootwormany such its reproductiondocument Cry3Bb1 a naturally-occurringowner wild typethis protein withof activity against under of Coleopteranrights insects,the produced bythis Bacillus thuringiensis (subsp. documentand/or of rights kumamotoensisto of ) fall use the This may distribution,and Cry3Bb1.pvzmir39 a variantrights sharing an aminoowner acid identity of >99% with the wild typesubject Cry3Bb1 protein be the violate cry3Bb1.pvzmir39 DNAcopy sequence encodingof the Cry3Bb1.pvzmir39 protein and CP4 EPSPS may 5-enolpyruvylshikimate-3-phosphate documentpublication, synthase from Agrobacterium It exploitation sp. thisstrain any CP4 cp4 epsps epsps codingpermission sequence from Agrobacterium sp. strain CP4 prohibited CTAB cetyltrimethylammoniumthe be bromide CTP chloroplastcommercial transit peptide Furthermore,any CTP2 Consequently,Arabidopsiswithout EPSPS transit peptide DNA deoxyribonucleic acid dATP deoxyadenosine triphosphate dCTP deoxycytosine triphosphate DDE daily dietary exposure DTT dithiothreitol dwt dry weight

1 Standard abbreviations, e.g., units of measure, will be used according to the format described in ‘Instructions to Authors’ in the Journal of Biological Chemistry.

Monsanto Company # 04-CR-109F Page 12 of 223 Abbreviations and Definitions (cont.)

E. coli Escherichia coli ECL enhanced chemiluminescence EDTA ethylenediaminetetraacetic acid ELISA enzyme-linked immunosorbent assay EPA Environmental Protection Agency EPSPS 5-enolpyruvylshikimate-3-phosphate synthase EU European Union FAS Foreign Agricultural Service FASTA algorithm used to find local high scoring alignments between a pair of protein or nucleotide sequences FDA Food and Drug Administration FFDCA Federal Food, Drug and Cosmetic Act FIFRA Federal Insecticide, Fungicide and Rodenticide Act and and regime. FMV figwort mosaic virus AG FR Federal Register FW fresh weight Bayer property protectionpublishing of contents fwt fresh weight of tissue its parties.data therefore GenBank a public genetic database maintained byand/or theor National Center for affiliates. Biotechnologyproperty Information intellectual thirdat the National Institutesmay of Health, its as Bethseda, MDthe of and is regulatory owner. a document HPLC high performanceany such liquid chromatography its reproductiondocument HRP horseradish peroxidaseowner this of under of IgA Immunoglobulinrightsthe A this documentand/or of rights IgE Immunoglobulinto of Efall use the This may distribution,and IgG Immunoglobulinrights G owner kb kilobasesubject be the violate kDa kilodaltoncopy of and LB mayleft borderdocumentpublication, It exploitation LOD limitthis of any detection LOQ limit of quantitationpermission prohibited mA milliamperethe be MAFF Ministrycommercial of Agricult ure, Forestry and Fisheries Furthermore,any MALDI-TOF MSConsequently, matrixwithout assisted laser desorption ionization time of flight mass spectrometry MHLW Ministry of Health, Labor and Welfare mM millimolar MO monogerm MOE margin of exposure MW molecular weight NASS National Agricultural Statistics Services NDF neutral detergent fiber NFDM nonfat dry milk NOEL no effect level OECD Organization for Economic Co-operation and Development

Monsanto Company # 04-CR-109F Page 13 of 223 Abbreviations and Definitions (cont.)

OD optical density ori-322 origin of replication for the ABI Agrobacterium derived from the broad host range plasmid RK2 ori-V a segment of pBR322 that provides the origin of replication for maintenance of plasmids in E. coli OSL overseason leaf OSR overseason root OSWP overseason whole plant PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction P-FMV 35S promoter from a modified figwort mosaic virus (FMV) PVDF polyvinylidene difluoride ract1 intron intron from the rice actin gene and and regime. RB right border AG ROP coding sequence for repressor of primer protein for maintenance of plasmid copy number inBayer E. coli property protectionpublishing of contents RB right border its parties.data therefore RNA ribonucleic acid and/oror affiliates. RT room temperatureproperty intellectualthird may its as rop a segment ofthe pBR322 of thatand represses the formation of RNA primer is regulatory owner. a document and is criticalany to suchthe maintenance and copyits number control of reproductiondocument plasmids in E. coliowner this of under of SD standard deviationrightsthe this documentand/or of rights SDS sodium dodecylto of sulfatefall use the This may distribution,and SE standard rightserror of the meanowner SGF simulatedsubject gastric fluid be the violate SIF simulatedcopy intestinal fluidof and sp. mayspecies document publication, It exploitation Taq Thermusthis any aquaticus, a thermophilic bacterium tahsp17 3’ 3’ nontranslatedpermission region of the wheat heat shock protein 17.3 prohibited containingthe the be polyadenylation sequence TBA tris-boratecommercial buffer with 0.25% (w/v) L-ascorbic acid Furthermore,any TBST Consequently, triswithout buffered saline with Tween T/C/R test/control/reference material TDF total detedgent fiber T-DNA transferred DNA of the Ti plasmid that integrates into the nuclear DNA of the plant during Agrobacterium-mediated transformation TMB 3,3’,5,5’-tetramethylbenzidene Tris tris(hydroxymethyl)-aminomethane TE buffer tris-EDTA buffer u units USDA- APHIS United States Department of Agriculture Animal and Plant Health Inspection Service

Monsanto Company # 04-CR-109F Page 14 of 223 Abbreviations and Definitions (cont.)

µg/g fwt microgram per gram fresh weight of tissue U.S. United States USC United States code wt weight

and and regime. AG

Monsanto Company # 04-CR-109F Page 15 of 223 NARRATIVE SUMMARY

Food and Feed Safety and Nutritional Assessment of MON 88017 Corn

Monsanto Company has developed, through the use of recombinant DNA techniques, MON 88017 corn plants that are tolerant to the action of the Roundup® family of agricultural herbicides and are protected from damage caused by corn rootworm (CRW) larval feeding. MON 88017 produces a 5-enolpyruvylshikimate-3-phosphate synthase protein from Agrobacterium sp. strain CP4 (CP4 EPSPS), which confers tolerance to glyphosate, the active ingredient in Roundup agricultural herbicides, and a modified Bacillus thuringiensis (subspecies kumamotoensis) Cry3Bb1 protein that selectively controls CRW species. The safety of both the CP4 EPSPS and Cry3Bb1 proteins has been assessed previously by FDA for other corn products. and and regime. Corn (Zea mays L.) is the largest crop grown in theAG United States in terms of acreage planted and net value. In 2002, its production covered 79.1 million acres that yielded Bayer property protectionpublishing nine billion bushels and had a net value of $21.of 2 billion. Weed controlcontents is essential is its cornfields, as weeds compete with the crop for sunlight,parties. waterdata and nutrients.therefore Failure to and/oror control weeds results in decreased yields andaffiliates. reduced crop quality. Introduction of property intellectualthird may MON 88017 will offer U.S. farmers the itsability to apply Roundup agricultural herbicides as the of and over the top of corn for broad-spectrumis weed controlregulatory with minimal owner.risk of crop injury. In a document any such its addition the use of glyphosate in conjunction with MONreproduction 88017document will allow the grower to owner this of utilize reduced tillage techniques, which provideunder significantof environmental benefits, such rightsthe this as reduced soil erosion, reduceddocumentand/or use of fossil fuels, and improvedrights soil quality. to of fall use of the This may distribution,and Corn yields are also negatively rightsimpacted by a numberowner of insect pests. One of the most subject pernicious in the U.S. cornbeltbe is corn rootworm.the violate Corn rootworm larvae damage corn by copy of feeding on the roots, reducing the ability of theand plant to absorb water and nutrients from may documentpublication, soil, and causing harvestingIt difficultiesexploitation due to plant lodging. Corn rootworm is the most significant insect pest problemthis any for corn production in the U.S. cornbelt from the permission standpoint of chemical insecticide usage,prohibited as over 16 million acres of corn were treated the with organophosphate, carbamate,commercial and be pyrethroid insecticides to control corn rootworm in 1998. Corn rootworm has been described as the billion-dollar pest complex based on Furthermore,any costs associated withConsequently, the applwithout ication of soil insecticides and crop losses due to pest damage. Introduction of MON 88017 will offer U.S. farmers a safe and effective alternative to the use of synthetic chemical insecticides for control of the corn rootworm pest.

Corn hybrids containing both the corn rootworm protection and glyphosate tolerance traits have been developed previously using traditional breeding techniques, i.e., two inbreds containing the individual traits were crossed to produce the stacked trait product. However, the traditional breeding process can be inefficient, requiring long development

® Roundup, Roundup Ready, and YieldGard are registered trademarks of Monsanto Technology LLC.

Monsanto Company # 04-CR-109F Page 16 of 223 times. MON 88017 was therefore developed using a vector that contains both the cp4 epsps and cry3Bb1 genes. This approach increases the efficiency of simultaneous introduction of both traits into new corn hybrids, thereby providing growers access to a variety of elite corn germplasms containing both traits.

MON 88017 was produced by Agrobacterium-mediated transformation of corn cells with plasmid vector PV-ZMIR39. This plasmid contains a gene encoding the CP4 EPSPS protein that provides tolerance to the action of Roundup agricultural herbicides and a gene encoding the Cry3Bb1 protein that has activity against corn rootworm. The Agrobacterium tumefaciens transformation vector is a disarmed, binary vector that contains both left and right transfer-DNA (T-DNA) border sequences to facilitate transformation. The DNA region that was integrated into the corn genome during the transformation process contains the cp4 epsps and cryBb1 gene expression cassettes. Molecular analyses of MON 88017 confirmed that single copies of the cp4 epsps and cry3Bb1 genes are integrated at a single locus in the corn genome with all expressionand and regime. elements intact and no plasmid bacterial backbone present.AG

Segregation analysis across ten generations confBayerirmed propertythe heritability protectionpublishing and stability of the of contents cp4 epsps and cry3Bb1 coding sequences. The results of this analysisits are consistent with parties.data therefore the finding of a single active site of insertion that segregates accordingand/oror to the Mendelian affiliates. laws of genetics. property intellectualthird may its as the of and is regulatory owner. a document The CP4 EPSPS protein in MON 88017any such is structurally homologousits to EPSPSs naturally reproductiondocument present in food crops (e.g., soybean and cornowner) and in microbialthis offood sources such as under of Baker’s yeast that have a long historyrights theof safe consumptionthis by humans and animals. The documentand/or of rights amino acid sequence of the CP4to EPSPS of protfall ein inuse MON 88017 is the same or >99% the This may distribution,and identical to the CP4 EPSPS proteinrights produced in Roundupowner Ready crops such as soybean, NK603 corn, cotton and canola,subject which have already completed the FDA consultation be the violate process. Roundup Ready cropscopy have been consof umed directly or as processed products by and humans and animalsmay since Roundupdocumentpublication, Ready soybeans were first commercialized in 1996. It exploitation The CP4 EPSPS protein isthis not any toxi c, as assessed by the history of safe use of the EPSPS class of proteins and as confirmed permissionby the lack of any treatment-related adverse affects in prohibited mice administered the CP4 EPSPSthe proteinbe by oral gavage. This lack of toxicity was expected based on the rapidcommercial degradation of the CP4 EPSPS protein and its enzymatic Furthermore,any activity in simulatedConsequently, mammalianwithout gastric and intestinal fluids. In addition, the CP4 EPSPS protein is not homologous to known protein toxins or allergens, is not glycosylated and is present at very low levels in MON 88017.

The mean levels of CP4 EPSPS protein in MON 88017 are 5.8 µg/g of dry weight in corn grain and 57 µg/g of dry weight in forage. These levels are comparable to levels found in Roundup Ready corn NK603, which has a history of safe consumption by humans and animals. Using a no observed effect level (NOEL) of 572 mg/kg body weight from the acute mouse gavage study, the margin of exposure for consumption of the CP4 EPSPS protein, based on the mean adult consumption of corn in the U.S., is calculated to be >3.6x105. This exposure calculation makes the conservative assumption that there is no loss of the CP4 EPSPS protein during the processing of corn grain into human food. It

Monsanto Company # 04-CR-109F Page 17 of 223 also assumes that 100% of the corn grain in human food is derived from MON 88017, which is highly unlikely considering the variety of commercial corn hybrids that exist in the marketplace.

Bacillus thuringiensis (B.t.) Cry proteins also have a long history of safe and widespread use. The Cry3Bb1 protein produced in MON 88017 is a member of the Cry3Bb class of proteins and shares >99% amino acid sequence identity with the wild type Cry3Bb1 protein contained in the topically applied commercial microbial product, Raven® Oil Flowable Bioinsecticide, and differs by only a single amino acid from the Cry3Bb1 variant produced in YieldGard® Rootworm corn (MON 863), which has completed the FDA consultation process. Cry3Bb1 had no effect when administered orally to mice at the highest dose tested of 1930 mg/kg. In vitro digestive fate studies have shown that the protein is rapidly degraded to small peptide fragments. Furthermore, the protein is not glycosylated in corn, and it lacks sequence similarity to known allergens and toxins. and and regime. The level of Cry3Bb1 protein in corn was determinedAG in multiple plant tissues collected from replicated field trials. The mean level of Cry3Bb1 protein in corn grain is 15 µg/g dry weight and 95 µg/g dry weight in forage. BayerUsing theproperty same protectionconservativepublishing assumptions of contents as described above, the margin of exposure for the Cry3Bb1 protein,its based on the mean parties.data therefore adult consumption of corn in the U.S., is calculated to be > 4.7and/or x 10or 5. affiliates. property intellectualthird may its as MON 88017 was shown to be compositionallythe of and equivalent to conventional corn with is regulatory owner. a document similar genetic background as wellany as tosuch other conventional corn itsvariet ies. A total of 77 reproductiondocument significant nutrients, anti-nutrients and secownerondary metabolitesthis wereof evaluated as part of under of the nutritional assessment of MONrights 88017the (68 in grain andthis nine in forage). documentand/or of rights Compositional analyses of the forageto of samplesfall includeduse proximates (protein, fat, ash, and the This may distribution,and moisture), acid detergent fiber (ADF),rights neutral detergentowner fiber (NDF), minerals (calcium, phosphorous), and carbohydratessubject by calculation. Compositional analyses of the grain be the violate samples included proximatescopy (protein, fat, asofh, and moisture), ADF, NDF, total dietary and fiber (TDF), amino mayacids, fattydocument acidspublication, (C8- C22), minerals (calcium, copper, iron, It exploitation magnesium, manganese, phosphorous,this any potassium, sodium, and zinc), vitamins (B1, B2, B6, E, niacin, and folic acid), anti-nutrienpermissionts (phytic acid and raffinose), secondary prohibited metabolites (furfural, ferulic acid,the anbed p-coumaric acid), and carbohydrates by calculation. Sixty-two componentscommercial were statistically assessed and 15 components were Furthermore,any below the limit ofConsequently, quantitation.without Results of the analysis showed that there were no statistically significant differences between MON 88017 and conventional corn for 232 of the 248 comparisons conducted. No statistically significant differences were found in forage. Statistically significant differences (p<0.05) were observed for only 16 comparisons in grain and all values fell within the 99% tolerance interval. Therefore it is unlikely that these differences are biologically meaningful. It is concluded, based on these data, that the forage and grain produced from MON 88017 are compositionally equivalent to the forage and grain produced from other commercial corn currently on the market.

® Raven is a registered trademark of Ecogen, Inc.

Monsanto Company # 04-CR-109F Page 18 of 223 Collectively, the data summarized in this document support a conclusion that food and feed products derived from MON 88017 are as safe and nutritious as those derived from conventional corn.

and and regime. AG

Monsanto Company # 04-CR-109F Page 19 of 223 PART II: SYNOPSIS OF CONSULTATION SUMMARY

SECTION 1. Name and address of the submitter

The submitter of this safety and nutritional assessment summary for corn MON 88017 is:

Monsanto Company 800 North Lindbergh Blvd. St. Louis, MO 63167

Communications with regard to this submission should be directed to , Regulatory Affairs Manager, at the Monsanto address. He can also be contacted by and telephone at . and regime. AG

Bayer property protectionpublishing SECTION 2. The subject of this summaryof and the plant species fromcontents which they its were derived parties.data therefore and/oror affiliates. property intellectualthird may The subject of this summary is corn MONits 88017 derived from a Monsanto proprietary as the of and corn inbred. is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of SECTION 3. Distinctive designations given to the subjectthe of this summary This may distribution,and rights owner subject The corn rootworm-protectedbe and glyphosate-tothe lerantviolate corn that is subject of this copy of summary has been designated MON 88017. Inand accordance with OECD’s “Guidance for may documentpublication, the Designation ofIt a Unique Identifierexploitation for Transgenic Plants,” MON 88017 has been assigned the unique identifierthis any MON-88Ø17-3. permission prohibited the commercial be Furthermore,any SECTION 4. IdentityConsequently, andwithout sources of the genetic material introduced into MON 88017

Agrobacterium-mediated transformation utilizing plasmid vector PV-ZMIR39 (Figure 1, Part IV) was used to generate MON 88017. This vector contains two expression cassettes: one coding for the native CP4 EPSPS protein from Agrobacterium sp. strain CP4 (Padgette et al., 1996), and the other coding for a variant of the wild-type Cry3Bb1 protein from Bacillus thuringiensis subsp. kumamotoensis. The genetic elements present in PV-ZMIR39 are listed in Table 1, Part IV.

The expression cassette for the coding sequence of the native CP4 EPSPS protein from Agrobacterium sp. strain CP4 (Padgette et al., 1996) consists of the promoter from the

Monsanto Company # 04-CR-109F Page 20 of 223 rice actin gene (McElroy et al., 1990), an intron from the rice actin gene (McElroy et al., 1991), the cp4 epsps coding sequence fused to a chloroplast transit peptide 2 (CTP2) sequence derived from Arabidopsis (Klee et al., 1987) and a 3’ nontranslated region of the nopaline synthase gene of Agrobacterium tumifaciens T-DNA that ends transcription and directs polyadenylation (Bevan et al., 1983).

The synthetic cry3Bb1 gene expression cassette that produces the Cry3Bb1 protein consists of the enhanced 35S promoter (Odell et al., 1985), 5’ untranslated leader of the wheat chlorophyll a/b/-binding protein (Lamppa, 1985), intron from the rice actin gene (McElroy et al., 1991), the synthetic cry3Bb1 coding sequence, and the 3’ nontranslated region of the coding sequence for wheat heat shock protein 17.3, which ends transcription and provides the signal for mRNA polyadenylation (McElwain and Spiker, 1989).

The molecular analyses described in Part IV demonstrate that MON 88017 contains a single copy of introduced DNA approximately 6.8 kb in size inserted at a singleand locus. and regime. This insert contains one intact copy each of the cp4AG epsps and cry3Bb1 gene expression cassettes. There are no detectable plasmid backbone sequences and no additional elements, linked or unlinked to intact cassettes,Bayer from transformationproperty protectionpublishing vector PV-ZMIR39. of contents its parties.data therefore These results support the conclusion that only the two expectedand/or full-lengthor proteins, CP4 affiliates. EPSPS and Cry3Bb1, are encoded byproperty the insert intellectual presentthird in MON 88017.may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of SECTION 5. The intended technicalrightsthe effect of MON this88017 documentand/or of rights to of fall use the MON 88017 corn plantsThis produce a CP4may Edistribution,PSPSand protein (5-enolpyruvylshikimate-3- rights owner phosphate synthase proteinsubject from Agrobacterium sp. strain CP4) that provides tolerance to be the violate the action of Roundup agriculturalcopy herbicidesof and a modified Bacillus thuringiensis and (subspecies kumamotoensismay ) documentCry3Bb1publication, protein that has activity against corn rootworm. It exploitation this any The CP4 EPSPS protein is structurallypermission and functionally similar to native plant EPSPS prohibited enzymes, but has a much reducedthe affinitybe for glyphosate (Padgette et al., 1996). Typically, glyphosate bindscommercial to the plant EPSPS enzyme and blocks the biosynthesis of aromatic aminoFurthermore, acids, therebyany depriving plants of these essential components Consequently,without (Steinrucken and Amrheim, 1980; Haslam, 1993). In Roundup Ready plants, requirements for growth and development are met by continued action of the CP4 EPSPS enzyme in the presence of glyphosate.

The Cry3Bb1 protein is selectively active against corn rootworm species and protects plants from damage due to corn rootworm feeding. MON 88017 is afforded a level of protection from corn rootworm feeding damage that is comparable or superior to that offered by currently available conventional insecticides. The Cry3Bb1 protein produced in MON 88017 (Cry3Bb1.pvzmir39) is a member of the Cry3Bb class of proteins and variants that shares >99% amino acid sequence identity with the wild type Cry3Bb1 protein and differs only by a single amino acid from the Cry3Bb1 variant produced in

Monsanto Company # 04-CR-109F Page 21 of 223 YieldGard® Rootworm corn (MON 863), which has completed the FDA consultation process. It is referred to as Cry3Bb1 in this submission.

SECTION 6. The applications and uses of MON 88017

Monsanto Company has developed, through the use of recombinant DNA techniques, corn plants that are tolerant to the action of the Roundup family of agricultural herbicides and protected from damage due to corn rootworm larval feeding. MON 88017 produces a CP4 EPSPS protein, which confers tolerance to glyphosate, and a modified Bacillus thuringiensis (subspecies kumamotoensis) Cry3Bb1 protein that is active against corn rootworm species. The cp4 epsps gene expressed in MON 88017 is identical to one of the cp4 epsps genes produced in Roundup Ready corn NK603, and the cry3Bb1 gene is a synthetic variant of the cry3Bb1 gene produced in YieldGard Rootworm corn.and and regime. AG One of the benefits of MON 88017 corn to growers will result from the use of Roundup herbicides to control weeds. Many benefits ofBayer Roundup property Ready protection technologypublishing have been of contents documented in studies of Roundup Ready soybeans (Gianessi and Carpenter,its 2000). The parties.data therefore benefits of the use of MON 88017 are likely to be similar to thoseand/oror of Roundup Ready affiliates. corn, which was commercialized in theproperty U.S. inintellectual 1998.third may its as the of and is regulatory owner. a document The benefits of Roundup Ready cornany include:such its reproductiondocument owner this of under of • Broad-spectrum weed control.rights the Roundup agriculturalthis herbicide controls both documentand/or of rights broadleaf weeds and grasses,to of including fall diffiusecult-to-control weed species (Franz et the This may distribution,and al., 1997). rights owner • Excellent crop safety.subject When used according to label recommendations, Roundup be the violate agricultural herbicidescopy c ontrol weedsof without injury to Roundup Ready corn and may documentpublication, plants. It exploitation • Minimal environmentalthis any impact. Roundup agricultural herbicides have been used permission for more than 30 years in variousprohibited applications. Glyphosate is recognized for its lack of soil persistence, lowthe risk of ground water contamination, and lack of commercial be toxicity to birds, mammals, fish, and invertebrates (Ahrens, 1994; Franz et al., Furthermore,any 1997; GiesyConsequently, et al., without2000). • Flexibility in treating for weed control. Since Roundup agricultural herbicides are applied onto the foliage of weeds after crop emergence, applications are only necessary if weed infestation reaches the threshold level for yield reductions. • Excellent fit with reduced-tillage systems. Benefits of conservation tillage include improved soil quality, improved water infiltration, reduced soil erosion and sedimentation of water resources, reduced runoff of nutrients and pesticides to surface water, improved wildlife habitat, increased carbon retention in soil, reduced fuel usage, and sustainable agricultural practices (Hebblethwaite, 1995; Reicosky et al., 1995). In addition, data collected during 1999-2001 have shown that the adoption of Roundup Ready corn can lead to a significant reduction in the levels

Monsanto Company # 04-CR-109F Page 22 of 223 of major pre-emergence corn herbicides in Illinois watersheds (Wauchope et al., 2001). • Cost effective weed control. The costs of weed control with Roundup agricultural herbicides are competitive with the cost of alternative weed-control options, especially in view of the high weed-control efficacy of glyphosate. Both large- and small-scale farmers can benefit equally from use of this technology. • Use of a herbicide with low risk to humans. Roundup agricultural herbicides pose a minimal health risk to humans (EPA, 1993; WHO, 1994).

Another major benefit from MON 88017 is protection from feeding damage by corn rootworm larvae. Corn is the largest crop grown in the U.S. in terms of acreage planted and net value. In 2002, corn production covered 79.1 million acres, yielding nine billion bushels of corn grain, at an average yield of 130 bu/A, and a total net value of $21.2 billion (NCGA, 2003). Corn yields are negatively impacted by a number of insect pests. and One of the most pernicious in the U.S. Cornbelt is corn rootwormand larvae,regime. which damage corn by feeding on the roots, reducing the ability ofAG the plant to absorb water and nutrients from soil, and causing harvesting difficulties due to plant lodging (Reidell, 1990; Spike and Tollefson, 1991). Corn rootwoBayerrm is theproperty most protection significantpublishing insect pest of contents problem for corn production in the U.S. from the standpoint of chemicalits insecticide parties.data therefore usage. Approximately 14 million acres of corn were treated inand/or 2000or with affiliates. property intellectualthird may organophosphate, carbamate and pyrethroidits insecticides to control corn rootworm as (Doane, 2001). Corn rootworm hasthe been of describedand as the billion-dollar pest complex, is regulatory owner. a document based on costs associated with the anyapplication such of soil insecticidesits and crop losses due to reproductiondocument pest damage (Metcalf, 1986). owner this of under of rightsthe this documentand/or rights to of fall use of In 2003, Monsanto commercialized YieldGard Rootwormthe corn (MON 863) that provides This may distribution,and an alternative and more effectiverights solution for theowner control of corn rootworm larvae based on biotechnology. The potentialsubject benefits of MON 88017 will be similar to those of MON be the violate 863 because MON 88017 containscopy a Cry3Bb1of protein that is equally efficacious against and may documentpublication, corn rootworm larvae.It In addition, growexploitationers will have the ability to apply Roundup agricultural herbicides overthis theany top of corn for broad-spectrum weed control. The rapid permission development of elite corn hybrids containingprohibited th ese traits is made possible by vector stack technology, i.e., the incorporationthe of multiple genes into a single transformation event. In commercial be traditional breeding methods, inbreds containing individual traits are crossed to produce Furthermore,any the desired stackedConsequently, hybrid.without This process can be inefficient, requiring long development times for the introduction of new hybrids into the market place. Vector stack technology increases the efficiency of the introduction of multiple traits into new hybrids, thereby providing the grower access to a variety of germplasms containing these traits. MON 88017 will offer growers many practical advantages over the use of chemical insecticides, including savings in time and money for application, labor, fuel, equipment, storage and disposal costs. In addition, MON 88017 is likely to be labeled for general use and will replace current reliance on restricted use products, providing growers and other occupational workers with greater safety, protect water bodies from run-off, and mitigate spray-drift and potential impacts on nontarget organisms (NTO), such as bird populations.

Monsanto Company # 04-CR-109F Page 23 of 223 The National Agricultural Statistics Service (NASS) of the U. S. Department of Agriculture (USDA) has compiled statistics of corn insecticide usage across 15 states, comprising 68.3 million acres of corn in 1999 (USDA-NASS, 2000). These statistics indicate that chemical insecticides registered for corn rootworm control were applied on over 30% of this corn acreage in 1999. Many of these insecticides have come under increased regulatory scrutiny because of concerns about worker safety, environmental risk, and exposure to infants and children. The introduction of MON 88017 will offer U.S. farmers a safe and effective alternative to the use of synthetic chemical insecticides for control of corn rootworm pests. Such a product offers the potential to significantly reduce the amount of chemical insecticides applied in the U.S. cornbelt, benefiting the environment and leading to a reduction in actual and potential adverse effects throughout the manufacturing, distribution, and use chain.

Based on the dual traits of glyphosate tolerance and corn rootworm protection, MON 88017 is expected to provide substantial benefits to growers by limiting yieldand losses from and regime. corn rootworm feeding damage and weed pressure,AG while at the same time reducing the risk to humans and the environment as a result of anticipated reductions in the use of chemical insecticides. Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its SECTION 7. Applications for which MONas 88017 is not suitable the of and is regulatory owner. a document any such its reproductiondocument Monsanto Company is aware of no food orowner feed uses ofthis conventional of corn that are not under of applicable to MON 88017. rightsthe this documentand/or of rights to of fall use 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 # 04-CR-109F Page 24 of 223 PART III: STATUS OF SUBMISSIONS TO OTHER REGULATORY AGENCIES

SECTION 1. Status of submission to USDA-APHIS

Monsanto will be requesting a Determination of Nonregulated Status for MON 88017 from USDA-APHIS in 2004, including all progenies derived from crosses between MON 88017 and other corn. Under regulations administered by USDA-APHIS (7 CFR 340), MON 88017 is currently considered a “regulated article.” Monsanto will continue to conduct all field tests for MON 88017 in strict compliance with USDA field regulations until a Determination of Nonregulated Status is obtained for MON 88017.

and and regime. AG SECTION 2. Status of submission to EPA Bayer property protectionpublishing of contents its Substances that are pesticides as defined under the Federalparties.data Insect icide, thereforeFungicide and and/oror Rodenticide Act (FIFRA) [7 U.S.C. §136(u)affiliates.] are subject to regulation by the U.S. property intellectualthird may its Environmental Protection Agency (EPA). as the of and is regulatory owner. a document any such its An application for registration of “Bacillus thuringiensisreproduction Cry3Bb1document Protein and the owner this of Genetic Material (Vector ZMIR39) Necessaunderry for itsof Production in Corn” has been rightsthe this documentand/or rights submitted to the EPA. An applicationto of forfall an exemptionuse of from the requirement of a tolerance for B.t. Cry3Bb1 proteins, pursuant to §408(d)the of the Federal Food Drug and This may distribution,and rights owner Cosmetic Act [21 U.S.C. 346subject a(d)], also was submitted to EPA (PP 7F4888). On May 11, 2001, EPA establishedbe a time-limited exemptionthe violate from the requirement of a tolerance copy of for Cry3Bb1 and the genetic material necessaryand for its production in all corn commodities may documentpublication, (EPA, 2001a). OnIt April 29, 2003 Monsantoexploitation petitioned the EPA to amend 40 CFR Part this 180 by removing the time limitationany for the exemption from the requirement of a permission tolerance for the plant-incorporated protectantprohibited Bacillus thuringiensis Cry3Bb1 protein the and the genetic material necessarycommercial for be its production in corn in or on field corn, sweet corn, and popcorn.Furthermore, In Octoberany 2003, a Federal Register notice was published announcing a public commentConsequently, period without(EPA, 2003b) to remove the time limitation.

Substances that are pesticidal as defined under the FIFRA (7U.S.C. section 136(u)), are subject to EPA’s regulatory authority. The initial registration for use of Roundup Ultra® Herbicide (EPA Reg. No. 524-475) over the top of Roundup Ready corn was granted by the EPA on March 28, 1997 (62 FR 17723-17730). Since that time a number of label amendments have been approved by the Agency and multiple glyphosate end-use products have been labeled for use in Roundup Ready corn. The most recent label for use in Roundup Ready corn, approved on June 23, 2003, for Roundup WeatherMAX®

® Roundup Ultra and Roundup WeatherMAX are registered trademarks of Monsanto Technology LLC.

Monsanto Company # 04-CR-109F Page 25 of 223 herbicide (EPA Reg. No 524-537), provides greater flexibility for in-crop applications. This new label allows increased over-the-top application rates and a wider window for applications with use of directed sprays up to 48-inch corn. EPA’s approval of the label was based on the review of supporting glyphosate residue data and the establishment of an increased tolerance level for glyphosate in field corn forage of 6 ppm (68 FR 39460, July 2, 2003).

Pursuant to section 408(d) of the Federal Food Drug and Cosmetic Act (FFDCA), 21 U.S.C. 346 a(d), the EPA previously has reviewed and established an exemption from the requirement of a tolerance for CP4 EPSPS and the genetic material necessary for the production of this protein in or on all raw agricultural commodities (40 CFR §180.1174).

SECTION 3. Status of submissions to foreign governments and and regime. AG Monsanto obtained an environmental approval from the Japanese Ministry of Agriculture, Forestry and Fisheries (MAFF) forBayer MON property88017 onprotection Aprilpublishing 23, 2003. of contents Regulatory submissions for import and production approvals will beits made to countries parties.data therefore that import U.S. corn grain and have regulatory approval processesand/oror in place. These will affiliates. include submissions to a number of foreipropertygn governmentintellectualthird regulatory mayagencies, including its as Japan’s MAFF (for use as animal feed),the of the Japaneseand Ministry of Health, Labor and is regulatory owner. a document Welfare (MHLW), as well as the Canadianany such Food Inspection Agencyits (CFIA) and Health reproductiondocument Canada. As appropriate, notifications of ownerimpor t will bethis made toof importing countries that under of do not have a formal approval process.rightsthe this documentand/or of rights to of fall use 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 # 04-CR-109F Page 26 of 223 PART IV: DEVELOPMENT OF MON 88017

SECTION 1. History and biology of corn

The history and biology of corn is summarized in OECD consensus document (OECD, 2003). Corn, Zea mays L., originated in Mexico and was grown as a food crop as early as 2700 B.C. (Salvador, 1997). It is now grown on more than 345 million acres globally (James, 2003a). The history of corn has been studied extensively, and multiple hypotheses for its origin and parentage have been advanced (Mangelsdorf, 1974). The preponderance of evidence supports the hypothesis that corn descended from teosinte (Galinat, 1988). Corn is the largest crop grown in the U.S. in terms of acreage planted and net value. In 2002, its production covered 79 million acres that yielded 9 billion and bushels and had a net value of $21.2 billion (NCGA, 2003). and regime. AG Corn is a highly productive crop, yielding an average of 130 bushels per acre in the U.S. Bayer property protectionpublishing during 2002 (NCGA, 2003). Its high yield maof kes it one of the most economicalcontents sources its of metabolizable energy for feeds, and of starch and sugarparties. datafor food and thereforeindustrial and/oror products. Approximately two-thirds of theaffiliates. corn produced in the U.S. is fed to livestock. property intellectualthird may Therefore, indirect consumption is muchits greater than direct consumption for humans. as the of and is regulatory owner. a document any such its Corn, the host plant, has been a staple of the humanreproduction diet fordocument centuries. Corn grain and its owner this of processed fractions are consumed in a multitudeunder of foodof and animal feed products. Corn rightsthe this forage is extensively consumeddocumentand/or as an animal feed by ruminants.rights Corn does not contain to of fall use of any known allergens or produce significant quantities ofthe toxins or anti-nutritional factors This may distribution,and warranting analytical or toxicologicalrights tests (Watson,owner 1982; White and Pollak, 1995). subject be the violate copy of and may documentpublication, 1.1. Scientific nameIt and taxonomicexploitation classification of corn this any permission Corn is a member of the tribe Maydae,prohibited which is included in the subfamily Panicoideae of the the grass family Gramineae.commercial The genera be included in the tribe Maydae include Zea and Tripsacum in theFurthermore, Westernany Hemisphere and Coix, Polytoca, Chionachne, Schlerachne, and Trilobachne Consequently,in Asia. withoutAlthough the Asian genera have been implicated by some as the origin of corn, the evidence is not as extensive and convincing as for the genera located in the Western Hemisphere.

There has been some fluctuation in Latin binomial designations of the species included in Zea in recent years (Doebley and Iltis, 1980). The genus Zea includes two subgenera: Luxuriantes and Zea. Corn (Zea mays L.) is a separate species within the subgenus Zea, along with three subspecies. All of the species within the genus Zea, except corn, are different species of teosinte. Until recently, the teosinte species were included in the genus Euchlaena rather than the genus Zea.

Monsanto Company # 04-CR-109F Page 27 of 223 The other genus included in the Maydae tribe is Tripsacum. Tripsacum includes 16 species with a basic set of 18 chromosomes (n = 18), and the various species of Tripsacum include multiples of 18 chromosomes, ranging from 2n = 36 to 2n = 108.

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.

Taxonomic classification of corn and its close relatives

Family - Gramineae Subfamily - Panicoideae Tribe - Maydae Western Hemisphere: I. Genus - Zea and and regime. A. Subgenus - Luxuriantes AG 1. Zea luxurians (2n = 20) 2. Zea perennis (2n = 40)Bayer property protectionpublishing of contents 3. Zea diploperennis (2n = 20) its parties.data therefore B. Subgenus - Zea and/oror affiliates. property intellectualthird may 1. Zea mays (2n =its 20) as Subspeciesthe of and is regulatory owner. a document 1. Z. maysany parviglumissuch (2n = 20) its reproductiondocument 2. Z. mays huehuetenangensisowner this (2n = of20) under of rightsthe this document3. and/orZ. mays mexicana (Schrad.) (2nrights = 20) to of fall use of II. Genus - Tripsacum the This may distribution,and Species—rights owner T. andersomiisubject (2n = 64) T. latifolium (2n = 36) be the violate T. australecopy (2n = 36) of T. percuvianum (2n = 72, 90, 108) and may documentpublication, It T. bravum (2nexploitation = 36, 72) T. zopilotense (2n = 36, 72) T. cundinamarcethis any (2n = 36) T. jalapense (2n = 72) permission T. dactyloides (2nprohibited = 72) T. lanceolatum (2n = 72) T. floridanumthe (2n = 36) T. fasciculatum (2n = 36) commercial be T. intermedium (2n = 72) T. maizar (2n = 36, 72) Furthermore,any Consequently, T. manisuroideswithout (2n = 72) T. pilosum (2n = 72) Asia: I. Genera— Chionachne (2n = 20) Schlerachne (2n = 20) Coix (2n = 10, 20) Trilobachne (2n = 20) Polytoca (2n = 20) Tribe—Andropogoneae I. Genus – Manisuris

Monsanto Company # 04-CR-109F Page 28 of 223 1.2. Growth and reproductive characteristics of corn

Corn is genetically one of the best developed and best characterized of the higher plants. Because of the separation of male and female inflorescence, the large number of seeds produced on the female inflorescence, the ease in handling (growing and hand pollinating), the nature of the chromosomes, and the low basic chromosome number (n = 10), corn has been accessible for study at all levels of genetics.

Corn evolved as an open-pollinated (cross-fertilizing) crop species and, until the 20th century, corn cultivars were what we designate today as open-pollinated corn varieties. 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 methods of selection were simple by present-day standards, but they were obviouslyand and regime. effective in developing races, varieties, and strains AGto satisfy their food, fuel, feed, and cultural needs. Hybridization occurred between varieties as cultures moved within the Western Hemisphere, releasing genetic variabilityBayer to developropertyp otherprotectionpublishing unique varieties. of contents its parties.data therefore The fundamental concepts for development of hybrid corn wereand/or definedor by 1920. Basic affiliates. property intellectualthird may studies on the genetic composition of a cornits variety were conducted to determine the as effects of self pollinating (or inbreeding,the of whichand is the opposite of outcrossing) within a is regulatory owner. a document corn variety (Shull, 1909). Becauseany corn such naturally cross- fertilizes,its the genetic reproductiondocument composition of each plant is not known. ownerContinuous self-pollinationthis of of individuals for under of rightsthe this seven to ten generations documentresultedand/or in pure lines (or inbred lines)rights within which every plant to of fall use of had similar traits. The correct interpretation of what occurredthe during inbreeding was This may distribution,and based on Mendelian genetics: therights heterozygous ownerloci were eliminated by inbreeding to homozygous loci of eithersubject one of the two alleles at each locus. The fixation of alleles in be the violate pure lines caused a general copyredu ction in vigorof and productivity. and may documentpublication, It exploitation It was found upon crossingthis two any pure lines that vigor was restored. If no selection occurred during inbreeding, the averagepermission perf ormance (e.g., grain yield) of all possible prohibited crosses was similar to performancethe of the original variety in which inbreeding was commercial be initiated. Some crosses, however, were better than the original open-pollinated variety Furthermore,any and could be reproducedConsequently, fromwithout th e 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 produce hybrids, evaluate hybrids to determine the best hybrid, and use pure-line parents to reproduce the superior hybrid and distribute it for use by growers (Shull, 1909).

1.3. History of corn development

Corn (Zea mays L.), or maize, is one of the few major crop species indigenous to the Western Hemisphere. Corn is grown in nearly all areas of the world and ranks third behind rice (Oryza sativa L.) and wheat (Triticum sp.) in total production. Corn has been studied extensively and it seems the probable domestication of corn was in southern Mexico more that 7,000-10,000 years ago. The putative parents of corn have not been

Monsanto Company # 04-CR-109F Page 29 of 223 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 known it today, cannot survive in the wild because the female inflorescence (the ear) restricts seed dispersal.

Corn originated in the highlands of Mexico 7,000 to 10,000 years ago. By the time Columbus visited the Western Hemisphere, corn was being grown by indigenous civilizations from Chile to southern Canada. Columbus noted the presence of corn on the north coast of Cuba in 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. Today, corn ranks third after wheat and rice as one of the world’s three leading food crops. However, unlike wheat and rice, the majority of corn produced in the U.S. is consumed by livestock. In the tropics and in the Southern Hemisphere cornand is a and regime. significant component of the human diet. AG

The original corn-growing areas did not includeBayer the north-centralproperty protection areapublishing (U.S. cornbelt) of of contents the United States. The highly productive U.S. cornbelt dent corns wereits derived after the parties.data therefore colonization of North America. The European settlers acceptedand/or theor local native affiliates. American varieties and incorporated themproperty with intellectual otherthird crops to providemay food, feed, and fuel its as for their survival. The current U.S.the cor ofnbelt dentand corns evolved during the gradual is regulatory owner. a document mingling of those settlements that spreadany such north and west from southeasternits North reproductiondocument America and those settlements that spreadowner south and westthis fromof northeastern North under of America. rightsthe this documentand/or of rights to of fall use the This may distribution,and The corn types grown in the norrightstheast are called ownernorthern flints; their origin is not clear, but races from the highlandssubject of Guatemala have similar ear morphology (Goodman and be the violate Brown, 1988). Northern flintscopy are largely eiofght-rowed with cylindrical ears, are early and maturing, and are short-staturedmay document publication,plants with tillers. The southern dent corns grown in the It exploitation southeast U. S. seemed tothis have any originated from the southeast coast of Mexico. Southern dent corns are characterized as havingpermission tall, late-maturing, non-tillered, poorly rooted prohibited plants with soft-textured white kernelsthe be on many-rowed, tapering ears. It seems the Tuxpeno race contributed commercialto the development of southern dents. The intentional and/or Furthermore,any unintentional crossingConsequently, betweenwithout the early northern flints and late southern dents led eventually to the highly productive U.S. cornbelt dent corns that are used extensively throughout the world today.

The origin of corn has been studied extensively, and hypotheses for the origin and for the parentage of corn have been advanced (Mangelsdorf, 1974). Hypotheses suggested for the origin of corn include the following: 1) cultivated corn is a descendent of pod corn; 2) corn originated by direct selection from teosinte; 3) corn, teosinte, and Tripsacum descended independently from a common, unknown ancestor; and 4) the tripartite theory: a) corn originated from pod corn, b) teosinte derived from a cross of corn and Tripsacum, and c) modern corn varieties evolved by corn intercrossing with teosinte or Tripsacum or both (Mangelsdorf, 1974).

Monsanto Company # 04-CR-109F Page 30 of 223

It has been suggested that modern corn originated from corn grass by a single-gene mutation causing ear development. Other suggestions have included Coix and species of the genus Manisuris in the tribe Andropogoneae for 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 use of molecular genetic markers.

Evidence has been reported to support the different hypotheses, but 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 distinguishing difference between corn and teosinte is the female inflorescence, or ear. Modern corn varieties have one to three lateral branches that terminate in an ear with eight to 24 kernel androws of 50 and regime. seeds, and the ear is enclosed in modified leaves or AGhusks. 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 by an induratedBayer glumeproperty (Goodman, protectionpublishing 1988). of contents its parties.data therefore and/oror affiliates. SECTION 2. Characterization of thpropertye vector intellectual usedthird in transformationmay its as the of and is regulatory owner. a document Plasmid vector PV-ZMIR39 (Figureany 1) such was used for the transformationits of corn cells to reproductiondocument produce MON 88017. It was constructedowner at Mo nsanto’sthis researchof laboratories in St. under of rightsthe this Louis, Missouri, using standarddocumentand/or molecular biology techniques.rights It is a disarmed, binary to of fall use of Agrobacterium tumefaciens transformation vector that ctheontains both the left and right This may distribution,and transfer-DNA (T-DNA) border rightssequences to facilitateowner transformation. The inserted DNA, the portion of plasmidsubject PV-ZMIR39 that integrated into the corn genome during the be the violate transformation process, containscopy the cp4 epspsof and cryBb1 gene expression cassettes. and may documentpublication, The specific geneticIt elements and originsexploitation of the various components used to construct plasmid vector PV-ZMIR39this anyare provided in Table 1. permission prohibited 2.1. The cp4 epsps gene and CP4the EPSPS protein commercial be

Furthermore,any The cp4 epsps codingConsequently, sequencewithout has the potential to provide high levels of tolerance to glyphosate when introduced into the plants (Padgette et al., 1996; OECD, 1999). Glyphosate binds to and blocks the activity of the native EPSPS, an enzyme of the aromatic amino acid biosynthetic pathway. In plants, the EPSPS enzyme is located in the chloroplast. Thus, in vector PV-ZMIR39, a chloroplast transit peptide 2 (CTP2) coding sequence was joined to the cp4 epsps coding sequence to target transport of the encoded protein to the chloroplast. The cp4 epsps coding sequence has been completely sequenced and encodes a 47.6 kDa protein consisting of a single polypeptide of 455 amino acids (Padgette et al., 1996). The targeted CTP2~CP4 EPSPS protein contains 531 amino acids and is approximately 55.8 kDa.

Monsanto Company # 04-CR-109F Page 31 of 223 2.1.a. The Arabidopsis thaliana EPSPS transit peptide (CTP2)

In the plant gene expression cassette, the cp4 epsps coding sequence is joined to a chloroplast transit peptide 2 (CTP2) isolated from the Arabidopsis thaliana epsps gene (Klee et al., 1987). This transit peptide directs the CP4 EPSPS protein to the chloroplast, the location of EPSPS in plants and the site of aromatic amino acid biosynthesis (Kishore and Shah, 1988). Transit peptides are typically cleaved and degraded from the full-length protein upon delivery to the plastid (Della-Cioppa et al., 1986).

2.1.b. The cp4 epsps regulatory sequences

As noted above, the CTP2-CP4 EPSPS coding sequence is under the control of the 5’ noncoding end of the rice actin 1 sequence (ract1) containing the promoter and first intron (McElroy et al., 1990) introduced upstream of the CTP2 sequence. The CP4 EPSPS sequence is joined to the 0.3-kb nopaline synthase 3’ nontranslated sequence,and and regime. NOS 3’, from Agrobacterium tumefaciens (Fraley etAG al., 1983), that provides the transcription termination and the mRNA polyadenylation signal. Bayer property protectionpublishing of contents 2.2. The cry3Bb1 gene and Cry3Bb1 protein its parties.data therefore and/oror affiliates. The Cry3Bb1 protein present in MONproperty 88017 intellectualis a memberthird of the Cry3Bbmay class of proteins its as that share >95% amino sequence homologythe of (Crickmoreand et al., 1998). It is a variant of the is regulatory owner. a document wild-type Cry3Bb1 protein isolatedany from such Bacillus thuringiensis (subsp.its kumamotoensis) reproductiondocument strain EG4691 (Donovan et al., 1992). Thownere amino acidthis sequences of of the Cry3Bb1 under of proteins present in MON 88017 andrights Bacillusthe thuringiensisthis (subsp. kumamotoensis) share documentand/or of rights 99.1% identity1 because they onlyto differof fallby six ofuse 652 amino acid residues. The amino the This may distribution,and acid sequences of the Cry3Bb1 rightsvariants present inowner MON 88017 and YieldGard Rootworm corn (MON 863) share 99.8%subject identity (differ by only one of 653 amino acid residues). be the violate The Cry3Bb1 proteins in MONcopy 863 and MONof 88017 have been extensively and characterized. may documentpublication, It exploitation this any The synthetic cry3Bb1 gene expressionpermission cassette that produces the Cry3Bb1 protein prohibited consists of the enhanced 35S promoterthe be (Odell et al., 1985), the 5’ untranslated leader of the wheat chlorophyll a/b/-bindingcommercial protein (Lamppa et al., 1985), and the intron from the Furthermore,any rice actin gene (McElroyConsequently, withoutet al ., 1991) joined to the synthetic cry3Bb1 coding sequence at the 5’ end. Joined to the 3’ end of the synthetic cry3Bb1 coding sequence is the 3’ nontranslated region of the coding sequence for wheat heat shock protein 17.3, which ends transcription and provides the signal for mRNA polyadenylation (McElwain and Spiker, 1989).

1 The wild-type Cry3Bb1 protein consists of 652 amino acid residues (Donovan et al., 1992). In MON 88017, the Cry3Bb1 protein (653 amino acid residues) contains an additional amino acid (alanine) at position 2, because it was necessary to create an Nco I restriction endonuclease site for the development of the plant gene expression plasmid vector PV-ZMIR39.

Monsanto Company # 04-CR-109F Page 32 of 223 2.3. T-DNA borders

Plasmid vector PV-ZMIR39 contains DNA sequences that are necessary for transfer of T- DNA into the plant cell. These sequences are termed the Right and Left Border regions. Within each of the regions is the border, a 24-26 bp sequence that generally defines the extent of the DNA that should be transferred into the plant genome. The Right Border present in PV-ZMIR39 is a 24 bp nucleotide sequence that was originally isolated from A. tumifaciens plasmid pTiT37 (Depicter et al., 1982). The Left Border in PV-ZMIR39 is a 25 bp nucleotide sequence from A. tumifaciens plasmid pTi5955, a derivative of plasmid pTiA6 (Barker et al., 1983).

2.4. Genetic elements outside the T-DNA borders

and The backbone region outside of the inserted DNA, which wasand not integratedregime. into the corn genome during transformation, contains two originsAG of replication necessary for replication and maintenance of the plasmid in bacteria, as well as a bacterial selectable Bayer property protectionpublishing marker gene, aad, which encodes an aminoglycoside-moof difying enzymecontents that is resistant to the action of the antibiotics spectinomycin and streptomycin. Detailedits descriptions of parties.data therefore all elements in the bacterial backbone region are presented in and/orTableor 1. 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 # 04-CR-109F Page 33 of 223 Figure 1. Plasmid map of vector PV-ZMIR39

Hind III 8 Xho I 15 EcoR I 394 LB P-ract ori-V ract1 intron CTP2 Pst I 2274 and and regime. AG cp4 epsps Pst I 2496 Bayer property protectionpublishing ROP of contents its PV-ZMIR39 parties.data therefore and/orEcoor R I 3027 12368 bpaffiliates. property intellectualthird may its NOS 3’ as the of and ori-322 is regulatory Hinowner.d III 3310 a document any such its reproductiondocument owner this ofPst I 3320 under of rightsthe this documentand/or rights P-e35S to of fall use of Pst I 8226 the EcoR V 3842 This may distribution,and rights owner Xba I 3963 aad subject wt CAB leader be the violate Pst I 7287 copy of ract1 intron and may documentpublication, It RB exploitation cry3Bb1 this any Pst I 6778 permission prohibited Xho I 6762 the commercial be Note: Sca I is a non-cutting enzyme Furthermore,tahsp17any 3’ Consequently,without EcoR I 6510

Monsanto Company # 04-CR-109F Page 34 of 223 Table 1. Summary of genetic elements in vector PV-ZMIR39

Genetic elements in cp4 epsps expression cassette Genetic Localizatio Bibliographic Element n (bp) Donor Function reference LB 12067- Octopine Ti Left border sequence Barker et al., 1983 (Left 12090 plasmid, essential for transfer of Border) pTi15955 T-DNA from the octopine Ti plasmid, pTi15955 Intervening 12091- Agrobacterium Polylinker Barker et al., 1983 sequence 12364 Intervening 12365-12 Synthetic Polylinker sequence and P-ract1 13 - 946 Rice actin gene Promoter and regime. McElroy et al., 1990 ract1 intron 947 - 1407 Rice actin gene IntronAG McElroy et al., 1991 Intervening 1408-1423 Synthetic Polylinker Bayer property protectionpublishing sequence of contents its CTP2 1424 -1651 Arabidopsis Chloroplastparties. datatransit thereforeKlee et al., 1987 and/oror thaliana affiliates.peptide sequence property intellectualthird may cp4 epsps 1652-3019 Agrobacteriumits Coding sequence for Padgette et al., 1996 as the of and sp. strainis CP4 the nativeregulatory CP4 EPSPSowner. a document any such its proteinreproduction document owner this of Intervening 3020-3031 Synthetic under Polylinker of rightsthe this documentand/or rights sequence to of fall use of NOS 3’ 3032-3287 Agrobacterium 3’nontranslatedthe region Bevan et al., 1983 This may distribution,and rights owner subjecttumefaciens of the nopaline be thesynthase violate (NOS) coding copy of sequenceand , which may documentpublication, It exploitation terminates transcription this any and directs permission prohibited polyadenylation the Intervening 3288-3320 commercialSynthetic be Polylinker sequence Furthermore,any Consequently,without

Monsanto Company # 04-CR-109F Page 35 of 223 Table 1 (cont.) Summary of genetic elements in vector PV-ZMIR39

Genetic elements in cry3Bb1 expression cassette Genetic Localizati Bibliographic Element on (bp) Donor Function reference P-e35S 3321-3933 Cauliflower Promoter with the Odell et al., 1985 mosaic virus duplicated enhancer Kay et al., 1987 region Intervening 3934-3957 Synthetic Polylinker sequence wt CAB 3958-4028 Wheat 5’ untranslated leader Lamppa et al., 1985 leader of the wheat chlorophyll a/b-binding protein and Intervening 4029-4056 Synthetic Polylinker and regime. sequence AG ract1 intron 4057-4517 Rice actin gene Intron McElroy et al., 1991 Bayer property protectionpublishing of contents its Intervening 4518-4533 Synthetic Polylinkerparties. data therefore and/oror sequence affiliates. property intellectualthird may cry3Bb1 4534-6495 Bacillus its Coding sequence for a Romano, C.P., 2002 as the of and thuringiensisis syntheticregulatory variant of owner. a document any such its subsp. Cry3Bb1reproduction proteindocument owner this of kumamotoensis under of rightsthe this Intervening 6496-6510document Syntheticand/or Polylinker rights to of fall use of sequence the This may distribution,and rights owner tahsp17 3’ 6511-6744 subject Wheat heat 3’nontranslated region McElwain and be shock protein theof theviolate coding sequence Spiker, 1989 copy of forand wheat heat shock may documentpublication, It exploitation protein 17.3, which this any ends transcription and permission prohibited directs polyadenylation the Intervening 6745-6840 commercialE. coli and be Polylinker and Depicker et al., 1982 sequence Furthermore,any synthetic synthetic RB 6841-6865Consequently, withoutNopaline Ti Right border sequence Depicker et al, 1982 (Right plasmid, essential for transfer of Border) pTiT37 T-DNA from the nopaline Ti plasmid, pTiT37

Monsanto Company # 04-CR-109F Page 36 of 223 Table 1 (cont.) Summary of genetic elements in vector PV-ZMIR39

Genetic elements for expression of the plasmid in E. coli Genetic Localizatio Bibliographic Element n (bp) Donor Function reference Intervening 6866-7350 Synthetic Polylinker Depicker et al, 1982 sequence Sutcliffe, 1978 Fling et al., 1985 aad 7351-8139 Bacterial Bacterial promoter and Fling et al., 1985 transposon Tn7 coding sequence for an GenBank accession aminoglycoside- X03043 modifying enzyme, 3’(9)-O- nucleotidyltransferase and from the transposonand regime. Tn7 AG Intervening 8140-8681 Synthetic Polylinker Fling et al., 1985 Bayer property protectionpublishing sequence of contentsSutcliffe, 1978 its ori-322 8682-9310 Plasmid Origin ofparties. replicationdata thereforeSutcliffe, 1978 pBR322 from from pBR322 forand/or or affiliates. property intellectualthird may E. coli its maintenance of plasmid as the of and is in E.regulatory coli owner. a document Intervening 9311-9727 Plasmidany such Portion of the plasmidits Sutcliffe, 1978 reproductiondocument of sequence pBR322 fromowner this under of rightsthe this documentE.and/or coli rights to of fall use of ROP 9728-9919 E. coli Coding sequencethe for Giza and Huang, This may distribution,and rights repressorowner of primer 1989 subject be theprotein violate for maintenance copy of ofand plasmid copy may documentpublication, It exploitation number in E. coli Intervening 9920-11182 thisPlasmid any Plasmid DNA Sutcliffe, 1978 permission sequence pBR322 fromprohibited the commercialE. coli be Intervening 11183-Furthermore,any E. coli Plasmid DNA Stalker et al., 1981 sequence 11430Consequently, without ori-V 11431- Agrobacterium, Origin of replication Stalker et al., 1981 11824 plasmid RK2 for Agrobacterium Intervening 11825- E. coli & Plasmid DNA Stalker et al., 1981 sequence 11910 Synthetic Intervening 11911- Agrobacterium DNA sequences Barker et al, 1983 sequence 12066

Monsanto Company # 04-CR-109F Page 37 of 223

Figure 2. Deduced amino acid sequence of the CP4 EPSPS protein produced in MON 88017

1 MAQVSRICNG VQNPSLISNL SKSSQRKSPL SVSLKTQQHP RAYPISSSWG

51 LKKSGMTLIG SELRPLKVMS SVSTACMLHG ASSRPATARK SSGLSGTVRI

101 PGDKSISHRS FMFGGLASGE TRITGLLEGE DVINTGKAMQ AMGARIRKEG

151 DTWIIDGVGN GGLLAPEAPL DFGNAATGCR LTMGLVGVYD FDSTFIGDAS

201 LTKRPMGRVL NPLREMGVQV KSEDGDRLPV TLRGPKTPTP ITYRVPMASA

251 QVKSAVLLAG LNTPGITTVI EPIMTRDHTE KMLQGFGANL TVETDADGVRand and regime. AG 301 TIRLEGRGKL TGQVIDVPGD PSSTAFPLVA ALLVPGSDVT ILNVLMNPTR

Bayer property protectionpublishing 351 TGLILTLQEM GADIEVINPR LAGGEDVADLof RVRSSTLKGV TVPEDRAPSMcontents its parties.data therefore 401 IDEYPILAVA AAFAEGATVM NGLEELRVKE SDRLSAVANGand/oror LKLNGVDCDE affiliates. property intellectualthird may its 451 GETSLVVRGR PDGKGLGNAS GAAVATHLDHas RIAMSFLVMG LVSENPVTVD the of and is regulatory owner. a document any such its 501 DATMIATSFP EFMDLMAGLG AKIELSDTKAreproduction A document owner this of under of rightsthe this documentand/or rights to of fall use of Sequence includes the CTP2 transit peptide (amino acidsthe 1-76 underlined). 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 # 04-CR-109F Page 38 of 223

Figure 3. Deduced amino acid sequence of the Cry3Bb1 protein produced in MON 88017

1 MANPNNRSEH DTIKVTPNSE LQTNHNQYPL ADNPNSTLEE LNYKEFLRMT

51 EDSSTEVLDN STVKDAVGTG ISVVGQILGV VGVPFAGALT SFYQSFLNTI

101 WPSDADPWKA FMAQVEVLID KKIEEYAKSK ALAELQGLQN NFEDYVNALN

151 SWKKTPLSLR SKRSQDRIRE LFSQAESHFR NSMPSFAVSK FEVLFLPTYA

201 QAANTHLLLL KDAQVFGEEW GYSSEDVAEF YRRQLKLTQQ YTDHCVNWYN

251 VGLNGLRGST YDAWVKFNRF RREMTLTVLD LIVLFPFYDI RLYSKGVKTEand and regime. AG 301 LTRDIFTDPI FLLTTLQKYG PTFLSIENSI RKPHLFDYLQ GIEFHTRLRP Bayer property protectionpublishing of contents 351 GYFGKDSFNY WSGNYVETRP SIGSSKTITS PFYGDKSTEPits VQKLSFDGQK parties.data therefore and/oror affiliates. 401 VYRTIANTDV AAWPNGKVYLproperty GVTKVDFSQYintellectualthird DDQKNETSTQmay TYDSKRNNGH its as the of and 451 VSAQDSIDQL PPETTDEPLEis KAYSHQLNYAregulatory ECFLMQDRRGowner. TIPFFTWTHR a document any such its reproductiondocument owner this of 501 SVDFFNTIDA EKITQLPVVK underAYALSSGASI of IEGPGFTGGN LLFLKESSNS rightsthe this documentand/or rights to of fall use of 551 IAKFKVTLNS AALLQRYRVR IRYASTTNLRthe LFVQNSNNDF LVIYINKTMN This may distribution,and rights owner 601 KDDDLTYQTFsubject DLATTNSNMG FSGDKNELII GAESFVSNEK IYIDKIEFIP be the violate copy of and 651 VQL may documentpublication, It exploitation this any permission The six amino acids that differ from theprohibited wild-type Cry3Bb1 protein are indicated in the underlined bold. The differencescommercial at bethe six positions are: 2A (insertion), H232R, S312L, N314T, E318K,Furthermore, Q349R.any Consequently,without

Monsanto Company # 04-CR-109F Page 39 of 223 SECTION 3. Characterization of the introduced genetic material

3.1 Molecular analysis – introduced DNA

Molecular analysis was performed to characterize the integrated DNA in MON 88017. Southern blot analysis was 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 both expression cassettes, and 4) stability of the inserted DNA during conventional breeding.

Detailed materials and methods for the molecular analysis are provided in Appendix I. A plasmid map illustrating the location of each element in PV-ZMIR39 is presentedand in and regime. Figure 1. Restriction enzyme sites and probes usedAG for Southern blot analysis are presented in Figures 4a and 4b. A schematic presentation of the DNA inserted into the genome of MON 88017, including restriction enzyme sites and expected restriction Bayer property protectionpublishing fragments, is provided in Figure 5. 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 # 04-CR-109F Page 40 of 223 Figure 4a. Schematic representation of the probes 1- 6 generated from the vector PV-ZMIR39 for Southern blot analyses

Hind III 8 Xho I 15 EcoRI 394 Region of LB Transferred DNA P-ract ori-V ract1 intron CTP2 Pst I 2274 Probe 1 cp4 epsps Probe 6 Pst I 2496 ROP Probe 2 PV-ZMIR39 and 12368 bp EcoR I 3027 and regime. AGNOS 3’ ori-322 Probe 3 Hind III 3310 Probe 5 Bayer Pst I property3320 protectionpublishing of contents Probe 4 P-e35S its parties.data therefore Pst I 8226 Xba I 3963 and/oror affiliates. aad property intellectualthirdwt CAB leader may its Pst I 7287 as ract1 intron the of and RB is regulatory owner. cry3Bb1 a document any such its reproductiondocument Pst I 6778 owner this of under of Xho I 6762 rightsthe this documentand/orNote: ScaI does not cut within PV-ZMIR39rights tahsp17 3’ to of fall use of the EcoThisR I 6510 may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any A circular map of the plasmid vector PV-ZMIR39permission used in Agrobacterium sp.-mediated transformation to produce MON 88017. Geneticprohibited ele ments are annotated in bold and restriction the sites (with positions relative commercialto the size be of the plasmid vector) are shown for enzymes used in the Southern analyses.Furthermore, DNAany probes were prepared by PCR amplification of plasmid PV-ZMIR39. The table describesConsequently, probes 1-6without used in the Southern analyses.

Probe Genetic Element(s) Start End Total Length (bp) 1 Partial LB + P-ract + partial CTP2 12080 1482 1771 2 CTP2 + cp4 epsps 1423 3019 1597 3 Partial cp4 epsps + NOS 3’ + P-e35S + 2991 4533 1543 wt CAB leader + ract1 intron 4 cry3Bb1 + tahsp17 3’ + RB 4534 6865 2332 5 Partial backbone 6866 9492 2627 6 Partial backbone 9465 12066 2602

Monsanto Company # 04-CR-109F Page 41 of 223 Figure 4b. Schematic representation of the probes 7- 12 generated from the vector PV-ZMIR39 for Southern blot analyses

Hind III 8 Xho I 15 EcoRI 394 LB Region of P-ract Transferred DNA ori-V ract1 intron CTP2 Pst I 2274 Probe 7 cp4 epsps Pst I 2496 ROP PV-ZMIR39 EcoR I 3027 regime.and 12368 bp Probe 8 and AG NOS 3’ ori-322 Probe 9 Hind III 3310

Probe 10Bayer propertyPst I 3320protectionpublishing of contents P-e35S its Probe 12 Probe 11 parties.data therefore Pst I 8226 Xba I 3963and/oror affiliates. aad property intellectualthird wt CAB leadermay its as Pst I 7287 the of and ract1 intron is regulatory owner. RB a document any such cry3Bb1 its reproductiondocument Pst I 6778 owner this of under of this Xho I 6762and/orrights the document to of fall of rights tahsp17 3’ Note: Sca I doesuse not cut within PV-ZMIR39 the This may distribution,and EcoR I 6510rights owner subject be the violate copy of and may documentpublication, It exploitation this A circular map of the plasmid vectorany PV-ZMIR39 used in Agrobacterium sp.-mediated permission transformation to produce MON 88017. Geneticprohibited ele ments are annotated in bold and restriction the sites (with positions relative commercialto the size be of the plasmid vector) are shown for enzymes used in the Southern analyses. DNA probes were prepared by PCR amplification of plasmid PV-ZMIR39. Furthermore,any The table describesConsequently, probes 7without – 12 used in the Southern analyses.

Probe Genetic Element(s) Start End Total Length (bp) 7 P-ract + ract1 intron 13 1407 1395 8 NOS 3’ 3032 3287 256 9 P-e35S 3321 3933 613 10 wt CAB leader + ract1 intron 3958 4533 576 11 cry3Bb1 4534 6510 1977 12 tahsp17 3’ 6511 6744 234

Monsanto Company # 04-CR-109F Page 42 of 223 Figure 5. Schematic representation of the insert present in MON 88017

A linear map of the inserted DNA from transformation vector PV-ZMIR 39 is shown. Genetic elements are annotated. Positions of the restriction sites for enzymes used in the Southern blot analyses are included for reference. Arrows indicate sites of the restriction digest. Sizes of predicted restriction fragments, calculated from the size of the linear map are identified. MON 88017 contains one copy of the insert at a single integration locus (Figure 6). and and regime. AG Direction of transcription Hind III 3612 Pst I 2798 Bayer property protectionpublishing Xho I 7065 Pst I 3622 of contents Pst I 2576 Xba I 4265 its parties.data therefore Hind III 310 and/oror Pst I 7080 Xho I 317 affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its Plant P-ract ract1 CTP2 cp4 epsps NOS 3’ P-e35S wtCABreproduction ract1document Cry3Bb1 tahsp17 3’ Plant owner this of genomic Intron under of leader intron genomic rightsthe this DNA documentand/or rights DNA to of fall use of the This may distribution,and rights owner Xba I >4.3 kb subject >2.8 kb be the violate copy of Xho I ~6.7 kb and may documentpublication, It exploitation this Hind III ~3.3. kb any >3.5 kb permission prohibited the >2.6 kb ~0.2 kb ~0.8commercial kb be ~3.5 kb Pst I Furthermore,any Consequently,without Pst I + Xho I ~2.3 kb ~0.2 kb ~0.8 kb ~3.5 kb

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3.1.a. Insert and copy number

The insert number (the number of integration sites of the introduced DNA in the corn genome) was evaluated by digesting the MON 88017 and conventional corn genomic DNA (control in Figure 6) with the restriction enzyme Sca I, which does not cleave within the plasmid PV-ZMIR39. This enzyme should release a restriction fragment containing the inserted DNA and adjacent plant genomic DNA. The number of restriction fragments detected indicates the number of inserts present.

The number of copies of the introduced DNA was determined by digesting the MON 88017 genomic DNA with Xba I, a restriction enzyme that cuts only once within PV-ZMIR39. The blot was probed simultaneously with four radiolabeled fragments of plasmid DNA that span the entire length of the insert (Probes 1, 2, 3 and 4, Figure 4a). If one copy of the introduced DNA is present, two bands should be produced, andrepresenting and regime. two border fragments. Each of these border fragmentsAG would contain a portion of the inserted DNA and flanking corn genomic DNA. Therefore, from this analysis it was possible to determine the number of copies of Bayerthe introduced property DNAprotection publishinginserted in MON of contents 88017. Plasmid PV-ZMIR39 DNA, previously digested with EcoRits I, was mixed with parties.data therefore control corn genomic DNA digested with Sca I, and then loadedand/or onor the gel to serve as a affiliates. property intellectualthird may positive hybridization control. its as the of and is regulatory owner. a document The results of these analyses are showany nsuch in Figure 6. Plasmid PV-ZMIR39its DNA, reproductiondocument previously digested with EcoR I, mixed withowner control cornthis genomicof DNA digested with under of rightsthe this Sca I, produced the expecteddocument and/orsize bands of approximately rights6.3 kb (faint), 3.5 kb and 2.6 to of fall use of kb, correlating to the sizes of PV-ZMIR39 EcoR I fragmentsthe (lanes 4 and 5). The This may distribution,and ~6.3 kb band produced a weakerrights signal, because ownera smaller portion of the target DNA sequence is present on thissubject ~6.3 kb EcoR I restriction fragment in comparison to the ~3.5 be the violate kb and ~2.6 kb fragments. copyMON 88017 genomicof DNA digested with Sca I (lanes 2 and and may documentpublication, 6) produced one bandIt at approximatelyexploitation 13 Kb . This result establishes that MON 88017 contains one insert locatedthis on any an approximately 13 Kb Sca I restriction fragment. MON 88017 DNA digested with Xbapermission I (lanes 3 and 7) produced two unique bands at prohibited approximately 7.4 kb and 5.5 kb,the representi ng two border fragments. In combination commercial be with the insert number analysis, this result establishes that MON 88017 contains one Furthermore,any copy of the introducedConsequently, DNAwithout at a single locus of integration.

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Figure 6. Southern blot analysis of MON 88017: insert and copy number LONG RUN SHORT RUN 1 2 3 4 5 6 7

40 20 15 10 40 8.1 7.1 20 6.1 15 5.1 4.1 10 and 3.1and regime. 8.1 AG 7.1 2.0 6.1 Bayer property1.6 protectionpublishing of contents its parties.data therefore 5.1 1.0and/oror affiliates. property intellectualthird may its as 4.1 the of and 0.5 is regulatory owner. a document any such its reproductiondocument owner this of under of 3.1 rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate 2.0 copy of and may documentpublication, It exploitation 1.6 this any permission prohibited the commercial be The blot was probed simultaneously with four 32P-labeled probes that span the full length of the Furthermore,any insert (Probes 1, 2,Consequently, 3, and 4,without Figure 4a). Each lane contains ~10 µg of digested genomic DNA isolated from grain. Lane designations are as follows: Lane 1: Control (Sca I) 2: MON 88017 (Sca I) 3: MON 88017 (Xba I) 4: Control (Sca I) spiked with PV-ZMIR39 (EcoR I) [0.5 copy] 5: Control (Sca I) spiked with PV-ZMIR39 (EcoR I) [1.0 copy] 6: MON 88017 (Sca I) 7: MON 88017 (Xba I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.

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3.1.b. cp4 epsps cassette intactness

MON 88017 corn genomic DNA was digested individually with Xho I to release the inserted DNA and with Hind III to release the cp4 epsps cassette. Control genomic DNA from conventional corn was digested with Xho I, or in some experiments with Hind III, to serve as a negative control. Plasmid PV-ZMIR39 DNA, previously digested with EcoR I, was mixed with control DNA digested with Xho I, and the mixture was then loaded on the gel to serve as a positive hybridization control. Individual Southern blots were probed with the rice actin promoter (P-ract) + ract1 intron, the CTP2 + cp4 epsps coding region, or the NOS 3’ polyadenylation sequence (Probes 7, 2, and 8, respectively, Figure 4b). The presence of a band representing the expected size of the full inserted DNA (Xho I digest, ~6.7 kb) or the cp4 epsps cassette (Hind III digest, ~3.3 kb) indicates that the cp4 epsps cassette and each of its elements are intact. For reference, the expected sizes of the full insert and cp4 epsps cassette upon restriction enzyme digestion are shownand in Figure and regime. 5. In these Southern analyses, the migration of the AG~6.7 kb Xho I fragment containing the entire insert was slightly slower than expected. This altered migration is likely due to the difference in salt concentrations between the testBayer DNA property sample protection and publishingthe molecular weight of contents marker (Sambrook, 2001). its parties.data therefore and/oror affiliates. 3.1.c p-ract + ract1 intron probe property intellectualthird may its as the of and is regulatory owner. a document The results of this analysis are shownany insuch Figur e 7. The Southern itsblot containing control, reproductiondocument plasmid and MON 88017 genomic DNA ownerdigested withthis XhoI orof HindIII was hybridized under of rightsthe this with the P-ract + ract introndocument probeand/or (Probe 7, Figure 4b). Controlrights corn genomic DNA to of fall use of digested with Xho I or Hind III (lanes 1 and 2, respectively)the showed faint endogenous This may distribution,and hybridization, indicating regionsrights of homology betweenowner the corn genome and the probe. Plasmid PV-ZMIR39 DNA,subject previously digested with EcoR I, mixed with control be the violate genomic DNA digested withcopy Xho I, producedof the expected size bands at approximately and may documentpublication, 3.5 kb and 2.6 kb (lanesIt 5 and 6). MONexploitation 88017 DNA digested with Xho I (lanes 3 and 7) produced a band correspondingthis any to the expected size of the insert (approximately 6.7 kb) permission in addition to the faint endogenous hybridizatiprohibited on pattern seen in the negative control (lane 1). MON 88017 DNA digestedthe with Hind III (lanes 4 and 8) produced bands at commercial be approximately 8.5 kb and 3.3 kb in addition to the faint endogenous hybridization pattern Furthermore,any seen in the controlConsequently, (lane 2).without The ~3.3 kb band corresponds to the expected size of an intact cp4 epsps cassette. The ~8.5 kb band corresponds to a border fragment predicted by hybridization of the ract1 intron portion of the probe to the ract1 intron target sequence present in the cry3Bb1 cassette. No unexplained bands were detected, indicating that MON 88017 does not contain any additional, detectable rice actin promoter or ract1 intron elements other than those associated with the intact cp4 epsps or cry3Bb1 cassettes.

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Figure 7. Southern blot analysis of MON 88017: p-ract + ract1 intron probe

LONG RUN SHORT RUN 1 2 3 4 5 6 7 8

40 20 40 15 20 10 15 8.1 7.1 10 6.1 8.1 5.1 and 7.1 and4.1 regime. 6.1 AG 3.1 5.1 Bayer property protectionpublishing of contents 4.1 2.0 its parties.data therefore and/or1.6or affiliates. property intellectualthird may 3.1 its as the of and 1.0 is regulatory owner. a document any such its reproductiondocument owner this of 2.0 0.5 under of this and/orrights the document fall of rights 1.6 to of use the This may distribution,and rights owner subject be the violate 1.0 copy of and may documentpublication, It exploitation this any permission prohibited 32 the The blot was probed with P-labeledcommercial rice be actin promoter and intron (Probe 7, Figure 4b). Each lane contains ~10 µg of digested genomic DNA isolated from grain. Lane designations are as Furthermore,any follows: Consequently,without Lane 1: Control (Xho I) 2: Control (Hind III) 3: MON 88017 (Xho I) 4: MON 88017 (Hind III) 5: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [0.5 copy] 6: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [1.0 copy] 7: MON 88017 (Xho I) 8: MON 88017 (Hind III) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.

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3.1.d. ctp2 + cp4 epsps probe

The results of this analysis are shown in Figure 8. The Southern blot containing control, plasmid and MON 88017 genomic DNA digested with XhoI or HindIII was hybridized with the CTP2 + cp4 epsps probe (Probe 2, Figure 4a). Control corn genomic DNA digested with Xho I or Hind III (lanes 1 and 2, respectively) showed no detectable hybridization bands, as expected for a negative control. Plasmid PV-ZMIR39 DNA previously digested with EcoR I, mixed with control DNA digested with Xho I, produced the expected size band at approximately 2.6 kb (lanes 5 and 6). MON 88017 genomic DNA digested with Xho I (lanes 3 and 8) produced a band corresponding to the expected size of the insert (approximately 6.7 kb). MON 88017 DNA digested with Hind III (lanes 4 and 7) produced a band at approximately 3.3 kb, corresponding to the expected size of an intact cp4 epsps cassette. No unexpected bands were detected, indicating that MON 88017 does not contain any additional, detectable CTP2 + cp4 epsps andcoding and regime. regions other than that associated with the intact cp4AG epsps cassette.

3.1.e. NOS 3’ probe Bayer property protectionpublishing of contents its parties.data therefore The results of this analysis are shown in Figure 9. The Southernand/or blotor containing control, affiliates. property intellectualthird may plasmid and MON 88017 genomic DNAits digested with XhoI or HindIII was hybridized as with the NOS 3’ probe (Probe 8, Figurethe of 4b). Controland corn genomic DNA digested with is regulatory owner. a document Xho I (lane 1) showed no detectableany hybridizat such ion bands, as expectedits for a negative reproductiondocument control. Plasmid PV-ZMIR39 DNA previouslyowner digestedthis with Ecoof R I, mixed with control under of rightsthe this DNA digested with Xho documentI, producedand/or the expected size bandrights at approximately 3.5 kb to of fall use of (lanes 4 and 5). MON 88017 DNA digested with Xho Ithe (lanes 2 and 6) produced a band This may distribution,and corresponding to the expected sizerights of the insert (approxiowner mately 6.7 kb). MON 88017 DNA digested with Hind IIIsubject (lanes 3 and 7) produced a band at approximately 3.3 kb, be the violate corresponding to the expectcopyed size of an intactof cp4 epsps cassette. No unexpected bands and may documentpublication, were detected, indicatingIt that MONexploitation 88017 does not contain any additional, detectable NOS 3’ polyadenylation elementsthis any other than that associated with the intact cp4 epsps permission cassette. prohibited the commercial be Furthermore,any Consequently,without

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Figure 8. Southern blot analysis of MON 88017: ctp2 + cp4 epsps probe LONG RUN SHORT RUN 1 2 3 4 5 6 7 8

40 20 15 10 8.1 6.1 7.1 40 5.1 20 4.1 15 3.1 2.0 10 1.6 8.1 1.0 7.1 and and regime. 6.1 AG 0.5 5.1 Bayer property protectionpublishing 4.1 of contents its parties.data therefore and/oror affiliates. 3.1 property intellectualthird may 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 rights to of fall use of 1.6 the This may distribution,and rights owner subject be the violate copy of 1.0 and may documentpublication, It exploitation this any permission The blot was probed with 32P-labeled CTP2prohibited + cp4 epsps (Probe 2, Figure 4a). Each lane contains the be ~10 µg of digested genomic DNAcommercial isolated from grain. Lane designations are as follows: Lane 1: ControlFurthermore, (Xho anyI) 2: ControlConsequently, (Hind III)without 3: MON 88017 (Xho I) 4: MON 88017 (Hind III) 5: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [0.5 copy] 6: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [1.0 copy] 7: MON 88017 (Hind III) 8: MON 88017 (Xho I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.

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Figure 9. Southern blot analysis of MON 88017: NOS 3’ probe

LONG RUN SHORT RUN 1 2 3 4 5 6 7

40 20 15 40 10 8.1 20 7.1 15 6.1 10 5.1 8.1 4.1 7.1 6.1 3.1 and and regime. 5.1 AG 2.0 4.1 Bayer property protectionpublishing of 1.6 contents its parties.data therefore 3.1 and/oror affiliates. property intellectualthird 1.0 may its as the of and is regulatory owner. a document any such 0.5 its 2.0 reproductiondocument owner this of under of rightsthe this 1.6 documentand/or rights to of fall use of the This may distribution,and rights owner subject 1.0 be the violate copy of and may documentpublication, It exploitation this any 32 permission The blot was probed with P-labeled NOSprohibited 3’ polyadenylation sequence (Probe 8, Figure 4b). Each lane contains ~10 µg of digestedthe genomic DNA isolated from grain. Lane designations are commercial be as follows: Furthermore,any Lane 1: ControlConsequently, (Xho I) without 2: MON 88017 (Xho I) 3: MON 88017 (Hind III) 4: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [0.5 copy] 5: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [1.0 copy] 6: MON 88017 (Xho I) 7: MON 88017 (Hind III) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.

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3.1.f. cry3Bb1 cassette intactness

MON 88017 genomic DNA was digested with Xho I to release the inserted DNA. Additionally, MON 88017 genomic DNA was digested with Pst I to release the cry3Bb1 cassette. Plasmid PV-ZMIR39 DNA previously digested with EcoR I, was mixed with control DNA digested with Xho I, and the mixture was then loaded on the gel to serve as a positive hybridization control. Control genomic DNA was digested with Xho I to serve as a negative control. Individual Southern blots were hybridized with the enhanced 35S promoter (P-e35S), the wt CAB leader + ract1 intron, the cry3Bb1 coding region, or the tahsp17 3’ polyadenylation sequence (Probes 9, 10, 11 and 12, respectively, Figure 4b). The presence of a band representing the expected size of the full insert (Xho I digest, ~6.7 kb) or the cry3Bb1 cassette (Pst I digest, ~3.5 kb) indicates that the cry3Bb1 cassette and each of its elements are intact. For reference, the expected sizes of the full insert and the cry3Bb1 cassette upon restriction enzyme digestion are shown in Figure 5. Similar to the cp4 epsps Southern analyses, occasionally the migration of the ~6.7 kb Xhoand I fragment and regime. containing the entire insert was slightly slower thanAG expected. This altered migration is likely due to the difference in salt concentrations between the test DNA sample and the molecular weight marker (Sambrook, 2001). Bayer property protectionpublishing of contents its parties.data therefore 3.1.g. p-e35S probe and/oror affiliates. property intellectualthird may its as The results of this analysis are shownthe ofin Figureand 10. The Southern blot containing control, is regulatory owner. a document plasmid and MON 88017 genomicany DNA such digested with XhoI or PstIits was hybridized with reproductiondocument the P-e35S probe (Probe 9, Figure 4b). Controlowner DNA thisdigested ofwith Xho I (lane 1) under of rightsthe this showed no detectable hybridizationdocumentand/or bands, as expected forrights the negative control. Plasmid to of fall use of PV-ZMIR39 DNA previously digested with EcoR I, mixedthe with control DNA digested This may distribution,and with Xho I, produced the expectedrights size band at approximatelyowner 3.5 kb (lanes 4 and 5). MON 88017 genomic DNAsubject was digested with Xho I (lanes 2 and 6) produced a band be the violate corresponding to the expectedcopy size of the insertedof DNA (approximately 6.7 kb). MON and may documentpublication, 88017 genomic DNAIt digested with exploitationPst I (lanes 3 and 7) produced a band at approximately 3.5 kb, correspondingthis any to the expected size of an intact cry3Bb1 cassette. No unexpected bands were detected,permission indicating that MON 88017 does not contain any prohibited additional, detectable enhanced the35S promoter elements other than that associated with the commercial be intact cry3Bb1 cassette. Furthermore,any Consequently,without 3.1.h. wt CAB leader + ract1 intron probe

The results of this analysis are shown in Figure 11. The Southern blot containing control, plasmid and MON 88017 genomic DNA digested with XhoI or PstI was hybridized with the wtCAB leader + ract intron probe (Probe 10, Figure 4b). Control corn genomic DNA digested with Xho I (lane 1) showed no detectable hybridization bands, as expected for a negative control. Plasmid PV-ZMIR39 DNA, previously digested with EcoR I, mixed with control DNA digested with Xho I, produced the expected size bands at approximately 3.5 kb and 2.6 kb (lanes 4 and 5). MON 88017 genomic DNA digested with Xho I (lanes 2 and 7) produced a band corresponding to the expected size of the insert (approximately 6.7 kb). MON 88017 genomic DNA digested with Pst I (lanes 3

Monsanto Company 04-CR-109F Page 51 of 223

and 6) produced bands at approximately 3.8 kb and 3.5 kb. The ~3.5 kb band corresponds to the expected size of the intact cry3Bb1 cassette. The ~3.8 kb band corresponds to a border fragment predicted by hybridization of the ract1 intron portion of the probe to the ract1 intron target sequence present in the cp4 epsps cassette. No unexpected bands were detected, indicating that MON 88017 corn does not contain any additional, detectable wt CAB leader or ract1 intron elements other than those associated with the intact cry3Bb1 or cp4 epsps cassettes.

3.1.i. cry3Bb1 probe

The results of this analysis are shown in Figure 12. The Southern blot containing control, plasmid and MON 88017 genomic DNA digested with XhoI or HindIII was hybridized with the cry3Bb1 probe (Probe 11, Figure 4b). Control corn genomic DNA digested with Xho I (lane 1) showed no detectable hybridization bands, as expected for the negative control. Plasmid PV-ZMIR39 DNA previously digested with EcoR I, mixedand with control and regime. DNA digested with Xho I, produced the expected sizeAG band at approximately 3.5 kb (lanes 4 and 5). MON 88017 genomic DNA digested with Xho I (lanes 2 and 6) produced a band corresponding to the expectedBayer size ofproperty the insert protection (approximatelypublishing 6.7 kb). of contents MON 88017 DNA digested with Pst I (lanes 3 and 7) produced a bandits at approximately parties.data therefore 3.5 kb, corresponding to the expected size of an intact cry3Bb1and/or cassette.or No unexpected affiliates. property intellectualthird may bands were detected, indicating that MONits 88017 does not contain any additional, as detectable cry3Bb1 coding regionsthe other of than thatand associated with the intact cry3Bb1 is regulatory owner. a document cassette. any such its reproductiondocument owner this of under of 3.1.j. tahsp17 3’ probe rightsthe this documentand/or rights to of fall use of the This may distribution,and The results of this analysis are shownrights in Figure 13.owner The Southern blot containing control, plasmid and MON 88017 subjectgenomic DNA digested with XhoI or PstI was hybridized with be the violate the tahsp17 3’ probe (Probecopy 12, Figure 4b). of Cont rol corn genomic DNA digested with and may documentpublication, Xho I (lane 1) showedIt no detectableexploitation hybridizat ion bands, as expected for a negative control. Plasmid PV-ZMIR39this any DNA previously digested with EcoR I, mixed with control permission DNA digested with Xho I, produced theprohibited expected size band at approximately 6.3 kb (lanes 4 and 5). MON 88017 genomicthe DNA digested with Xho I (lanes 2 and 6) commercial be produced a band corresponding to the expected size of the insert (approximately 6.7 kb). Furthermore,any MON 88017 DNAConsequently, digestedwithout with Pst I (lanes 3 and 7) produced a band at approximately 3.5 kb, corresponding to the expected size of an intact cry3Bb1 cassette. A small area of hybridization was present in lane 3 directly above the expected hybridization signal. This spot does not span the entire lane, and does not appear in the short run of the same sample in lane 7, indicating that this signal is an artifact. No unexpected bands were detected, indicating that MON 88017 does not contain any additional, detectable tahsp17 3’ polyadenylation elements other than that associated with the intact cry3Bb1 cassette.

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Figure 10. Southern blot analysis of MON 88017: p-e35S probe

LONG RUN SHORT RUN 1 2 3 4 5 6 7

40 20 15 40 10 20 8.1 15 7.1 6.1 10 5.1 8.1 4.1 7.1 6.1 3.1 regime.and 5.1 and AG 2.0 4.1 1.6 Bayer property protectionpublishing of contents its 3.1 parties.data 1.0 therefore and/oror affiliates. property intellectualthird may its as 0.5 the of and is regulatory owner. 2.0 a document any such its reproductiondocument of 1.6 owner this under of rightsthe this documentand/or rights to of fall use of the This may distribution,and 1.0 rights owner subject be the violate copy of document and may publication,exploitation 0.5 It this any permission prohibited the be The blot was probed with 32P-labeledcommercial enhanced 35S promoter (Probe 9, Figure 4b). Each lane contains ~10 µgFurthermore, of digestedany genomic DNA isolated from grain. Lane designations are as follows: Lane 1: ControlConsequently, (Xho I) without 2: MON 88017 (Xho I) 3: MON 88017 (Pst I) 4: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [0.5 copy] 5: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [1.0 copy] 6: MON 88017 (Xho I) 7: MON 88017 (Pst I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.

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Figure 11. Southern blot analysis of MON 88017: wtCAB leader + ract1 intron probe

LONG RUN SHORT RUN 1 2 3 4 5 6 7

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

5.1 Bayer property protection2.0 publishing of contents its parties.data 1.6 therefore 4.1 and/oror affiliates. property intellectualthird may its as 1.0 the of and 3.1 is regulatory owner. a document any such its reproductiondocument owner this of0.5 under of rightsthe this documentand/or of rights 2.0 to of fall use the This may distribution,and rights owner 1.6 subject be the violate copy of and may documentpublication, It exploitation 1.0 this any permission prohibited the commercial be Furthermore,any The blot was probedConsequently, with 32P-labeledwithout wheat CAB leader + rice actin intron (Probe 10, Figure 4b). Each lane contains ~10 µg of digested genomic DNA isolated from grain. Lane designations are as follows: Lane 1: Control (Xho I) 2: MON 88017 (Xho I) 3: MON 88017 (Pst I) 4: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [0.5 copy] 5: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [1.0 copy] 6: MON 88017 (Pst I) 7: MON 88017 (Xho I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.

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Figure 12. Southern blot analysis of MON 88017: cry3Bb1 probe

LONG RUN SHORT RUN 1 2 3 4 5 6 7

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

5.1 Bayer property 2.0protectionpublishing of contents 1.6 its 4.1 parties.data therefore and/oror affiliates. property intellectualthird may its 1.0 as 3.1 the of and is regulatory owner. a document any such its reproductiondocument owner this 0.5of under of rightsthe this documentand/or rights 2.0 to of fall use of the This may distribution,and rights owner 1.6 subject be the violate copy of and may documentpublication, It exploitation 1.0 this any permission prohibited the be The blot was probed with 32P-labeledcommercial cry3Bb1 coding region (Probe 11, Figure 4b). Each lane contains ~10 µgFurthermore, of digestedany genomic DNA isolated from grain. Lane designations are as follows: Lane 1: ControlConsequently, (Xho I) without 2: MON 88017 (Xho I) 3: MON 88017 (Pst I) 4: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [0.5 copy] 5: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [1.0 copy] 6: MON 88017 (Xho I) 7: MON 88017 (Pst I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.

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Figure 13. Southern blot analysis of MON 88017: tahsp17 3’ probe LONG RUN SHORT RUN 1 2 3 4 5 6 7

40 20 40 15 20 10 15 8.1 7.1 10 6.1 8.1 5.1 7.1 4.1 6.1 3.1 and 5.1 and regime. AG 4.1 2.0 1.6 Bayer property protectionpublishing 3.1 of contents its parties.1.0data therefore and/oror affiliates. property intellectualthird may its 0.5 2.0 as the of and is regulatory owner. 1.6 a document any such its reproductiondocument owner this of under of rightsthe this 1.0 documentand/or rights to of fall use of the This may distribution,and rights owner subject 0.5 be the violate copy of document and may publication,exploitation It this any The blot was probed with 32P-labeled tahsp17 3’ polyadenylation sequence (Probe 12, Figure 4b). permission Each lane contains ~10 µg of digested genomicprohibited DNA isolated from grain. Lane designations are the as follows: commercial be Lane 1: ControlFurthermore, (Xho anyI) 2: MON 88017Consequently, (Xhowithout I) 3: MON 88017 (Pst I) 4: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [0.5 copy] 5: Control (Xho I) spiked with PV-ZMIR39 (EcoR I) [1.0 copy] 6: MON 88017 (Xho I) 7: MON 88017 (Pst I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.

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3.2. Analysis for backbone fragments

For this analysis, MON 88017 and control DNA samples were digested with Xba I, which cuts only once within PV-ZMIR39. The blot was hybridized simultaneously with two radiolabeled probes that span the entire backbone sequence in PV-ZMIR39 (Probes 5 and 6, Figure 4a). The MON 88017 samples were obtained from the same generation used to test for stability of the insert during breeding (Figure 16). These generations are shown on the MON 88017 breeding tree (Figure 15). Because of expected cross-hybridization of the probes to the molecular weight marker bands, these lanes were trimmed from the blot prior to hybridization. Aligning these lanes to the blot after hybridization allowed for appropriate annotation of the molecular weight marker band sizes on the film.

The results of this analysis are shown in Figure 14. Plasmid PV-ZMIR39 DNA previously digested with EcoR I and mixed with digested control corn genomicand DNA and regime. (lanes 3 and 4) produced one expected size band at AGapproximately 6.3 kb. The control LH198 DNA (lane 1) produced a diffuse hybridization signal below 1.6 kb, which is likely the result of bacterial contamination of thisBayer source property of grain. protection Aspublishing expected, the of contents second source of control DNA (lane 2) did not produce any detectableits hybridization. parties.data therefore Genomic DNA from plants representing six generations of MONand/or or88017 (lanes 5 through affiliates. property intellectualthird may 11) also showed no detectable hybridizationits bands. These results establish that the as MON 88017 does not contain any thedetectable of backboneand sequences from the is regulatory owner. a document transformation vector PV-ZMIR39.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 04-CR-109F Page 57 of 223

Figure 14. Southern blot analysis of MON 88017: backbone analysis

1 2 3 4 5 6 7 8 9 10 11

40 40 20 20 15 15 10 10 8.1 8.1 7.1 7.1 6.1 6.1 5.1 5.1 4.1 4.1 and and regime. AG 3.1 3.1 Bayer property protectionpublishing of contents its 2.0 parties.data therefore and/oror 2.0 affiliates. 1.6 property intellectualthird may its 1.6 as the of and is regulatory owner. a document any such its reproductiondocument 1.0 owner this of under of 1.0 rightsthe this documentand/or rights to of fall use of the This may distribution,and 0.5 rights owner subject 0.5 be the violate copy of and may documentpublication, It exploitation this any permission32 The blot was probed simultaneously with prohibitedtwo P-labeled probes that span the backbone in PV-ZMIR39 (Probes 5 and 6, Figurethe 4a). Each lane contains ~10 µg of digested genomic DNA commercial be isolated from grain. Lane designations are as follows: Lane 1: ControlFurthermore, LH198any (Xba I) Consequently,without 2: Control LH59 x LH198 (Xba I) 3: Control LH59 x LH198 (Xba I) spiked with PV-ZMIR39 (EcoR I) [0.5 copy] 4: Control LH59 x LH198 (Xba I) spiked with PV-ZMIR39 (EcoR I) [1.0 copy] 5: MON 88017 [LH198BC0F1 x LH59]F2 (Xba I) 6: MON 88017 LH198BC0F1 (Xba I) 7: MON 88017 LH198BC0F2 (Xba I) 8: MON 88017 LH198BC1F1 (Xba I) 9: MON 88017 LH198BC1F3 x LH59(H) (Xba I) 10: MON 88017 LH59 x LH198BC3F3(H) (Xba I) 11: MON 88017 HC33 x LH59BC2F3(H) (Xba I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.

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3.3. Insert stability in multiple breeding generations

In order to determine generational stability of the integrated DNA, a Southern blot analysis to determine the number of copies of integrated transgenes was performed on multiple generations from the breeding tree of MON 88017. For reference, the breeding tree of MON 88017 is presented in Figure 15. The specific generations tested are indicated in bold. The [LH198BC0F1 x LH159]F2 generation, which was used as a test substance in the molecular characterization analyses described in Sections 3.1 – 3.4 of this Part IV, was included as a reference in this analysis. For this analysis, MON 88017 and conventional corn genomic DNA samples were digested with Xba I, which digests only once within PV-ZMIR39. The blot was probed simultaneously with four radiolabeled probes that span the entire length of the insert (Probes 1, 2, 3 and 4, Figure 4a). and and regime. The results of the Southern blot analysis are shown AGin Figure 16. Plasmid PV-ZMIR39 DNA previously digested with EcoR I, mixed with digested control DNA, produced the expected size bands of approximately 6.3 kb, 3.5Bayer kb andproperty 2.6 kb, protection correlatingpublishing to the sizes of of contents PV-ZMIR39 EcoR I fragments (lanes 3 and 4). The ~6.3 kb band producedits a weaker parties.data therefore signal because a smaller portion of the target insert sequence isand/or presentor on this ~6.3 kb affiliates. property intellectualthird may EcoR I restriction fragment in comparisonits to the ~3.5 kb and ~2.6 kb fragments. The as previously characterized generationthe (lane of 5) producedand the expected size bands of is regulatory owner. a document approximately 7.4 kb and 5.5 kb thatany had such b een produced in the previousits copy number reproductiondocument analysis (Figure 6). In addition, six generationsowner of MONthis 88017of (lanes 6 through 11) under of rightsthe this produced the same size bandsdocumentand/or as the previously characterizedrights ge neration (~7.4 kb and to of fall use of ~5.5 kb). These results demonstrate that the expected Southernthe fingerprint of the This may distribution,and MON 88017 insert has been maintainedrights across thownere branches of the breeding tree that were tested. Therefore, the stabilitysubject of the integrated DNA in MON 88017 has been be the violate established over multiple generations.copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company 04-CR-109F Page 59 of 223

Figure 15. Breeding tree of MON 88017

a R0 x LH198

LH198BC0F1 ⊗ x LH59

x LH198

LH198BC1F1 [LH198BC0F1 x LH59]F1(H) LH198BC0F2 ⊗ bc ⊗ x LH59 ⊗ [LH198BC0F x LH59]F LH198BC1F2 LH198BC2F1 1 2 LH198BC0F3 ⊗ bc ⊗ LH59BC1F regime.and 1 LH198BC1F3 and LH198BC3F1 x LH59 LH198BC0F4 AG

⊗ LH59BC2F1 x LH59 ⊗ Bayer property protectionpublishing of contents LH198BC3F2 LH59BC2F its 2 ⊗ parties.data therefore ⊗ LH59 x LH198BC1F3 (H) and/oror affiliates. propertyLH198BC3F intellectualthird may LH59BC2F3 its 3 xas Tester1 x Tester2 the of and is regulatory owner. LH59 x LH198BC3Fa document HC33 x LH59BC2F any such 3(H) its 3(H) reproductiondocument of owner⊗ this under of rightsthe this documentand/or of rights to of[LH59 fall x LH198BC3Fuse 3(H)]F1 the This may distribution,and rights owner subject R0 - originally transformed plant; ⊗ - self-pollinated; (H) - hybrid; bc = backcross be the violate copy of and may documentpublication, It exploitation this any permission prohibited the Data commercialMON be 88017 Generation Molecular analysis: [LH198BC0F1 x LH59]F2 Furthermore,any Insert stability (Southern.Consequently, blot): without generations bolded Protein stability (western blot): generations bolded 2002 T/C/R trials: LH59 x LH198BC3F3(H) seed; [LH59 x LH198BC3F3(H)]F1 grain Expression/composition: LH59 x LH198BC3F3(H) seed; [LH59 x LH198BC3F3(H)]F1 grain

Monsanto Company 04-CR-109F Page 60 of 223

Figure 16. Stability of integrated DNA in MON 88017

1 2 3 4 5 6 7 8 9 10 11

40 40 20 20 15 15 10 10 8.1 8.1 7.1 7.1 6.1 6.1 5.1 5.1

4.1 4.1 and and regime. AG 3.1 3.1 Bayer property protectionpublishing of contents its parties.data therefore 2.0 and/oror 2.0 affiliates. property intellectualthird may 1.6 its 1.6 as the of and is regulatory owner. a document any such its reproductiondocument owner this of 1.0 under of 1.0 rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner 0.5 subject 0.5 be the violate copy of and may documentpublication, It exploitation 32 The blot was probed simultaneouslythis any with four P-labeled probes that span the full length of the inserted DNA (Probes 1, 2, 3, and 4, Figurepermission 4a). Each lane contains ~10 µg of digested genomic prohibited DNA isolated from grain. Lane designationsthe be are as follows: Lane 1: Control LH198 (Xbacommercial I) 2: ControlFurthermore, LH59any x LH198 (Xba I) 3: ControlConsequently, LH59 x withoutLH198 (Xba I) spiked with PV-ZMIR39 (EcoR I) [0.5 copy] 4: Control LH59 x LH198 (Xba I) spiked with PV-ZMIR39 (EcoR I) [1.0 copy] 5: MON 88017 [LH198BC0F1 x LH59]F2 (Xba I) 6: MON 88017 LH198BC0F1 (Xba I) 7: MON 88017 LH198BC0F2 (Xba I) 8: MON 88017 LH198BC1F1 (Xba I) 9: MON 88017 LH198BC1F3 x LH59(H) (Xba I) 10: MON 88017 LH59 x LH198BC3F3(H) (Xba I) 11: MON 88017 HC33 x LH59BC2F3(H) (Xba I) Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.

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3.4. Organization of the insert in MON 88017

The organization of the elements within the insert in MON 88017 was confirmed using PCR analysis by 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, as well as the results of the PCR analyses, are shown in Figures 17 and 18.

The control reactions containing no template DNA (Figure 17: lanes 4, 7, 10 and 14; Figure 18: lanes 4, 8 and 11) did not generate PCR products with any of the primer sets, as expected. The control reactions containing DNA from conventional corn (Figure 17: lanes 3, 6, 9 and 13) did not generate PCR products with four out of the seven primer sets. The primer set used to generate Products E, F and G did amplify PCR products from the control DNA (Figure 18: lane 3, 7 and 10); however, none of these products were of the expected size for the primer set. The same PCR products were also generated and using MON 88017 genomic DNA as a template (Figure 18: laneand 2, 6 andregime. 9). The generation of these PCR products is likely the resultAG of nonspecific amplification of sequences in the corn genome due to one or both of the PCR primers in the primer set. Bayer property protectionpublishing of contents its The plasmid PV-ZMIR39 was used as a positive controlparties. indata the three PCRtherefore analyses (Products C-E) that amplified products containing only insertedand/or DNAor rather than the affiliates. property intellectualthird may genomic DNA flanking the insert. In theseits three analyses, amplification from genomic as the of and DNA from MON 88017, as wellis as the plasmid PV-ZMIR39,regulatory generatedowner. the expected size a document PCR products of 1.0 kb for Productany C (Figuresuch 17: lanes 8 and 11),its 2.0 kb for Product D reproductiondocument (Figure 17: lanes 12 and 15) and 2.3 kb forowner Pr oduct Ethis (Figure 18:of lanes 2 and 5). In the under of rightsthe this amplification of Productdocument E usingand/or plasmid PV-ZMIR39 as templaterights DNA, faint, lower to of fall use of molecular weight products were visible that were most thelikely due to nonspecific This may distribution,and amplification (Figure 18: lane 5).rights Am plificationowner from MON 88017 genomic DNA also generated the expected sizesubject PCR products of 1.0 kb for Product A (Figure 17: lane 2); be the violate 0.9 kb for Product B (Figurecopy 17: lane 5); 2.3of kb for Product F (Figure 18: lane 6); and and may documentpublication, 2.2 kb for Product ItG (Figure 18: laneexploitation 9). The generation of the predicted size PCR products from MON 88017this establishes any that the arrangement or linkage of elements in the permission insert is the same as those in plasmid PV-ZMIprohibited R39 and that the elements within each gene the cassette are arranged as depictedcommercial in bethe schematic of the insert in Figure 5.

Furthermore,any Consequently,without

Monsanto Company 04-CR-109F Page 62 of 223

Figure 17. Overlapping PCR analyses across the insert in MON 88017: Part I

Product A Product B Product C Product D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

2.5 kb 2.5 kb 2.0 kb 2.0 kb 1.5 kb 1.5 kb 1.0 kb 1.0 kb 0.5 kb 0.5 kb

and and regime. AG A: 1.0 kb C: 1.0 kb B: 0.9 kb D: 2.0 kb Bayer property protectionpublishing of contents its 5’ Flank parties.data therefore 3’ Flank LB and/oror RB P-ract CTP2 P-e35S

3’ NOS propertyaffiliates. third may

cp4 epsps cp4 intellectual its 3’ tahsp17 ract1 intron ract1 as intron ract1 the of and leader wt CAB is regulatory owner. a documentits any such cry3Bb1.pvzmir39 reproductiondocument PCR analyses demonstrating the linkage of theowner indi vidual geneticthis elementsof within the insert in under of MON 88017 were performed on DNArights extractedthe from grain ofthis MON 88017 and conventional documentand/or of rights corn. The expected product size forto eachof ampliconfall isuse highlighted in the illustration of the insert the in MON 88017 that appearsThis at the bottommay of thedistribution, figure.and 20 µl of each of the PCR reactions were rights owner loaded on the gel. subject be the violate Lane designation is as follows:copy of 1: 500 bp DNA ladder (Life Technologies) and may documentpublication, 2: MON 88017It genomic DNA exploitation this 3: control LH59 x LH198any DNA permission 4: control no DNA template prohibited the 5: MON 88017 genomiccommercial DNA be 6: control LH59 x LH198 DNA Furthermore,any 7: control noConsequently, DNA templatewithout 8: MON 88017 genomic DNA 9: control LH59 x LH198 genomic DNA 10: control no DNA template 11: plasmid PV-ZMIR39 DNA 12: MON 88017 genomic DNA 13: control LH59 x LH198 genomic DNA 14: control no DNA template 15: plasmid PV-ZMIR39 DNA 16: 500 bp DNA ladder (Life Technologies) Symbol denotes sizes obtained from MW markers on ethidium bromide stained gel.

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Figure 18. Overlapping PCR analyses across the insert in MON 88017: Part II

Product E Product F Product G 1 2 3 4 5 6 7 8 9 10 11 12

3.0 kb 3.0 kb 2.5 kb 2.5 kb 2.0 kb 2.0 kb

0.5 kb 0.5 kb

and and regime. AG

F: 2.3 kb E: 2.3 kb Bayer property protectionG: 2.2publishing kb of contents its parties.data therefore 5’ Flank and/oror 3’ Flank LB affiliates. may RB P-ract CTP2 property intellectualthird P-e35S its 3’ NOS

cp4 epsps as the of 3’ tahsp17 ract1 intron ract1 and regulatory intron ract1 owner. is document wt CAB leader CAB wt a any such its reproductiondocument owner this cry3Bb1.pvzmir39 of under of rightsthe this documentand/or of rights to of fall use the PCR analyses demonstratingThis the linkagemay of the distribution, indiandvidual genetic elements within the insert in rights owner MON 88017 were performedsubject on plasmid PV-ZMIR39 DNA, conventional corn and MON 88017 be the violate genomic DNA extracted fromcopy grain. The expectedof product size for each amplicon is highlighted in the illustration of the insert in MON 88017 that andappears at the bottom of the figure. 20 µl of may documentpublication, each of the PCR productsIt were loaded onexploitation the gel. this Lanes designation is as follows:any permission 1: 500 bp DNA molecular weight ladderprohibited (Life Technologies) the 2: MON 88017 genomiccommercial DNA be 3: control LH59 x LH198 genomic DNA Furthermore,any 4: control noConsequently, DNA templatewithout 5: plasmid PV-ZMIR39 DNA 6: MON 88017 genomic DNA 7: control LH59 x LH198 genomic DNA 8: control no DNA template 9: MON 88017 genomic DNA 10: control LH59 x LH198 genomic DNA 11: control no DNA template 12: 500 bp DNA molecular weight ladder (Life Technologies) Symbol denotes sizes obtained from MW markers on ethidium bromide stained gel.

Monsanto Company 04-CR-109F Page 64 of 223 3.5. Inheritance of corn rootworm protection and glyphosate tolerance traits in MON 88017

Chi square analysis of Mendelian inheritance data over ten generations was performed to determine the heritability and stability of the cry3Bb1 and cp4 epsps genes in MON 88017. Expected and observed segregation frequencies of MON 88017 progeny that are positive for the corn rootworm (CRW)-protected and glyphosate-tolerant phenotypes are presented in Table 2. Plants were identified as positive for the CRW- protected phenotype based on the detection of the Cry3Bb1 protein by ELISA. Because the cp4 epsps and cry3Bb1 genes are linked in the vector used for transformation, and are expected to have identical segregation ratios in the progeny of MON 88017, the results were, also applicable to inheritance of the glyphosate tolerant trait in MON 88017.

The breeding history of the ten generations evaluated for Mendelian inheritanceand (Figure and regime. 15) was as follows: AG

• The LH198BC0F generation was derived Bayerfrom cross-pollinatingproperty protectionpublishing the original 1 of contents transformed plant with an inbred line, LH198 its parties.data therefore and/or • The LH198BC0F2 generation was derived from self-pollinatingor individual affiliates. property intellectualthird may LH198BC0F1 plants its as • The LH198BC1F1 generation wasthe ofderived andfrom backcrossing LH198BC0F1 plants to is regulatory owner. a document conventional inbred line LH198any such its reproductiondocument of • The LH198BC2F1 generation was derivedowner from backcrossingthis LH198BC1F1 plants to under of this conventional inbred line LH198and/orrights the document to of fall of rights • use The LH198BC3F1 generation was derived from backcrossingthe LH198BC2F1 plants to This may distribution,and conventional inbred line LH198rights owner subject • The LH198BC3F2 generation was derivedthe from violate self-pollinating individual be copy LH198BC3F1 plants of and • may documentpublication, The LH198BC0FIt 1xLH59 generationexploitation was derived from crossing LH198BC0F1 plants to conventional inbredthis line any LH59 • The LH59BC1F generation waspermission derived from backcrossing LH198BC0F xLH59 1 prohibited 1 plants to conventional inbredthe line LH59 commercial be • The LH59BC2F1 generation was derived from backcrossing LH59BC1F1 plants to Furthermore,any conventional Consequently,inbred linewithout LH59 • The LH59BC2F2 generation was derived from self-pollinating individual LH59BC2F1 plants

Genotype frequency was compared by means of a Chi square test (Little and Hills, 1978). The Chi square value (χ2) was computed as follows:

χ2 = Σ [ ( |o – e |- 0.5 )2 / e ]

where, o = observed frequency of the genotype; e = expected frequency of the genotype; and 0.5 = Yates correction factor for analysis with one degree of freedom (df).

Monsanto Company 04-CR-109F Page 65 of 223 Results from the Mendelian heritance analysis are summarized in Table 2. A chi square value of at least 3.84 is needed to indicate statistical difference (p≤0.05). With only two exceptions, all χ2 values were less than the critical value of 3.84, thus indicating no significant differences between observed and expected frequency for the CRW-protected /glyphosate-tolerant phenotypes in eight breeding generations of MON 88017. Although the results obtained for the LH198BC1F1 generation were significant at p≤0.05 (Chi square = 3.84, 1 df), they were not significant at p≤0.01 (Chi square = 6.63). The results obtained for the LH198BC0F1 x LH59 generation are attributed to gamete selection caused by Roundup agricultural herbicide application to plants of the previous (LH198BC0F1) generation. Roundup agricultural herbicide application was necessary to obtain non-segregating seed for purposes of field evaluation. Gamete selection caused by glyphosate application has been previously observed for Roundup Ready corn NK603 (USDA petition 00-011-01p).

and Table 2. Comparison of expected and observed segregationand frequencies regime. for progeny of MON 88017 plants AG

a Bayer property protectionapublishing Observed of Expected contents its parties.data therefore and/oror 2 Generation + - affiliates. + - χ property intellectualthird may its as the of and † LH198BC0F1 21 is 14 regulatory 17.5 17.5owner. 1.03 a documentits † LH198BC0F2 53 12any such reproduction 48.75 16.25 1.15 this documentof # LH198BC1F1 21 9 owner 15 15 4.03 under of LH198BC F 10 rights15 the 12.5this 12.5 0.64† 2 1 documentand/or rights to of fall use of † LH198BC3F1 8 5 6.5 6.5 0.31 the † LH198BC3F2 This21 3 may distribution,and 18 6 1.39 rights owner * LH198BC0F1xLH59 29 subject 0 14.5 14.5 27.03 the violate † LH59 BC1F1 be7 copy 5 6 6 0.08 of † LH59 BC2F1 8 5 and 6.5 6.5 0.31 may documentpublication, LH59 BC F It 35 13exploitation 36 12 0.03† 2 2 this any permission a Symbol (+) denotes number of plantsprohibited that are positive for the two traits; (-) denotes number the of plants that are negativecommercial for the be two traits. † ≤ Not significantFurthermore, at p any0.05 (Chi square = 3.84, 1 df). # Significant atConsequently, p≤0.05 without(Chi square = 3.84, 1 df); but not significant at p≤0.01 (Chi square = 6.63). * Significant at p≤0.05 (Chi square = 3.84, 1 df) and p≤0.01 (Chi square = 6.63).

The results of this analysis are consistent with the finding of a single locus of insertion of the cry3Bb1 and cp4 epsps genes that segregate according to Mendel’s laws of genetics. The stability of the insert has been demonstrated through seven generations of cross- fertilization and three generations of self-pollination.

Results of the Southern blot analysis performed on multiple generations from the breeding tree of MON 88017 to determine insert stability were discussed in Section 3.3 and presented in Figure 16.

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3.6. Summary of the molecular characterization of MON 88017

MON 88017 was produced by Agrobacterium sp.-mediated transformation of a corn with plasmid vector PV-ZMIR39, which contains both the cp4 epsps and cry3Bb1 gene expression cassettes. MON 88017 contains one copy of the introduced DNA at a single integration locus on an approximately 13 kb Sca I restriction fragment. No additional elements from transformation vector PV-ZMIR39, linked or unlinked to the intact cassettes, were detected in the genome of MON 88017. PCR analyses were performed to determine the 5’ and 3’ insert-to-plant junctions and confirm the organization of the elements within the insert. Insert stability analysis demonstrated that the expected Southern blot fingerprint of MON 88017 insert was maintained across the branches of the breeding tree that were tested, thereby confirming the stability of the integrated DNA over multiple generations. No backbone sequences from the transformation vector PV-ZMIR39 were detected in the tested generations. These data support the conclusion and that only the two expected full-length proteins, Cry3Bb1 and andCP4 EPSPS,regime. are encoded by the insert in MON 88017. AG

Bayer property protectionpublishing of contents its parties.data therefore SECTION 4. Other data and information about the developmentand/oror of MON 88017 affiliates. property intellectualthird may its as All relevant information regardingthe development of and of MON 88017 is described in Parts II – is regulatory owner. VII of this summary. 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 04-CR-109F Page 67 of 223

PART V: PRESENCE OF GENES THAT ENCODE RESISTANCE TO ANTIBIOTICS

No genes that encode resistance to an antibiotic were inserted into the genome of MON 88017. Molecular characterization data presented in Part IV Section 3 confirms the absence of the add gene encoding an antibiotic resistance marker.

and and regime. AG

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PART VI: CHARACTERIZATION OF THE PROTEINS INTRODUCED INTO MON 88017

SECTION 1. Identity and function of the CP4 EPSPS and Cry3Bb1 proteins present in MON 88017

MON 88017 contains a 5-enolpyruvylshikimate-3-phosphate synthase protein from Agrobacterium spp. strain CP4 (CP4 EPSPS). The cp4 epsps coding region encodes a 47.6 kDa protein consisting of a single polypeptide of 455 amino acids (Padgette et al., 1996). The CP4 EPSPS protein is structurally and functionally similar to plant EPSPS enzymes, but has a much reduced affinity for glyphosate (Padgette et al., 1996). and Typically, glyphosate binds to the plant EPSPS enzyme and blocksand the biosynthesisregime. of aromatic amino acids, thereby depriving plants of theseAG essential components (Steinrucken and Amrhein, 1980; Haslam, 1993). In Roundup Ready plants, however, requirements for Bayer property protectionpublishing growth and development are met by the continuedof action of the CP4 EPSPScontents enzyme in the presence of glyphosate. A safety assessment of the CP4 EPSPS proteinits has been parties.data therefore described previously in the literature (Harrison et al., 1996). and/oror affiliates. property intellectualthird may its as The Cry3Bb1 protein expressed inthe MON of 88017and is a member of the Cry3Bb class of is regulatory owner. a document proteins that share >95% amino sequenceany such hom ology (Crickmore itset al., 1998). It is a reproductiondocument variant of the wild-type Cry3Bb1 protein ownerisolated fromthis Bacillus of thuringiensis (subsp. under of rightsthe this kumamotoensis) strain EG4691documentand/or (Donovan et al., 1992) andrights a variant of the Cry3Bb1 to of fall use of protein present in commercial YieldGard Rootworm cornthe (MON 863). The amino acid This may distribution,and sequence identity between the Cry3Bb1rights proteinsowner presen t in MON 88017, YieldGard Rootworm corn, and in Bacillussubject thuringiensis (subsp. kumamotoensis) ranges between be the violate 99.1-99.8%. The amino acidcopy sequence of theof Cry3Bb1 protein present in MON 88017 and may documentpublication, and Bacillus thuringiensisIt (subsp. kumamotoensisexploitation ) shares 99.1% identity (differs by six of 652 amino acid residues).this any The Cry3Bb1 protein present in MON 88017 and permission YieldGard Rootworm corn share 99.8%prohibited amino acid identity (differs by only one of 653 amino acid residues). YieldGardthe Rootworm corn has been granted full regulatory commercial be clearance in the U.S. FDA consultation was completed in December 2001. In 2001 EPA Furthermore,any established an exemptionConsequently, withoutfrom the requirement of a tolerance for the Cry3Bb1 protein and the genetic material necessary for its production in field corn, sweet corn and popcorn (40CFR §180.1214). Non-regulated status was granted by USDA in October 2002, and EPA registration was granted in February 2003. Thus, the Cry3Bb1 protein, as expressed in MON 88017, has been well characterized and its safety for human and animal consumption has been established.

1.1. Characterization of the CP4 EPSPS protein produced in MON 88017

The CP4 EPSPS protein was purified from MON 88017 grain and from E. coli culture that produced the CP4 EPSPS protein. A panel of analytical tests was used to identify, characterize and compare the plant- and E. coli-produced CP4 EPSPS proteins: (1)

Monsanto Company 04-CR-109F Page 69 of 223

western blot analysis and densitometry, (2) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry, (3) matrix- assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS), (4) N-terminal sequence analysis, (5) glycosylation analysis, and (6) CP4 EPSPS activity assay.

These data provide a detailed characterization of the CP4 EPSPS protein isolated from grain of MON 88017 and establish the equivalence of the plant-produced CP4 EPSPS protein to the E. coli-produced CP4 EPSPS protein. The materials and methods for the analytical tests are provided in Appendix II. The results are summarized in the following sections.

1.1.a. Western blot analysis to confirm the identity of the CP4 EPSPS protein

A protein band migrating at an approximate molecular weight of 45 kDa on SDS-PAGE gel was identified as the CP4 EPSPS protein by western blot analysis using anda goat anti- and regime. CP4 EPSPS antibody (Figure 19, Lanes 9-14). ThisAG serum previously was shown to be specific for the CP4 EPSPS protein and therefore was used to confirm the identity of the plant-produced CP4 EPSPS protein. One immunoreactiveBayer property band protection migratingpublishing at of contents approximately 45 kDa was observed in both the E. coli- and plant-producedits CP4 EPSPS parties.data therefore samples, confirming the identity of the CP4 EPSPS protein producedand/oror in MON 88017. affiliates. property intellectualthird may its as 1.1.b. Molecular weight and puritythe ofof the CP4and EPSPS protein is regulatory owner. a document any such its reproductiondocument Molecular weight and purity of the plant-ownerproduced CP4this EPSPS of protein were estimated under of rightsthe this using densitometric analysisdocument ofand/or a Colloidal Brilliant Blue Grights stained SDS-polyacrylamide to of fall use of gel (Figure 20). Out of three bands observed on the SDS-polyacrylamidethe gel, the 45kDa This may distribution,and band was identified as CP4 EPSPSrights protein by westernowner blot analysis. The predicted molecular weight of the CP4subject EPSPS protein, based on the deduced amino acid sequence, be the violate is 47.7 kDa. The apparent molecularcopy weightof of the plant-produced CP4 EPSPS protein, and may documentpublication, estimated by comparisonIt to molecularexploitation weight markers on the SDS-polyacrylamide gel, was 45 kDa (Figure 19) andthis theany plant- and E. coli-produced proteins co-migrated on a permission Tris-glycine buffered 4-20% polyacrylamideprohibited ge l (Figure 20). The purity value for the plant-produced CP4 EPSPS proteinthe calculated as the average of three separate loads was commercial be 45.5%. Furthermore,any Consequently,without The molecular weight of the plant-produced CP4 EPSPS protein was further confirmed using MALDI-TOF mass spectrometry. The average mass (MH+), determined from three separate spectral acquisitions, was 47,447.25 daltons. This value compares well with the predicted mass for CP4 EPSPS, 47,730 daltons.

Monsanto Company 04-CR-109F Page 70 of 223

Figure 19. Western blot analysis demonstrating equivalence of CP4 EPSPS proteins produced in MON 88017 and E. coli

112 3 4 5 6 7 8 9 011 1213 14 15

kDa 250 150 100 75

and 50 and regime. AG 37 Bayer property protectionpublishing 25 of contents its parties.data therefore 20 and/oror affiliates. 15 property intellectualthird may its as the of and 10 is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of Samples of the plant- and E. coli-produced CP4 EPSPS proteinsthe (1, 2, and 3 ng) were separated This may distribution,and using SDS-PAGE and transferred rightsto a PVDF membrane.owner The membrane was incubated with goat subject anti-CP4 EPSPS polyclonalbe antiserum to detect thethe presence violate of CP4 EPSPS protein. The dots (Lanes 1 and 15) represent thecopy location of the molecularof weight markers (MWM) on the and may documentpublication, membrane. A 10 secIt exposure is shown.exploitation this any Lane Sample permission Amount (ng) 1 Bio-Rad Precision Plus Dualprohibited Color MWM --- the be 2 E. coli-producedcommercial CP4 EPSPS 1 3 Furthermore,E. coli-producedany CP4 EPSPS 1 4 E.Consequently, coli-producedwithout CP4 EPSPS 2 5 E. coli-produced CP4 EPSPS 2 6 E. coli-produced CP4 EPSPS 3 7 E. coli-produced CP4 EPSPS 3 8 loading buffer --- 9 plant-produced CP4 EPSPS 1 10 plant-produced CP4 EPSPS 1 11 plant-produced CP4 EPSPS 2 12 plant-produced CP4 EPSPS 2 13 plant-produced CP4 EPSPS 3 14 plant-produced CP4 EPSPS 3 15 Bio-Rad Precision Plus Dual Color MWM

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Figure 20. Molecular weight and purity of the plant-produced CP4 EPSPS protein demonstrated by SDS-PAGE analysis

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

kDa 200.0 116.3 97.4 66.2

and and regime. 45.0 AG

Bayer property protectionpublishing 31.0 of contents its parties.data therefore and/oror 21.5 affiliates. property intellectualthird may its 14.4 as the of and is regulatory owner. 6.5 a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of 1, 2 and 3 µg total protein of plant-produced CP4 EPSPS proteinthe sample were separated by This may distribution,and rights owner electrophoresis on a 4-20% gradientsubject polyacrylamide gel under reducing and denaturing conditions. The stained gelbe was analyzed using densitometrythe violate to determine the purity and copy of molecular weight of the CP4 EPSPS protein. and may documentpublication, It exploitation Lane Samplethis any Amount (µg) 1 empty permission --- prohibited 2 empty the be --- 3 loadingcommercial buffer --- 4 Furthermore, loadingany buffer --- 5 Consequently, Bio-Radwithout Broad Range MWM --- 6 plant-produced CP4 EPSPS 1 7 plant-produced CP4 EPSPS 2 8 plant-produced CP4 EPSPS 3 9 loading buffer --- 10 loading buffer --- 11 empty --- 12 empty ---

Monsanto Company 04-CR-109F Page 72 of 223

1.1.c. MALDI-TOF MS tryptic map mass analysis

MALDI-TOF mass spectrometry analysis was performed on the CP4 EPSPS protein produced in MON 88017. The protein band identified as CP4 EPSPS was excised from the gel, chemically reduced, alkylated, digested with trypsin, and analyzed using MALDI-TOF MS. The ability to identify a protein using this method is dependent upon matching a sufficient number of observed tryptic peptide fragment masses with theoretical tryptic peptide fragment masses. With sufficient mass accuracy, four tryptic peptides may be sufficient to identify a protein (Jiménez et al., 1998). Peptides were considered to match when a difference in molecular weight of less than one dalton was found between the observed and theoretical fragment masses. Matches were made without consideration for potential natural amino acid modifications. For the 45kDa protein, a total of 22 observed peptide mass fragments matched the theoretical tryptic peptide mass map for the CP4 EPSPS protein. and and regime. AG The amino acid sequence of the plant-produced CP4 EPSPS protein was deduced from the coding region of the cp4 epsps gene presentBayer in MONproperty 8801 7.protection Apublishing coverage map was of contents generated using the identified masses from the MALDI-TOF MS trypticits mass analysis. parties.data therefore Approximately 55.2% (251 of 455 amino acids) of the expectedand/or proteinor sequence was affiliates. property intellectualthird may identified (Figure 21). The predicted molits ecular weight of the full-length CP4 EPSPS as protein, based on the deduced aminothe acidof sequence,and is 47.7 kDa. Using the above is regulatory owner. a document criteria, the 45kDa plant-produced anyprotein such band was identified asits CP4 EPSPS protein. reproductiondocument owner this of under of rightsthe this Figure 21. MALDI-TOFdocument MSand/or coverage map of the CP4rights EPSPS protein sequence to of fall use of produced in MON 88017 the This may distribution,and rights owner subject be the violate 1 MLHGASSRPA TARKSSGLSGcopy TVRIPGDKSIof SHRSFMFGGL ASGETRITGL and 51 LEGEDVINTGmay KAMQAMGARI documentpublication, RKEGDTWIID GVGNGGLLAP EAPLDFGNAA It exploitation 101 TGCRLTMGLV GVYDFDSTFIthis GDASLTKRPM GRVLNPLREM GVQVKSEDGD 151 RLPVTLRGPK TPTPITYRVPany MASAQVKSAV LLAGLNTPGI TTVIEPIMTR permission 201 DHTEKMLQGF GANLTVETDA DGVRTIRLEGprohibited RGKLTGQVID VPGDPSSTAF 251 PLVAALLVPG SDVTILNVLMthe NPTRTGLILTbe LQEMGADIEV INPRLAGGED 301 VADLRVRSST LKGVTVPEDRcommercial APSMIDEYPI LAVAAAFAEG ATVMNGLEEL 351 RVKESDRLSAFurthermore, VANGLKLNGVany DCDEGETSLV VRGRPDGKGL GNASGAAVAT 401 HLDHRIAMSFConsequently, LVMGLVSENPwithout VTVDDATMIA TSFPEFMDLM AGLGAKIELS 451 DTKAA

1.1.d. N-terminal sequence analysis

An SDS-PAGE analysis of the plant-produced CP4 EPSPS protein revealed three protein bands (migrating at approximately 45, 48, and 55 kDa, Figure 22), each representing greater than 10% of the total protein in the plant-produced CP4 EPSPS sample. The identity of these bands was evaluated using N-terminal sequence analysis (Table 3). The observed amino acid sequence for the 45 kDa protein band matched the expected N-terminal amino acid sequence of the CP4 EPSPS protein. Two sequences, both

Monsanto Company 04-CR-109F Page 73 of 223

consistent with the N-terminus of the CP4 EPSPS protein were observed in the plant- produced CP4 EPSPS protein sample. The primary sequence originates at residue 2 (leucine) and matches the expected N-terminal amino acid sequence of the CP4 EPSPS protein from residues 2 through 16 (Table 3). It is not uncommon for the initiator methionine to be removed from proteins in eukaryotic organisms by an endogenous methionine aminopeptidase (Arfin and Bradshaw, 1988). The secondary sequence starts at residue 4 (glycine) and matches the expected N-terminal amino acid sequence of the CP4 EPSPS protein from residues 4 through 18 (Table 3). The loss of several N-terminal amino acid residues may be due to protease action when plant cells are homogenized. Despite the ragged N-terminus, the sequence data confirm the identity of the 45 kDa protein in the plant-produced sample as the CP4 EPSPS protein.

The N-terminal sequence of the 48 kDa protein band was obtained and the sequence did not match that of the CP4 EPSPS protein. The sequence obtained was used to search the non-redundant public database and the PIR Protein database but no matchesand were and regime. identified. Only five amino acid residues were obtainedAG from the N-terminal sequence analysis of the 55 kDa protein and therefore no positive identification could be made for this protein. Bayer property protectionpublishing of contents its parties.data therefore Table 3. N-terminal sequence of the CP4 EPSPS protein producedand/oror in MON 88017 affiliates. property intellectualthird may its as the of and MW Observed Sequenceis regulatory Identityowner. a document any such its reproductiondocument Major Sequence owner this Residuesof 2-16 of under of LHGASSRPATARKS(S)rightsthe this CP4 EPSPS documentand/or rights 45 kDa to of fall use of Secondary Sequence the Residues 4-18 of This may distribution,and GASSRPATARKS(S)G(L)rights owner CP4 EPSPS subject 48 kDa GGFKVTRISEGPVKXbe the violate Not Identified copy of and may documentpublication, 55 kDa It (G)LIDGXXXXXXXXXXexploitation Not Identified this any permission Sequences obtained for each proteinprohibited band were compared with the known sequence for the CP4 EPSPS and/or againstcommercial protein be sequence databases. The ( ) denotes tenuous amino acid designationsFurthermore,any and Xs refer to undesignated amino acids. Amino acid residues 1-18 (using the Consequently,IUPAC-IUBwithout amino acid codes) of the CP4 EPSPS protein are MLHGASSRPATARKSSGL.

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1.1.e. Functional activity

The specific activity of the plant- and E. coli-produced CP4 EPSPS proteins was estimated concurrently using a phosphate release assay. The specific activity of the plant-produced protein was 7.1 U/mg of CP4 EPSPS, and the specific activity of the E. coli-produced CP4 EPSPS protein was 6.0 U/mg. This confirmed that the functional activity of the CP4 EPSPS produced in MON 88017 is similar to that of the E. coli- produced CP4 EPSPS.

1.1.f. Glycosylation analysis

Many eukaryotic proteins are post-translationally modified with carbohydrate moieties (Rademacher et al., 1988). These carbohydrate moieties may be complex branched and polysaccharide structures or simple monosaccharides. In contrast,and prokaryoticregime. organisms such as E. coli lack the necessary organelles and biochemicalAG pathways required for protein glycosylation. To test if post-translational glycosylation of the plant-produced Bayer property protectionpublishing CP4 EPSPS protein occurred, this protein wasof analyzed for covalentlycontents bound carbohydrate moeities. The E. coli-produced CP4 EPSPS protein wasits analyzed parties.data therefore concurrently as a negative control in this experiment. Transferrinand/oror protein was analyzed affiliates. property intellectualthird may as a positive control. The positive controitsl (transferrin) was clearly detected in a as the of and concentration-dependent manneris at loadings of 0.5regulatory and 1 µ g/lane (Figureowner. 22, Lanes 2-3). a document No bands were observed in the E. coliany - suchor plant-produced proteinits samples (Lanes 4-7). reproductiondocument Several dots present on the blot were consowneridered to be thisnon-specific of background because under of rightsthe this of their form and location.document Becauseand/or no band was observedrights at the expected molecular to of fall use of weight for the plant- or E. coli-produced CP4 EPSPS proteins,the it was concluded that the This may distribution,and proteins were not glycosylated. 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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Figure 22. Glycosylation analysis of CP4 EPSPS protein produced in MON 88017

1 2 3 4 5 6 7 8 9 10

kDa 214

118 92

and and regime. 36 AG 29 Bayer property protectionpublishing of contents its parties.data therefore 21 and/oror affiliates. property intellectualthird may its 7 as 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 Proteins were separatedThis by SDS-PAGE undermay distribution, reducingand and denaturing conditions and transferred rights owner to a PVDF membrane. The dotssubject (Lanes 1 and 8) represent the location of the molecular weight be the violate markers (MWM) on the membrane.copy A three minuteof exposure is shown. and may documentpublication, It exploitation Lane Samplethis Amount (µg) 1 Bio-Radany Prestained Broad Range MWM --- permission 2 transferrin prohibited 0.5 the 3 transferrincommercial be 1.0 4 E. coli-produced CP4 EPSPS 0.5 Furthermore,any 5 Consequently,E. coliwithout -produced CP4 EPSPS 1.0 6 plant-produced CP4 EPSPS 0.5 7 plant-produced CP4 EPSPS 1.0 8 Bio-Rad Prestained Broad Range MWM --- 9 empty --- 10 empty

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1.1.g. Conclusions for characterization of the CP4 EPSPS protein produced in MON 88017

A panel of analytical tests was used to characterize and compare the plant- and E. coli- produced CP4 EPSPS proteins. The equivalence of the plant-produced CP4 EPSPS protein to the E. coli-produced CP4 EPSPS protein was evaluated by comparing their molecular weights, immunoreactivity with anti-CP4 EPSPS antibodies, glycosylation status and functional activity.

The plant-produced CP4 EPSPS protein was identified as a 45 kDa band using western blot analysis. The N-terminal sequence of this 45 kDa protein band was consistent with the N-terminal sequence of the CP4 EPSPS protein. MALDI-TOF mass spectrometry analysis of the plant-produced CP4 EPSPS protein after trypsin digestion yielded peptide masses encompassing 55.2% of the deduced amino acid sequence (251/455and amino acids) and regime. of the plant-produced CP4 EPSPS. The purity of theAG plant-produced CP4 EPSPS protein was 45.5%. The approximate molecular weight of the protein band identified as the plant-produced CP4 EPSPS protein, estimatedBayer by comparison property protectionto molecularpublishing weight of contents markers on a Colloidal Brilliant Blue G stained SDS-polyacrylamideits gel, was 45 kDa. parties.data therefore The molecular weight of the plant-produced CP4 EPSPS, as determinedand/oror using the mass affiliates. property intellectualthird may average (MH+) molecular weight using itsMA LDI-TOF MS, was 47,447.25 Da. The plant- as produced CP4 EPSPS protein wasthe not of glycosylated and . The functional activity of the plant- is regulatory owner. a document and E. coli-produced CP4 EPSPS proteinsany such was determined using itsa phosphate release reproductiondocument assay and shown to be similar. The specificowner activity ofthis the plant-of and E. coli-produced under of rightsthe this CP4 EPSPS proteins wasdocument 7.1 and/orU/mg and 6.0 U/mg, respectively.rights to of fall use of the This may distribution,and These results lead to the conclusionrights that the plant-producedowner CP4 EPSPS protein is equivalent to the E. coli-producedsubject CP4 EPSPS protein. be the violate copy of and may documentpublication, It exploitation 1.2. Characterization ofthis Cry3Bb1 any protein produced in MON 88017 permission prohibited The Cry3Bb1 protein was purifiedthe from the grain of MON 88017 and from E. coli commercial be cultures that express the Cry3Bb1 protein. Proteins of three molecular weights were Furthermore,any identified as the full-lengthConsequently,without Cry3Bb1 protein and protease-cleaved products of Cry3Bb1 (Figure 23). The Cry3Bb1 protein (predicted molecular weight of 74.4 kDa) is processed in corn to produce peptides with apparent molecular weights of ~66 and ~55 kDa. The ~55 kDa peptide is thought to correspond to the B.t. tryptic core protein described in the literature (Schnepf et al., 1998). Four immunoreactive bands migrating at approximately 77, 66, 55, and 46 kDa were observed in the sample purified from MON 88017 grain. The immunoreactive band of 46 kDa may represent a degradation product of Cry3Bb1 protein but was not considered for further analysis because it was smaller than the expected size of the tryptic core protein.

A panel of analytical tests was used to characterize the plant-produced Cry3Bb1 protein: (1) western blot analysis and densitometry, (2) SDS-PAGE and densitometry, (3)

Monsanto Company 04-CR-109F Page 77 of 223

MALDI-TOF mass spectrometry, (4) glycosylation analysis, and (5) a Cry3Bb1 bioactivity assay. The equivalence of the plant-produced Cry3Bb1 protein to the E. coli- produced Cry3Bb1 protein was evaluated by comparing their full-length molecular weights, immunoreactivity with anti-Cry3Bb1 antibodies, glycosylation status and functional activity.

The materials and methods used in these experiments are provided in Appendix II. Results obtained from the analyses conducted to characterize the Cry3Bb1 protein produced in MON 88017 are discussed in the following sections.

Figure 23. Polypeptides derived from the full-length Cry3Bb1 protein

158159 652 and and regime. Cry3Bb1 ProteinAG 74.4 kDa Bayer property protectionpublishing 67.7of kDa contents its parties.data therefore 56.8 kDa and/oror affiliates. property intellectualthird may its 10.9 kDa as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of The amino acid positions are shown onrights topthe and correspond tothis residue numbers obtained from N- documentand/or rights terminal sequence analysis of a homologousto of fallCry3Bb1 use protein. of The predicted molecular weights the for each polypeptide areThis shown. may distribution,and rights owner subject be the violate copy of and may documentpublication, 1.2.a. Western blotIt analysis to confexploitationirm the identity of the Cry3Bb1 protein this any permission Western blot analysis was performed usingprohibited a goat anti-Cry3Bb1 serum. That serum was the shown to be specific for Cry3Bb1commercial protein be and was used to confirm the identity of the plant-producedFurthermore, Cry3Bb1any protein. Four immunoreactive bands migrating at approximately 77,Consequently, 66, 55,without and 46 kDa were observed (Figure 24, lanes 9-14) in the plant- produced Cry3Bb1 sample. Full-length Cry3Bb1 protein with predicted molecular weight of 74.4 kDa is processed in corn to produce peptides with apparent molecular weight ~66 kDa and ~ 55 kDa (Figure 23). The immunoreactive band at 46 kDa may represent a degradation product of the Cry3Bb1 protein. This band was not analyzed because it was smaller than the expected size of the tryptic core determined for homologous Cry3Bb1 proteins.

The plant- and E. coli-produced Cry3Bb1 proteins were loaded in duplicate at three purity-corrected protein amounts: 1, 2, and 3 ng (Figure 24). The percent difference in the immunoreactivity between the E. coli- and plant-produced Cry3Bb1 protein was calculated based on densitometric analysis of the bands for two different exposures (15

Monsanto Company 04-CR-109F Page 78 of 223

and 30 sec) of the western blot. The Cry3Bb1 antibody recognizes the plant-produced full-length protein and several smaller protein fragments that represent proteolytically cleaved forms of this protein. Bands greater than 55 kDa were included in the densitometric analysis of the plant-produced sample. The average difference in immunoreactivity between the plant- and E. coli-produced Cry3Bb1 proteins was 9%.

1.2.b. Molecular weight and purity of the Cry3Bb1 protein

The molecular weight and purity of the plant-produced Cry3Bb1 protein were determined using densitometric analysis of a Colloidal Brilliant Blue G stained SDS-polyacrylamide gel (Figure 25). The predicted molecular weight of the full-length Cry3Bb1 protein, based on the deduced amino acid sequence, is 74.6 kDa. The full-length Cry3Bb1 protein in the plant-produced sample had an estimated molecular weight of 77.2 kDa. The apparent molecular weight of the full-length E. coli-produced Cry3Bb1 protein, on the same gel, was estimated to be 77.7 kDa (Lane 7). The molecular weightsand of the and regime. additional protein fragments, previously identified asAG Cry3Bb1 protein (Section 1.2.a), were estimated to be 66.2 and 55.4 kDa. The purity of the plant-produced Cry3Bb1 protein was calculated as the sum of the three Bayerprotein bandpropertys (77.2, protection 66.2,publishing and 55.4 kDa) of contents and the average from three separate loads (1, 2, and 3 µg) of total proteinits (Lanes 4-6). parties.data therefore The average purity of the plant-produced Cry3Bb1 protein wasand/or 66.1%.or 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 04-CR-109F Page 79 of 223

Figure 24. Western blot analysis demonstrating equivalence of plant- and E.coli- produced Cry3Bb1 protein

112 3 4 5 6 7 8 9 011 1213 14 15

kDa

250 150 100 75 Cry3Bb1

37 and and regime. AG 25

20 Bayer property protectionpublishing 15 of contents its parties.data therefore 10 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 Samples of the E. coli- and plant- producedrightsthe Cry3Bb1 proteinsthis (1, 2, and 3 ng) were separated documentand/or of rights using SDS-PAGE and transferred toto a PVDFof fall memb rane.use The membrane was incubated with goat the anti-Cry3Bb1 antibodiesThis to detect the presencemay distribution, ofand Cry3Bb1 protein. The dots (Lanes 1 and 15) rights owner represent the location of the subjectmolecular weight markers (MWM) on the membrane. A 15 sec be the violate exposure is shown. copy of and may documentpublication, Lane SampleIt exploitation Amount (ng) this 1 Bio-Rad Precisionany Plus Dual Color MWM --- permission 2 E. coli-produced Cry3Bb1prohibited protein 1 the 3 E. coli-producedcommercial Cry3Bb1 be protein 1 4 E. coli-produced Cry3Bb1 protein 2 Furthermore,any 5 E.Consequently, coli-producedwithout Cry3Bb1 protein 2 6 E. coli-produced Cry3Bb1 protein 3 7 E. coli-produced Cry3Bb1 protein 3 8 2× loading buffer --- 9 plant-produced Cry3Bb1 protein 1 10 plant-produced Cry3Bb1 protein 1 11 plant-produced Cry3Bb1 protein 2 12 plant-produced Cry3Bb1 protein 2 13 plant-produced Cry3Bb1 protein 3 14 plant-produced Cry3Bb1 protein 3 15 Bio-Rad Precision Plus Dual Color MWM ---

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Figure 25. Molecular weight and purity of Cry3Bb1 protein demonstrated by SDS- PAGE analysis

1 2 3 4 5 6 7 8 9 10

kDa 200.0 116.25 97.4 66.2 Cry3Bb1

and 45.0 and regime. AG

31.0 Bayer property protectionpublishing of contents its parties.data therefore 21.5 and/oror affiliates. property intellectualthird may 14.4 its as the of and 6.5 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 1, 2, and 3 µg of total Thisprotein from the plant-producedmay distribution,and Cry3Bb1 protein sample and 1 µg of total rights owner proten from the E. coli-producedsubject Cry3Bb1 protein sample were separated by electrophoresis on a be the violate 4-20% gradient polyacrylamidecopy gel under reduciofng and denaturing conditions. The stained gel was analyzed using densitometry to determine the andpurity and molecular weight of the Cry3Bb1 may documentpublication, It exploitation protein. this any permission Lane Sample prohibited Amount (µg) the 1 empty commercial be --- 2 empty --- Furthermore,any 3 Bio-RadConsequently, Broadwithout Range MWM --- 4 plant-produced Cry3Bb1 protein 1 5 plant-produced Cry3Bb1 protein 2 6 plant-produced Cry3Bb1 protein 3 7 E. coli-produced Cry3Bb1 protein 1 8 Bio-Rad Broad Range MWM --- 9 empty --- 10 empty ---

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1.2.c. MALDI-TOF MS tryptic mass map analysis

The identity of the plant-produced Cry3Bb1 protein was also assessed using MALDI- TOF mass spectrometry. The ability to identify a protein using this method depends upon matching a sufficient number of observed to expected (theoretical) mass fragments. With sufficient mass accuracy, four tryptic peptides may be enough to identify a protein (Jiménez et al., 1998). Peptides were considered to match when a difference in molecular weight of less than one dalton was found between the observed and theoretical fragment mass. Matches were made without consideration for potential natural amino acid modifications.

The identity of each of the three protein bands (~77, ~66 and ~55 kDa) observed in western blot analysis (Figure 24) was confirmed. For the 77 kDa protein, 20 observed peptide mass fragments were matched to the theoretical tryptic peptide massand map for the and regime. full-length Cry3Bb1 protein. For the 66 kDa proteinAG fragment, 20 observed peptide mass fragments were matched to the theoretical tryptic peptide mass map for the Cry3Bb1 protein. For the 55 kDa protein fragment, 15 observedBayer property peptide protection masspublishing fragments were of contents matched to the theoretical tryptic peptide mass map for the Cry3Bb1its protein. The parties.data therefore identified masses were used to assemble a combined coverageand/or mapor (Figure 26) for the affiliates. property intellectualthird may three bands identified as Cry3Bb1 proteinits in the western blot analysis. In total, as approximately 44% (287 of 653 aminothe of acids) ofand the expected protein sequence was is regulatory owner. a document identified. any such its reproductiondocument owner this of under of rightsthe this Figure 26. MALDI-TOFdocument MSand/or coverage map of the Cry3Bb1rights protein sequence to of fall use of produced in MON 88017 the This may distribution,and rights owner 1 MANPNNRSEH DTIKVTPNSEsubject LQTNHNQYPL ADNPNSTLEE LNYKEFLRMT be the violate 51 EDSSTEVLDN STVKDAVGTGcopy ISVVGQILGVof VGVPFAGALT SFYQSFLNTI and 101 WPSDADPWKAmay FMAQVEVLIDdocumentpublication, KKIEEYAKSK ALAELQGLQN NFEDYVNALN It exploitation 151 SWKKTPLSLR SKRSQDRIREthis LFSQAESHFR NSMPSFAVSK FEVLFLPTYA 201 QAANTHLLLL KDAQVFGEEWany GYSSEDVAEF YRRQLKLTQQ YTDHCVNWYN permission 251 VGLNGLRGST YDAWVKFNRFprohibited RREMTLTVLD LIVLFPFYDI RLYSKGVKTE the 301 LTRDIFTDPI FLLTTLQKcommercial beYG PTFLSIENSI RKPHLFDYLQ GIEFHTRLRP 351 GYFGKDSFNYFurthermore, anyWSGNYVETRP SIGSSKTITS PFYGDKSTEP VQKLSFDGQK 401 VYRTIANTDVConsequently, AAWPNGKVYLwithout GVTKVDFSQY DDQKNETSTQ TYDSKRNNGH 451 VSAQDSIDQL PPETTDEPLE KAYSHQLNYA ECFLMQDRRG TIPFFTWTHR 501 SVDFFNTIDA EKITQLPVVK AYALSSGASI IEGPGFTGGN LLFLKESSNS 551 IAKFKVTLNS AALLQRYRVR IRYASTTNLR LFVQNSNNDF LVIYINKTMN 601 KDDDLTYQTF DLATTNSNMG FSGDKNELII GAESFVSNEK IYIDKIEFIP 651 VQL

Shaded regions correspond to tryptic peptide masses that were identified from the 77, 66, and 55 kDa bands, analyzed separately, using MALDI-TOF MS. The gray shaded sequence corresponds to the region identified from the 77 kDa band. The black shaded sequence corresponds to additional sequence identified from the 66 kDa band. No additional amino acids were identified from the 55 kDa band that were not observed in the 77 or 66 kDa bands.

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1.2.d. Functional activity of the Cry3Bb1 protein

The biological activity of E. coli- and plant-produced Cry3Bb1 proteins was estimated in a diet-incorporated bioassay. E. coli- and plant-produced Cry3Bb1 proteins were incorporated into a diet and fed to larvae of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, a susceptible insect. Biological activity was measured as an LC50, which is defined as the estimated lethal concentration of protein (µg/ml diet) required to kill 50% of the CPB larvae. The LC50 values for CPB when fed the plant- and E. coli-produced Cry3Bb1 proteins were 0.414 µg Cry3Bb1 protein/mL diet and 0.554 µg Cry3Bb1 protein/mL diet, respectively. Statistical analysis demonstrated that the two LC50 values were not different (p>0.05), leading to the conclusion that the E. coli- and plant-produced Cry3Bb1 proteins are functionally equivalent.

1.2.e. Glycosylation analysis of the Cry3Bb1 protein and and regime. Many eukaryotic proteins are post-translationally modifiedAG with carbohydrate moieties (Rademacher et al., 1988). These carbohydrate moieties may be complex branched Bayer property protectionpublishing polysaccharide structures or simple monosaccharides.of In contrast, prokaryoticcontents organisms its such as E. coli lack the necessary organelles and biochemicalparties.data pathwaystherefore required for and/oror protein glycosylation. To test if post-translationalaffiliates. glycosylation of the plant-produced property intellectualthird may its Cry3Bb1 protein occurred, this protein wasas analyzed for covalently bound carbohydrate the of and moieties (Figure 27). The E. coliis-produced Cry3Bb1regulatory protein and transferrinowner. protein were a document any such its analyzed concurrently as negative and positive controls,reproduction respectively.document The positive owner this of control (transferrin) was clearly detected in undera concentration-dependent of manner at loadings rightsthe this documentand/or rights of 0.25 and 0.5 µg/lane (Lanes 2-3).to of A bandfall at ~25use kDaof was observed in the E. coli- produced protein sample (Lanes 4-5) and no band was observedthe in the plant-produced This may distribution,and rights owner Cry3Bb1 sample (Lanes 6-7).subject The band observed in the E. coli-produced Cry3Bb1 sample was at a lower molecularbe weight than the Cry3Bb1violate protein. This band likely copy of represents a naturally biotinylated protein that andco-purified with the E. coli-produced may documentpublication, It exploitation Cry3Bb1 protein (Choi-Rheethis et al., 2003), because it would bind to the streptavidin-HRP conjugate and be detected byany the ECL system. No band was observed at the expected permission molecular weights for the Cry3Bb1 protein;prohibited th erefore, it was concluded that it was not the glycosylated. commercial be Furthermore,any Consequently,without 1.2.f. Conclusions of the characterization of the Cry3Bb1 protein produced in MON 88017 corn

A panel of analytical tests was used to characterize and compare the plant- and E. coli- produced Cry3Bb1 proteins: (1) western blot analysis and densitometry, (2) SDS-PAGE and densitometry, (3) MALDI-TOF mass spectrometry, (4) glycosylation analysis, and (5) a Cry3Bb1 activity assay. The equivalence of the plant-produced Cry3Bb1 protein to the E. coli-produced Cry3Bb1 protein was evaluated by comparing their full-length molecular weights, immunoreactivity with anti-Cry3Bb1 antibodies, glycosylation status, and functional activity.

Monsanto Company 04-CR-109F Page 83 of 223

Protein bands migrating at approximate molecular weights of 77, 66, and 55 kDa were identified as plant-produced Cry3Bb1 protein fragments using western blot analysis with anti-Cry3Bb1 antibody. The purity, calculated as the sum of the percent optical density of the three bands identified as Cry3Bb1 protein, was 66.1%. The approximate molecular weights of each of the three Cry3Bb1 protein bands identified in the plant-produced Cry3Bb1 protein sample, estimated by comparison to molecular weight markers on a Colloidal Brilliant Blue G stained SDS-polyacrylamide gel, were 77, 66, and 55 kDa. MALDI-TOF mass spectrometry analysis of these bands after trypsin digestion yielded peptide masses consistent with the peptide masses expected for the Cry3Bb1 protein. Together, masses identified for each protein band yielded 44% overall coverage (287 out of 653 amino acids) of the peptide sequence of the plant-produced Cry3Bb1 protein. The plant-produced Cry3Bb1 protein was not glycosylated. The functional activities of the plant- and E. coli - produced Cry3Bb1 proteins determined by insect bioassay were not significantly different (p>0.05), leading to the conclusion that the proteins are functionally equivalent. and and regime. AG Based on the results of these tests it was concluded that the plant- and E. coli-produced Cry3Bb1 proteins are physicochemically and functionallyBayer property equivalent. 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

Monsanto Company 04-CR-109F Page 84 of 223

Figure 27. Glycosylation analysis of the Cry3Bb1 protein produced in MON 88017

1 2 3 4 5 6 7 8 9 10 kDa

214

118 92

52.2

and 35.7 and regime. AG 28.9 Bayer property protectionpublishing of contents 20.8 its parties.data therefore and/oror affiliates. 6.8 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 Proteins were separated using SDS-PAGEto of underfall reducinguse of a nd denaturing conditions and transferred to a PVDF membrane. The dots in lanes 1 and the8 represent the location of the This may distribution,and rights owner molecular weight markers (MWM)subject on the membrane. A 10 minutes exposure is shown. be the violate copy of Lane Sample and Amount (µg) may documentpublication, 1 It Bio-Rad Prestainedexploitation Broad Range MWM --- 2 transferrinthis any 0.25 3 transferrin permission 0.5 prohibited 4 E. coli-producedthe be Cry3Bb1 protein 0.25 5 E. colicommercial-produced Cry3Bb1 protein 0.5 6 Furthermore, plant-producedany Cry3Bb1 protein 0.25 without 7 Consequently, plant-produced Cry3Bb1 protein 0.5 8 Bio-Rad Prestained Broad Range MWM --- 9 empty --- 10 empty

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SECTION 2. Levels of the CP4 EPSPS and Cry3Bb1 proteins in MON 88017

The levels of the CP4 EPSPS and Cry3Bb1 proteins in various tissues of MON 88017 were estimated using enzyme-linked immunosorbent assay (ELISA). The materials and methods for the ELISA analysis, as well as a description of the tissue types, are provided in Appendix III. To produce the tissues for analysis, MON 88017 and conventional corn hybrids were planted at three field locations during the 2002 growing season. The sites were located in the major corn-growing region of the United States. A randomized complete block design with three replications was used at all sites. Pollen, silk, forage, forage root, grain, stover and young leaf were collected at appropriate times of plant development. Cry3Bb1 and CP4 EPSPS protein levels in these tissues are presented in Table 4. The following tissues were collected over the season from the V2 to the R6 vegetative growth stages: overseason leaf samples (OSL 1 – 4), overseason whole plant (OSWP 1-4), and overseason root (OSR 1-4). The levels of CP4 EPSPS andand Cry3Bb1 and regime. proteins in overseason tissues are presented in TablesAG 5 and 6. Limits of detection and quantitation are presented in Table 7. Bayer property protectionpublishing of contents The mean CP4 EPSPS protein levels in analyzed tissues collected acrossits three sites parties.data therefore ranged during the season from 150-220 µg/g dwt in leaf and 70-150and/oror µg/g dwt in root affiliates. property intellectualthird may (Table 5). The mean CP4 EPSPS proteiitsn levels across three sites were 220 µg/g dwt in as young leaf, 390 µg/g dwt in pollen,the 57 of µ g/g dwtand in forage, and 5.8 µg/g dwt in grain is regulatory owner. a document (Table 4). CP4 EPSPS levels wereany not such assessed in whole plant, silkits and stover. In reproductiondocument general, levels of the CP4 EPSPS proteinowner declined duringthis the growingof season. under of rightsthe this documentand/or rights to of fall use of The mean Cry3Bb1 protein levels in analyzed tissues collectedthe across the three sites This may distribution,and ranged from 260-570 µg/g dwt rightsin leaf, 220-500 µownerg/g dwt in the whole plant, and 100-370 µg/g dwt in root tissues harvestedsubject throughout the growing season (Table 6). The mean be the violate levels across three sites werecopy 570 µg/g dwt inof young leaf, 25 µg/g dwt in pollen, 95 µg/g and may documentpublication, dwt in forage, 380It µ g/g dwt in silk, exploitation130 µg/g dwt in forage root, 15 µg/g dwt in grain, and 88 µg/g dwt in stoverthis (Table any 4). permission prohibited the commercial be

Furthermore,any Consequently,without

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Table 4. Levels of the Cry3Bb1 and CP4 EPSPS proteins in tissues of MON 88017

Cry3Bb1 CP4 EPSPS Mean (SD)2 [range]3 Mean (SD)2 [range]3 Tissue Growth (µg/g dwt) (µg/g fwt) (µg/g dwt) (µg/g fwt) Type1 Stage Young V2-V3 570 (170) 76 (23) 220 (30) 30 (5.3) leaf (14-22 DAP) [230-820] [28-110] [160-260] [19-36] Pollen R1 25 (4.2) 14 (2.5) 390 (85) 220 (43) (62-69 DAP) [17-32] [11-20] [210–470] [130-280] Silk R1 380 (65) 37 (5.6) (62-69 DAP) [300-500] [30-45] NM NM Forage R4-R6 and (early dent) 95 (19) 27 (5.5) and57 (7.6) regime. 16 (2.1) (97-124 DAP) [75-130] [22-39]AG [42-69] [12-19] Forage R4-R6 Bayer property protectionpublishing root (early dent) 130 (29) of 21 (3.1) 70 (20) contents 11 (2.8) its (97-124 DAP) [98-170] [17-27]parties. data [47-110] therefore [6.6-15] Grain R6 15 (3.6) 13(3.1) and/or5.8or (0.97) 5.1 (0.89) affiliates. property intellectualthird may (133–146 DAP) [10-22] its [8.7-19] [4.1-7.1] [3.7-6.3] as the of and Stover R6 is regulatory owner. a document any such its (after harvest) 88 (13) 30 (4.4)reproduction document NM NM owner this of (133-147 DAP) [71-110] under[25-39] of rightsthe this documentand/or of rights 1 to of fall use Description of the tissue types is provided in Appendix III.the Limits of detection and quantitation This may distribution,and are described in Table 7. rights owner 2 The mean and standard deviationsubject were calculated across sites and replicates. (n=9) 3 be the violate Minimum and maximum valuescopy were determinedof for each tissue type across sites. document and may publication,exploitation DAP = days after plantingIt this any n = number of samples permission SD = standard deviation. The mean and SDprohibited were calculated across sites (n=9) the NM = not measured commercial be Furthermore,any Consequently,without

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Table 5. Levels of the CP4 EPSPS protein (µg/g dwt) in overseason tissues of MON 88017

Tissues1,2 V2-V3 V5 V8 V11-V17 R4-R6 (n=9) (14-22 DAP) (26-34 DAP) (40-45 DAP) (55-62 DAP) (97-124and DAP) and regime. AG Leaf Mean (SD) 220 (30) 190 (26) 170 (37) 150 (19) NA Range 160-260 130-250 140-240Bayer property 120-170protectionpublishing of contents its parties.data therefore Root Mean (SD) 150 (34) 110 (29) 100 (30) and/or 97 (18)or 70 (20) affiliates. property intellectualthird may Range 110-220 74-160 its 62-160 72-130 47-110 as the of and is regulatory owner. 1 a document Growth stages V2-V3, V5, V8 and V11-V17 correspond to overseasonany such tissue samples -1, -2, -3its and -4, respectively (e.g., OSL-1, etc.). The root reproductiondocument samples collected at the R4-R6 and R6 growth stages correspond to forageowner root and thissenescent of root, respectively. The DAP values were estimated under of from information in the production report rightsthe this documentand/or of rights 2Description of tissue types is provided in Appendix III. Litomits ofof detectionfall anduse quantitation are described in Table 7. the This may distribution,and rights owner DAP = days after planting. subject be the violate NA = not applicable. copy of dwt = dry weight tissue and may documentpublication, It exploitation SD = standard deviation this any permission prohibited the commercial be

Furthermore,any Consequently,without

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Table 6. Levels of the Cry3Bb1 protein (µg/g dwt) in overseason tissues of MON 88017

Tissues1 V2-V3 V5 V8 V11-V17 R4-R6 R6 (n=9) (14-22 DAP) (26-34 DAP) (40-45 DAP) (55-62 DAP) (97-124 DAP) (133-147 DAP)

Leaf Mean (SD) 570 (170) 430 (58) 310 (45) 260 (44) NA NA Range 230-820 310-510 240-380 190-340 and and regime. AG Whole plant Mean (SD) 500 (64) 380 (170) 310 (48) 220 (23) NA NA Range 410-590 150-600 230-380Bayer property 190-250protectionpublishing of contents its parties.data therefore Root Mean (SD) 370 (80) 250 (71) 210 (78) 180and/or (37)or 130 (29) 100 (19) affiliates. property intellectualthird may Range 240-510 190-420 its 150-410 110-230 98-170 77-140 as the of and is regulatory owner. 1 a document Growth stages V2-V3, V5, V8 and V11-V17 correspond to over-seasany suchon tissue samples -1, -2, its-3 and -4, respectively (e.g., OSL-1, etc.). The root reproductiondocument samples collected at the R4-R6 and R6 growth stages correspond to forageowner root andthis senescent of root, respectively. under of rightsthe this documentand/or of rights DAP = days after planting to of fall use the NA = not applicable This may distribution,and rights owner dwt = dry weight tissue subject be the violate SD = standard deviation copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table 7. Limits of detection and quantitation for the ELISA of Cry3Bb1 and CP4 EPSPS proteins

Cry3Bb1 CP4 EPSPS LOD LOQ LOD LOQ Tissue1 (µg/g fwt) (µg/g fwt) (µg/g fwt) (µg/g fwt) Leaf 0.0073 0.044 0.090 0.17 Root 0.032 0.040 0.050 0.10 Pollen 0.020 0.039 0.12 0.18 Silk 0.012 0.041 NM NM Forage 0.010 0.047 0.11 0.21 Grain 0.0097 0.051 0.18 0.28 and and regime. 1 Description of the tissue types is provided in AppendixAG III. Whole plant and stover were analyzed using the forage assay. Senescent root was analyzed using the root assay. Bayer property protectionpublishing of contents LOD = limit of detection. its parties.data therefore LOQ = limit of quantitation. and/oror affiliates. NM = not measured. 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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SECTION 3. Estimate of dietary exposure

Use of corn as food and animal feed is discussed in Part VII, Section 1. Humans and animals will be exposed to CP4 EPSPS and Cry3Bb1 proteins through dietary intake of food and feed derived from MON 88017. The quantity of corn consumed on a daily basis by humans and livestock, as well as the levels of CP4 EPSPS and Cry3Bb1 proteins in corn, are necessary to derive an estimate of daily dietary exposure (DDE). Humans consume only corn grain or processed food products derived from corn grain. Livestock consume both corn grain and forage.

The mean adult consumption of corn grain (endosperm fraction) in the United States is 0.27 g/kg body weight/day. This consumption figure is based on the CSFII 1994-1998 food consumption surveys for adults in the United States (DEEM US™ data base (acute and version 7.77) – Exponent, Inc.). and regime. AG Consumption of corn is much higher in livestock. The daily consumption of corn for the Bayer property protectionpublishing young pig is 37 g/kg body wt/day (assumingof 60% dietary inclusion rate)contents and 23 g/kg body wt/day for the finishing pig (assuming 75% dietary inclusion rate).its The four-week parties.data therefore old broiler consumes 57 g/kg body wt/day when the inclusionand/or rateor of corn is 60% of the affiliates. property intellectualthird may diet. The lactating dairy cow (~570 kg bodyits wt) producing 37 kg of milk per day as consumes about 4.4 kg of corn perthe day of and 10.4and kg of corn silage per day or 7.7g/kg body is regulatory owner. a document wt/day and 18.2 g/kg body wt/day,any respectively such (Ouellet et al., 2003).its Daily dietary reproductiondocument exposure (DDE) is computed as follows: owner this of under of rightsthe this documentand/or rights to of fall use of DDE = Corn consumption (g/kg) x Cry3Bb1 or CP4 EPSPSthe protein concentration (µg/g) This may distribution,and rights owner Using upper bound estimatessubject of human and animal daily corn consumption, it is possible be the violate to calculate the margin of exposurecopy (MOE) offor these proteins. The margin of exposure is and may documentpublication, defined as the ratioIt of the no observedexploitation effect level (NOEL) derived from toxicology tests to the estimate of human thisand any animal daily dietary exposure (DDE). The MOE is permission computed as follows: prohibited the commercial be MOE = NOEL (µg/kg) ÷ DDE (µg/kg) Furthermore,any Consequently,without The exposure calculation makes the conservative assumption that there is no loss of the introduced protein during the processing of corn grain into human food. It also assumes that 100% of the corn grain ending up in human food is derived from the MON 88017, which is highly unlikely given the variety of commercial corn hybrids that exist in the market place.

3.1. Estimated dietary exposure to the CP4 EPSPS protein

The mean level of CP4 EPSPS protein in MON 88017 grain is 5.8 µg/gram (dwt) (range 4.1-7.1) and 57 µg/gram (dwt) (range 42 – 69) in forage (Table 4, Section 2, Part VI). The highest dose of CP4 EPSPS protein that was administered by gavage to mice, 572

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mg/kg (572,000 µg/kg), produced no adverse effects; therefore the no observed effect level (NOEL) in the CP4 EPSPS mouse gavage study is 572,000 µg/kg.

A dairy cow consumes both corn grain and silage, and therefore the amount of CP4 EPSPS needs to be considered from both sources. CP4 EPSPS in corn silage (40% grain and 60% forage) would have a mean CP4 CPSPS concentration of (5.8 µg/g x 40% + 57 µg/g x 60%) = 36.5 µg/g. The high end of the range is calculated to be (7.1µg/g x 40% + 69 µg/g x 60%) = 44.2 µg/g CP4 EPSPS. A summary of the calculated margins of exposure for humans and livestock for CP4 EPSPS protein produced in MON 88017 is presented in Table 8.

3.2. Estimated dietary exposure to the Cry3Bb1 protein

The mean level of Cry3Bb1 protein in corn grain is 15 µg/gram (dwt) (range 10 - 22) and 95 µg/gram (dwt) (range 75 – 130) in forage. The highest dose of Cry3Bb1and protein that and regime. was administered by gavage to mice, 1930 mg/kg, producedAG no adverse effects; therefore, the NOEL in the Cry3Bb1 mouse gavage study is 1,930,000 µg/kg. Bayer property protectionpublishing of contents For dairy cow, the amount of consumed Cry3Bb1 was considered fromits both corn grain parties.data therefore and silage. Cry3Bb1 in corn silage (40% grain and 60% forage)and/or wouldor have a mean affiliates. property intellectualthird may Cry3Bb1 concentration of (15 µg/g x 40%its + 95 µg/g x 60%) = 63 µg/g. The high end of as the range is calculated to be (22 µg/gthe xof 40% + and130 µg/g x 60%) = 86.8 µg/g Cry3Bb1. A is regulatory owner. a document summary of the calculated marginsany of esuchxposure to Cry3Bb1 proteinits produced in MON reproductiondocument 88017 by humans and farm animals is presentedowner in Tablethis 8. of under of rightsthe this documentand/or rights to of fall use of the 3.3. Margins of exposureThis may distribution,and rights owner subject be the violate Large margins of exposure copywere noted for CP4of EPSPS protein for humans (364,331) and and may documentpublication, livestock (806.8- 4,287),It and for Cry3Bb1exploitation for humans (470,731) and livestock (1,529 – 5,594) (Table 8). These calculatedthis any margins of exposure indicate that there is virtually no permission risk to human and animal health that willprohibited be associated with dietary exposure to food and feed products derived from MONthe 88017. commercial be Furthermore,any Consequently,without

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Table 8. Margins of exposure for dietary consumption of CP4 EPSPS and Cry3Bb1 proteins in MON 88017 when used as food or animal feed.

Adult Broiler Young Finishing Dairy Parameter Human Chicken Pig Pig Cow Daily grain consumption 0.27 57 37 23 7.7 (g/kg body wt) Daily forage consumption NA NA NA NA 18.2 (g/kg body wt) CP4 EPSPS DDE 0.0016 0.331 0.215 0.133 0.709 (mg/ kg body wt/day) (0.0019) 1 (0.405) (0.263) (0.163) (0.859) MOE 364,331 1,730 2,665 4,287 806.8 and (301,053) (1,413) and(2,177) regime. (3,503) (666) AG Cry3Bb1 DDE 0.0041 0.855 0.555 0.345 1.262 Bayer property protectionpublishing (mg/ kg body wt/day) (0.0059)of (1.254) (0.814)contents (0.506) (1.749) its MOE 470,731 2,257parties. data 3,477 therefore5,594 1,529 and/oror (327,119)affiliates. (1,539) (2,371) (3,814) (1,103) 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 SECTION 4. Assessment of the potential for allergenicitythe of the CP4 EPSPS and This may distribution,and Cry3Bb1 proteinsrights produced in MONowner 88017 subject be the violate copy of This assessment of the allergenic potential of aand protein addresses the following questions, may documentpublication, which identify characteristicsIt of knownexploitation allergens: this any permission 1. Is the protein from a known allergenicprohibited source? the 2. Does the protein representcommercial a relatively be large portion of the total protein in MON 88017?Furthermore, any 3. Is the proteinConsequently, structurallywithout similar, based on amino acid sequence, to known allergens? 4. Is the protein resistant to digestion in simulated mammalian digestive fluid?

In the following sections, these four questions are addressed in detail for the CP4 EPSPS and Cry3Bb1 proteins produced in MON 88017. General information on the methods used in assessing the last two questions – structural similarity to known allergens and stability in simulated digestive fluids- is provided below.

1 Numbers calculated using high end of the expression range given in parentheses.

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Structural similarity to known allergens

In order to assess potential similarity to allergens, bioinformatic analyses were performed on the CP4 EPSPS and Cry3Bb1 proteins expressed in MON 88017. The comparisons were performed using the allergen (AD4) database.

Exposure to allergens in foods may cause sudden, severe, life-threatening reactions in susceptible individuals. Gliadins are suspected to cause celiac disease (gluten-sensitive enteropathy) and are also considered important immunologically active proteins. Screening the amino acid sequences of proteins introduced into plants by modern biotechnology for similarity to sequences of known allergens and gliadins is one of many assessments performed to evaluate product safety. Similarly, the amino acid sequences of introduced proteins are also screened against known toxins as well as all known proteins in publicly available genetic databases (see Section 5 of Part VI). and and regime. The FASTA algorithm can be used to evaluate the extentAG of sequence similarity between a query protein sequence and a database sequence. In principle, if two proteins share sufficient linear sequence similarity, they also Bayerwill shareproperty three-dime protectionpublishingnsional structure and, of contents therefore, functional homology. By definition, homologous proteinsits share secondary parties.data therefore structure and common three-dimensional folds (Pearson, 2000).and/or Becauseor the degree of affiliates. similarity between homologs varies widely,property thintellectuale datathird need to be carefullymay evaluated in its as order to maximize their value to thethe allergy of assessmentand process. is regulatory owner. a document any such its reproductiondocument The bioinformatics assessment is used to owneridentify similaritiesthis betwof een the query protein under of and known or clinically cross-reactiverightsthe allergens. Whilethis related (homologous) proteins documentand/or of rights may share only 25% amino acidto identity of fallin a 200use amino acid overlap (Pearson, 2000), the This may distribution,and this is not generally sufficient torights indicat e IgE-mediatedowner cross-reactivity (Aalberse et al., 2001). Indeed, allergenic subjectcross-reactivity caused by proteins sharing conformational or be the violate linear epitopes with knowncopy allergens is rareof at 50% identity and typically requires >70% and amino acid identity mayacross thedocument fullpublication, length of the protein sequences (Aalberse, 2000). Such It exploitation high levels of identity arethis readily any detected using FASTA. Additionally, proteins closely related to gliadins or glutenins (thepermission proteins that trigger celiac disease, a non-IgE prohibited mediated allergic disorder) can thebe easilybe identified using FASTA. It is possible that proteins structurally unrelatedcommercial to allergens and gliadins may still contain smaller Furthermore,any immunologicallyConsequently, significantwithout epitope s. For this comparison an immunologically relevant sequence was defined as eight linearly contiguous, identical amino acids (Metcalfe et al., 1996). A second bioinformatics tool (IDENTITYSEARCH) was used to specifically identify short linear polypeptide matches to known or suspected allergens. The results of the bioinformatics analyses of CP4 EPSPS and Cry3Bb1 amino acid sequences are described below in Sections 4.1.c. and 4.2.c, respectively.

Stability in simulated digestive fluids

A factor that increases the likelihood of allergic oral sensitization to proteins is the stability of the protein to gastrointestinal digestion. Protein allergens tend to be stable to the peptic and acidic conditions of the digestive system if they are to reach and pass

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through the intestinal mucosa to elicit an allergenic response (Astwood et al., 1996; Metcalfe et al., 1996). A relationship between digestibility in simulated gastric fluid (in vitro pepsin digestibility) and food safety has been previously demonstrated using a digestion model (Astwood et al., 1996). Simulated intestinal fluid is frequently used for in vitro studies to assess the digestibility of food components (Yagami et al., 2000; Okunuki et al., 2002).

Recently, the pepsin digestibility assay protocol was standardized by the International Life Science Institute (ILSI) based on results obtained from an international, multi- laboratory ring study (Thomas et al., in press). This test showed that the results of in vitro pepsin digestion assays are reproducible when standard protocols were followed. Using these protocols, the pepsin digestion assay was used to assess the susceptibility of the CP4 EPSPS and Cry3Bb1 proteins to pepsin digestion in vitro. In addition, in vitro susceptibility of these proteins to pancreatin was assessed for stability in simulated intestinal fluid according to methods described in the United States Pharmacopeiaand (1995). and regime. The stability of the CP4 EPSPS and Cry3Bb1 proteinsAG in simulated digestive fluids is discussed in Sections 4.1.d. and 4.2.d., respectively. Bayer property protectionpublishing of contents its parties.data therefore 4.1. Assessment of the potential for allergenicity of the CP4and/or EPSPSor protein affiliates. property intellectualthird may its 4.1.a. Source of the CP4 EPSPS protein as the of and is regulatory owner. a document any such its reproductiondocument As described in Part VI, Section 1, the cp4owner epsps codingthis sequenceof was obtained from a under of naturally occurring, glyphosate-degradingrightsthe bacterium andthis has been identified by the documentand/or of rights American Type Culture Collectionto asof an fallAgrobacterium use species. Agrobacterium species the are not known humanThis or animal pathogensmay distribution, andand are not commonly allergenic. A number rights owner of food crops have been improvedsubject by addition of a single functional gene from be the violate Agrobacterium tumefacienscopy (FAO/WHO, 1991)of and there are no reports of allergies to and Agrobacterium speciesmay (Sectiondocument publication,5.3, Part VI). Furthermore, according to FAO/WHO It exploitation (2001), there is no knownthis population any of individuals sensitized to bacterial proteins. Therefore, it can be concluded thatpermission the CP4 EPSPS protein is not from a known prohibited allergenic source. the commercial be 4.1.b. ProportionFurthermore, of totalany protein – CP4 EPSPS Consequently,without

Data presented in Section 2 of Part VI show that the overall mean level of CP4 EPSPS protein in MON 88017 grain is 5.8 µg/g (dwt). The mean % dry weight of total protein in MON 88017 grain is 12.51% (Table 16). The percent of CP4 EPSPS protein in one gram of MON 88017 grain is calculated as follows:

5.8 µg/g ÷ 125,100 µg x 100% = 0.0046362 %

Therefore, the CP4 EPSPS protein represents a very small portion of total protein in MON 88017 grain.

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4.1.c. Bioinformatics analyses of sequence similarity of the CP4 EPSPS protein produced in MON 88017 to allergens

Using the bioinformatics analyses described above, potential structural similarities between the CP4 EPSPS protein and proteins in the allergen database were evaluated using the FASTA sequence alignment tool. Nine identified proteins were ranked according to their degree of similarity. The highest similarity was to a small portion of the Dermatophagoides farinae allergen Der f 2 (Accession No. AAB30829). This alignment demonstrated 30.5% identity over an 82 amino acid (aa) window with a high E-score of 0.41. Moreover, the alignment contained four gaps, and the longest stretch of contiguous amino acid identities consisted of five amino acids. The length of the overlap is relatively short (18%) when compared to the full length (455 aa) of the CP4 EPSPS protein. Consequently, no structural and/or functional homology between the CP4 EPSPS protein and the Der f 2 allergen can be inferred. It is unlikely that allergenic and cross-reactivity will be observed even when there is ≥ 50% identityand acrossregime. the entire length of the protein (Aalberse, 2000). Therefore, itAG is highly unlikely that cross- reactivity exists between the CP4 EPSPS protein and the Der f 2 allergen. Inspection of Bayer property protectionpublishing the remaining eight alignments did not showof any significant similaritiescontents between the CP4 its EPSPS protein and aligned allergens. No immunologicallyparties.data relevant sequencestherefore (eight and/oror contiguous amino acid identities) were deteaffiliates.cted when the CP4 EPSPS protein sequence property intellectualthird may was compared to the AD4 sequence database.its as the of and is regulatory owner. a document any such its reproductiondocument 4.1.d. Stability of the CP4 EPSPS proteinowner in simulatedthis digestiveof fluids under of rightsthe this documentand/or rights to of fall use of Harrison et al. (1996) demonstrated that the CP4 EPSPSthe protei n is rapidly degraded in This may distribution,and simulated digestive fluids. Therights half-life for CP4owner EPSPS was less than 15 seconds in the subject gastric system and less thanbe 10 minutes in thethe intestinal violate system, based on western blot copy of analysis. Therefore, if any of the CP4 EPSPS andprotein were to survive in the gastric may documentpublication, system, it would beIt rapidly degradedexploitation in the intestine. As a comparison, 50% of solid food has been estimated tothis empty any from the human stomach in two hours, while 50% permission liquid empties in approximately 25 minutesprohibited (Sleisenger and Fordtran, 1989). Based on the this information, CP4 EPSPScommercial protein be is expected to degrade rapidly in the mammalian digestive tract.Furthermore, any Consequently,without Subsequent experiments were performed to assess the in vitro digestibility of the CP4 EPSPS protein in simulated gastric fluid (SGF). As with the previous study (Harrison et al., 1996), the CP4 EPSPS protein used was produced in and purified from E. coli. Digestibility was assessed by three methods, including SDS-PAGE gel staining, western blot analysis, and EPSPS enzymatic activity assay.

The results of these experiments demonstrate that the E. coli-produced CP4 EPSPS protein was rapidly digested after incubation in SGF. The SDS-PAGE Colloidal Blue gel staining method demonstrated that at least 98% of the E. coli-produced CP4 EPSPS protein was digested in SGF within 15 seconds (Figure 28). No protein bands due to degradation of the CP4 EPSPS protein were observed. Western blot analysis (Figure 29)

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confirmed that greater than 95% of the immunoreactive E. coli-produced CP4 EPSPS protein was digested in SGF within 15 seconds. Similarly, it was demonstrated that the EPSPS activity was reduced to <10% within 15 seconds of incubation of the CP4 EPSPS protein in SGF (Table 9). In summary, and in complement to the earlier study, the results of the SDS PAGE, western blot, and functional assays demonstrate that the E. coli- produced CP4 EPSPS protein is rapidly degraded in simulated gastric fluid.

and and regime. AG

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Figure 28. Digestion of CP4 EPSPS protein in simulated gastric fluid demonstrated by SDS PAGE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

MW ( kD ) 200

116.3 97.4 66.3 E. coli- p roduced 55.4 CP4 EPSPS protein

Pepsin 36.531

21.5 and and regime. 14.4 AG

6 Bayer property protectionpublishing 3.5 of contents its 2.5 parties.data therefore and/oror affiliates. property intellectualthird may its as the of and Proteins were separated by SDS-PAGEis using a 10→20%regulatory polyacrylamide owner. gradient in a tricine a document any such its buffered gel. Proteins were detected by staining with Brilliantreproduction Bluedocument G stain. E. coli-produced owner this of CP4 EPSPS protein was loaded at 500 ng per launderne based ofon pre-digestion concentration. rightsthe this documentand/or rights to of fall use of Lane Description the Incubation time This may distribution,and rights owner 1 Molecular weightsubject markers be the violate 2 Experimentalcopy control without pepsinof (P0) 0 s and 3 Experimentalmay controldocumentpublication, without CP4 EPSPS (N0) 0 s It exploitation 4 CP4 EPSPSthis protein in SGF, T = 0 0 s 5 CP4 EPSPS proteinany in SGF, T = 1 15 s permission 6 CP4 EPSPS protein in SGF,prohibited T = 2 30 s the 7 CP4 EPSPS commercialprotein in beSGF, T = 3 1 min 8 Furthermore, CP4 EPSPSany protein in SGF, T = 4 2 min 9 CP4Consequently, EPSPSwithout protein in SGF, T = 5 4 min 10 CP4 EPSPS protein in SGF, T = 6 8 min 11 CP4 EPSPS protein in SGF, T = 7 15 min 12 CP4 EPSPS protein in SGF, T = 8 30 min 13 CP4 EPSPS protein in SGF, T = 9 60 min 14 Experimental control without CP4 EPSPS (N9) 60 min 15 Experimental control without pepsin (P9) 60 min

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Figure 29. Digestion of CP4 EPSPS protein in simulated gastric fluid demonstrated by western blot analysis

121 2 3 4 5 6 7 8 9 10 11 12 13 144 15 MW (kD)

200 116.3 66.3 E. coli-produced CP4 EPSPS protein 36.5 21.5 14.4

6 and and regime. 3.5 AG

Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its Proteins were separated by SDS-PAGE using aas 10 →20% polyacrylamide gradient in a tricine the of and buffered gel. E. coli-produced CP4is EPSPS protein wasregulatory loaded at 1 ng perowner. lane based on 90% a document purity and pre-digestion concentration.any such its reproductiondocument of owner this under of this Lane Descriptionand/or rights the Incubation Time document to fall of rights 1 Molecular weight markersof use the 2 ExperimentalThis control withoutmay distribution, pepsinand (P0) 0 sec rights owner 3 Experimentalsubject control without CP4 EPSPS (N0) 0 sec be the violate 4 CP4 EPSPS proteincopy in SGF, T =of 0 0 sec and 5 CP4 EPSPSmay proteindocumentpublication, in SGF, T = 1 15 sec It exploitation 6 CP4 EPSPSthis protein in SGF, T = 2 30 sec 7 CP4 EPSPS proteinany in SGF, T = 3 1 min permission 8 CP4 EPSPS protein in SGF,prohibited T = 4 2 min the 9 CP4 EPSPS commercialprotein in beSGF, T = 5 4 min 10 CP4 EPSPS protein in SGF, T = 6 8 min Furthermore,any 11 CP4Consequently, EPSPSwithout protein in SGF, T = 7 15 min 12 CP4 EPSPS protein in SGF, T = 8 30 min 13 CP4 EPSPS protein in SGF, T = 9 60 min 14 Experimental control without CP4 EPSPS (N9) 60 min 15 Experimental control without pepsin (P9) 60 min

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Table 9. Specific activity of E. coli-produced CP4 EPSPS protein after digestion in simulated gastric fluid

Specific Activity Sample (Units/mg protein) Experimental control without pepsin incubated 4.92 for 0 seconds Experimental control without pepsin incubated 2.10 for 60 minutes E. coli-produced CP4 EPSPS protein in SGF 5.63 incubated for 0 seconds E. coli-produced CP4 EPSPS protein in SGF 0.27 incubated for 15 seconds and E. coli-produced CP4 EPSPS protein in SGF and 0.15regime. incubated for 30 seconds AG E. coli-produced CP4 EPSPS protein in SGF 0.15 Bayer property protectionpublishing incubated for 60 seconds of contents its Experimental control without CP4 EPSPS parties.data 0.02therefore and/oror incubated for 0 seconds affiliates. property intellectualthird may its Experimental control without CP4as EPSPS 0.05 the of and incubated for 60 minutesis regulatory owner. a document any such its Buffer Blank reproductiondocument 0.01 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 4.2. Assessment of the potential for allergenicityand of the Cry3Bb1 protein may documentpublication, It exploitation 4.2.a. Source of the Cry3Bb1this any protein permission prohibited the The Cry3Bb1 protein is a variantcommercial of be the wild-type Cry3Bb1 protein isolated from Bacillus thuringiensis (subsp.Furthermore, kumamotoensisany ) strain EG4691 (Donovan et al., 1992). Bacillus thuringiensis subspeciesConsequently, kumamotoensiswithout is a spore-forming, gram-positive bacterium that is found naturally in soil. B.t. strains have been used commercially in the U.S. since 1958 to produce microbial-derived products with insecticidal activity (EPA, 1988). There are no known reports of allergies to Bacillus thuringiensis species.

4.2.b. Proportion of total protein – Cry3Bb1

The mean level of Cry3Bb1 protein in corn grain is 15 µg/g (dwt). The mean % dry weight of total protein in MON 88017 grain is 12.51% (Table 16). The percent of Cry3Bb1 protein in one gram of MON 88017 grain is calculated as follows:

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15 µg/g ÷ 125100 µg x 100% = 0.01199 %

Therefore, the Cry3Bb1 protein represents a very small portion of total protein in MON 88017 grain.

4.2.c. Bioinformatics analysis of sequence similarity of the Cry3Bb1 protein produced in MON 88017 to allergens

Using the methods described above, potential structural similarities shared between the Cry3Bb1 protein and proteins in the allergen database were evaluated using the FASTA sequence alignment tool. Six identified proteins were ranked according to their degree of similarity. None of these proteins had an E-score of less than 1x10-5. The best similarity observed was to the Anisakis simplex muscle tropomyosin allergen, Ani s 3 (Accession No. Q9NAS5). In this alignment, the overlap of 120 aa contained four gaps, showed only and 27.5% identity, and had an E-score of 1.1. The length of the overlapand wasregime. relatively short when compared to the full-length (653 aa) Cry3Bb1AG protein. The longest stretch of contiguous amino acid identities consisted of three amino acids. Consequently, no Bayer property protectionpublishing structural and/or functional homology betweenof the Cry3Bb1 protein andcontents the Ani s 3 allergen can be inferred. As allergenic cross-reactivity is unlikely toits be observed even parties.data therefore when there is ≥ 50% identity across the entire length of the proteinand/oror (Aalberse, 2000), it is affiliates. property intellectualthird may highly unlikely that cross-reactivity existsits between the Cry3Bb1 protein and the Ani s 3 as the of and allergen. Inspection of the remainingis five alignmentsregulatory did not show owner.any significant a document similarities between the Cry3Bb1 pranyotein such and aligned allergens. its reproductiondocument owner this of under of rightsthe this No immunologically relevantdocumentand/or sequences (eight contiguousrights amino acid identities) were to of fall use of detected when the Cry3Bb1 protein sequence was comparedthe to the AD4 sequence This may distribution,and database. rights owner subject be the violate 4.2.d. Stability of the Cry3Bb1copy protein inof simulated digestive fluids and may documentpublication, It exploitation Using the ILSI pepsin digestionthis any protocol described above (Section 4, Thomas et al., in permission press)) in vitro digestibility of the Cry3Bb1prohibited protein was assessed in simulated gastric the (SGF) and simulated intestinalcommercial (SIF) be fluids. SGF contains the proteolytic enzyme pepsin. Digestibility in SGF was assessed using Colloidal Brilliant Blue G stained SDS Furthermore,any polyacrylamide gelsConsequently, and westernwithout blot analysis. SIF contains a mixture of proteolytic enzymes (including trypsin) known as pancreatin. Digestibility in SIF was assessed using western blot analysis.

The Cry3Bb1 protein was rapidly digested when incubated in SGF. At least 98% of the full-length (~75 kDa) Cry3Bb1 protein was digested within 15 seconds in SGF when analyzed using Colloidal Brilliant Blue G stained SDS polyacrylamide gels (Figure 30). A proteolytic fragment (migrating at the dye front) was observed at several of the initial time points, but was absent after four minutes of incubation. At least 99.8% of the full- length Cry3Bb1 protein was digested within 15 seconds in SGF and no proteolytic fragments were observed for samples evaluated by western blot analysis (Figure 31).

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The full-length Cry3Bb1 protein was also rapidly digested when incubated in SIF. At least 99.5% of the full-length Cry3Bb1 protein was digested within 1 minute in SIF when evaluated by western blot analysis (Figure 32). As expected, protease-resistant fragments were observed throughout the 24-hour time course. Proteolytic (trypsin) conversion of the full-length protoxin to an active toxin has been described for all members of the Cry1, Cry2, and Cry3 families (EPA, 2001b, 2002, 2003a). These results suggest that the Cry3Bb1 protein will be readily digestible in the mammalian digestive tract.

and and regime. AG

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Figure 30. Digestion of the Cry3Bb1 protein in simulated gastric fluid demonstrated by SDS-PAGE analysis

Lane: 12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 kDa 200 116 97 66 Cry3Bb1

45 Pepsin 31 22

7 and and regime. AG

Bayer property protectionpublishing of contents its Proteins were separated by SDS-PAGE using a 10-20% polyacrylamideparties.data gradienttherefore Tris-tricine buffered gel and visualized by staining with Colloidal Brilliant Blue G stain.and/or Cry3Bb1or protein was loaded at affiliates. property intellectualthird may 500 ng per lane based on pre-digestion concentrationits and purity values. as the of and is regulatory owner. a document Lane Description Incubation anyTime such Lane Description its Incubation Time reproductiondocument 1 MWM* --- owner 14 SGF-2this T0 of 0 sec under of 2 500 ng SGF-2 T0 --- rightsthe 15 SGF-2this T1 15 sec documentand/or rights 3 200 ng SGF-2 T0 ---to of fall 16 use SGF-2 of T2 30 sec the 4 100 ng SGF-2This T0 --- may distribution, 17 and SGF-2 T3 1 min rights owner 5 50 ng SGF-2 T0 subject --- 18 SGF-2 T4 2 min 6 25 ng SGF-2 T0 be --- 19 the violate SGF-2 T5 4 min copy of 7 10 ng SGF-2 T0 --- 20 and SGF-2 T6 8 min may documentpublication, 8 5 ng SGF-2It T0 --- exploitation 21 SGF-2 T7 15 min 9 2.5 ng SGF-2 T0 this any --- 22 SGF-2 T8 30 min permission 10 1 ng SGF-2 T0 --- prohibited 23 SGF-2 T9 60 min 11 MWM* ---the be 24 SGF-2 N60 60 min 12 SGF-2 N0 commercial 0 sec 25 SGF-2 P60 60 min 13 SGF-2 Furthermore,P0 any 0 sec 26 MWM* --- Consequently,without

*MWM (molecular weight markers) = Bio-Rad SDS-PAGE Standards, Broad Range

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Figure 31. Digestion of the Cry3Bb1 protein in simulated gastric fluid demonstrated by western blot analysis

Lane: 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 26

kDa 207 119 99

38 and and regime. 29 AG 20

Bayer property protectionpublishing 7 of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document Proteins were separated by SDS-PAGEany using such a 10-20% polyacrylamideits gradient in a Tris-tricine reproductiondocument buffered gel. Proteins were transferred to PVDFowner for westernthis blot analysis.of Immunoreactivity was under of detected by ECL; the above image is rightsa 5 secthe exposure. this documentand/or rights to of fall use of the Lane Description This Incubation Timemay distribution,Laneand Description Incubation Time rights owner 1 MWM* subject --- 14 SGF-2 T0 0 sec 2 10 ng SGF-2 T0 be --- 15the violate SGF-2 T1 15 sec copy of 3 5 ng SGF-2 T0 --- 16 and SGF-2 T2 30 sec may documentpublication, 4 1 ng SGF-2It T0 --- exploitation 17 SGF-2 T3 1 min this 5 0.5 ng SGF-2 T0 any --- 18 SGF-2 T4 2 in permission 6 0.2 ng SGF-2 T0 --- prohibited 19 SGF-2 T5 4 min the 7 0.1 ng SGF-2 T0 commercial --- be 20 SGF-2 T6 8 min 8 0.05 ng SGF-2 T0 --- 21 SGF-2 T7 15 min Furthermore,any 9 0.02 ng SGF-2Consequently, T0 without --- 22 SGF-2 T8 30 min 10 0.01 ng SGF-2 T0 --- 23 SGF-2 T9 60 min 11 MWM* --- 24 SGF-2 N60 60 min 12 SGF-2 N0 0 sec 25 SGF-2 P60 60 min 13 SGF-2 P0 0 sec 26 MWM* ---

*MWM (molecular weight markers) = Bio-Rad Prestained SDS-PAGE Standards, Broad Range

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Figure 32. Digestion of Cry3Bb1 protein in simulated intestinal fluid demonstrated by western blot analysis

Lane: 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 26

kDa 207 119 99

38 regime.and 29 and AG 20

Bayer property protectionpublishing 7 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 Proteins were separated by SDS-PAGE usingowner a 10- 20% polyacrylamidethis of gradient in a Tris-tricine under of buffered gel. Proteins were transferredrights tothe PVDF for western thisblot analysis. Immunoreactivity was documentand/or of rights detected by ECL; the above image tois a 10of secfallond exposure.use The dots (Lanes 1, 10, and 26) the represent the location ofThis the molecular weightmay distribution, markersand on the membrane. rights owner subject be the violate Lane Description Incubationcopy Time of Lane Description Incubation Time 1 MWM* --- and 14 SIF T1 1 min may documentpublication, 2 10 ng SIF T0It --- exploitation 15 SIF T2 5 min this 3 5 ng SIF T0 any --- 16 SIF T3 15 min permission 4 1 ng SIF T0 --- prohibited 17 SIF T4 30 min the 5 0.5 ng SIF T0 commercial --- be 18 SIF T5 1 hr 6 0.2 ng SIF T0 --- 19 SIF T6 2 hr Furthermore,any 7 0.1 ng SIFConsequently, T0 without --- 20 SIF T7 4 hr 8 0.05 ng SIF T0 --- 21 SIF T8 6 hr 9 0.01 ng SIF T0 --- 22 SIF T9 12 hr 10 MWM* --- 23 SIF T10 24 hr 11 SIF N0 0 min 24 SIF N24 24 hr 12 SIF P02 0 min 25 SIF P24 24 hr 13 SIF T0 0 min 26 MWM* ---

*MWM (molecular weight markers) = Bio-Rad Prestained SDS-PAGE Standards, Broad Range

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4.3. Discussion and conclusions

The data and information provided in this section addressed the questions important to an assessment of allergenic potential of a protein. There are no reports of allergies to either of the donor organisms – Agrobacterium or Bacillus thuringiensis species. Thus, CP4 EPSPS and Cry3Bb1 proteins are not from known, commonly allergenic sources. The CP4 EPSPS and Cry3Bb1 proteins represent no more than 0.00144% and 0.01199 % of the total protein in the grain of MON 88017, respectively. Therefore, the CP4 EPSPS and Cry3Bb1 proteins would constitute a very small portion of the total protein present in feed and food derived from MON 88017. Bioinformatics analyses demonstrated that the CP4 EPSPS and Cry3Bb1 proteins do not share structurally or immunologically relevant amino acid sequence similarities with known allergens. Thus, it is highly unlikely that the CP4 EPSPS or Cry3Bb1 proteins contain immunologically cross-reactive allergenic epitopes. Digestive fate experiments conducted with E. coli-produced CP4and EPSPS and and regime. Cry3Bb1 demonstrated that both proteins are rapidlyAG digested in simulated digestive fluids, a characteristic shared among proteins with a history of safe consumption. Thus, it is concluded that MON 88017 does not poseBayer a significant property allergenic protectionpublishing risk. of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as SECTION 5. Safety assessment theof theof CP4 andEPSPS and Cry3Bb1 proteins is regulatory owner. a document any such its reproductiondocument The previous section described an assessmentowner of the potentialthis forof allergenicity of the CP4 under of rightsthe this EPSPS and Cry3Bb1 proteinsdocumentand/or in MON 88017 based on therights sources of the proteins, their to of fall use of proportion of the total protein in MON 88017, a comparisonthe of their sequence to known This may distribution,and allergenic proteins, and their stabilityrights in simulatedowner digestive fluids. In this section, safety assessments of the CP4 EPSPSsubject and Cry3Bb1 proteins are provided, including an be the violate evaluation of the structural copysimilarity of the ofCP4 EPSPS and Cry3Bb1 to known proteins and may documentpublication, and toxins, acute oralIt toxicity by mouseexploitation gavage, the safety of the donor organisms (Agrobacterium and Bacillusthis any thuringiensis, respectively), the similarity of CP4 EPSPS to EPSPSs derived from food sourcespermission with a l ong history of safe consumption, and the prohibited history of safe use of B.t. proteinsthe are discussed. commercial be Furthermore,any 5.1. Safety assessmentConsequently, ofwithout the CP4 EPSPS protein 5.1.a. Structural similarity of CP4 EPSPS to known proteins and toxins

Potential structural similarities shared between the CP4 EPSPS protein and proteins in the ALLPEPTIDES database were evaluated using the FASTA sequence alignment tool. Although the FASTA program directly compares amino acid sequences (i.e., primary protein structure), the alignment data may be used to infer higher order structural similarities (i.e., secondary and tertiary protein structures). Proteins that share a high degree of similarity throughout the entire length are often homologous. Homologous proteins share secondary structure and common three-dimensional folds. Identified proteins were ranked according to their degree of similarity. The most significant

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alignment was to the CP4 EPSPS protein found in genetically enhanced Glycine max (Accession No. AY125353), demonstrating 100.0% identity over a 455 aa overlap window with an E-score of 1.4 e-165. This result was expected as the CP4 EPSPS proteins in Roundup Ready soybean and MON 88017 are identical. All the remaining alignments with significant E-scores (i.e., < 1 e-5) were to other members of the EPSPS protein family and, therefore, do not present a risk of adverse biological activity toward humans and animals.

Potential structural similarities shared between the CP4 EPSPS protein and proteins in the toxin database were also evaluated using the FASTA sequence alignment tool. Identified proteins were ranked according to their degree of similarity. The most significant alignment was to the Bacillus cereus sphingomyelinase c precursor protein (Accession No. P11889), demonstrating only 28.2% identity over a 131 aa overlap window with an – E-score of 0.26. These data demonstrate that the CP4 EPSPS protein is highly unlikely to share any structural homology to any known toxin proteins. and and regime. AG Results of the FASTA sequence alignments demonstrated a lack of structurally relevant similarity between the CP4 EPSPS protein andBayer any knownproperty toxic protection or publishingpharmacologically of contents active proteins relevant to human or animal health. its parties.data therefore and/oror affiliates. 5.1.b. Acute oral toxicity study withproperty the CP4 intellectual EPSPSthird protein may its as the of and is regulatory owner. a document An oral acute toxicity study was conductedany such with E. coli-producedits CP4 EPSPS protein reproductiondocument (Harrison et al., 1996). The E. coli-producedowner CP4 EPSPSthis proteinof has been shown to be under of rightsthe this equivalent to the plant-produceddocumentand/or CP4 EPSPS present in MONrights 88017 (Section 1, Part VI). to of fall use of Acute administration was considered appropriate to assessthe the potential toxicity of CP4 This may distribution,and EPSPS protein because toxic proteinsrights generally actowner via acute mechanisms (Sjobad et al., 1992; Pariza and Foster, 1983).subject The no observed effect level (NOEL) for oral toxicity of be the violate CP4 EPSPS in mice was 572copy mg/kg, the highestof dose tested (Harrison et al., 1996). and may documentpublication, There were no significantIt differencesexploitation in body weight, cumulative body weight, or food consumption between thethis vehicle any or bovine serum albumin protein control groups and the CP4 EPSPS-treated groups. permission prohibited the commercial be Furthermore,any 5.1.c. Safety of theConsequently, donorwithout organism – Agrobacterium sp. strain CP4

Agrobacterium sp. strain CP4 was chosen as the donor organism because this bacterium exhibited tolerance to glyphosate by producing a naturally occurring glyphosate-tolerant EPSPS (Padgette et al., 1996). The bacterial isolate, CP4, was identified by the American Type Culture Collection as an Agrobacterium species. Agrobacterium species are not known human or animal pathogens and are not commonly allergenic (FAO/WHO, 1991). Furthermore, according to FAO/WHO (2001), there is no known population of individuals sensitized to bacterial proteins.

Agrobacterium sp. strain CP4 has been reviewed previously as a part of the safety assessment of the donor organism during Monsanto consultations with the FDA

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regarding Roundup Ready soybean (Glycine max) (1994), canola (Brassica napus) (1995), cotton (Gossypium hirsutum) (1995), NK603 corn (Zea mays) (2000), and sugarbeet line 77 (Beta vulgaris) (1998).

5.1.d. Similarity of CP4 EPSPS to EPSPSs derived from food sources with a long history of safe consumption

The CP4 EPSPS protein present in MON 88017 is substantially similar to EPSPSs consumed in a variety of food and feed sources. As shown in Table 10, the CP4 EPSPS protein is homologous to EPSPSs naturally present in food crops (e.g., soybean and corn) and in microbial food sources such as Baker’s yeast (Saccharomyces cerevisiae), which have a history of safe human consumption (Padgette et al., 1996; Harrison et al., 1996). The similarity of the CP4 EPSPS protein to EPSPSs in a variety of foods and extensive human and animal consumption of the family of EPSPS proteins support the lack of health concerns. and and regime. AG The ubiquitous presence of homologous EPSPS enzymes in plant material used as food and feed establishes that EPSPS proteins and theirBayer enzymeproperty activity protection publishingpose no hazards for of contents humans and animals. Furthermore, the EPA has established an exemptionits from the parties.data therefore requirement of a tolerance for residues of CP4 EPSPS and theand/or geneticor material necessary affiliates. property intellectualthird may for its production in all plants (40 CFR §180.1174).its as the of and is regulatory owner. a document any such its Table 10. Comparison of the deduced amino acidreproduction sequencedocument of native CP4 EPSPS to owner this of that of other EPSPSs under of rightsthe this documentand/or rights to of fall use of Soybean Corn E.the coli S. cerevisiae This may distribution,and rights owner subject(food/ (food/ (present in (Baker’s yeast) be feed) feed)the violatehuman gut) copy of CP4 EPSPS and may documentpublication, % sequence identityIt 26 exploitation24 26 30 this % sequence similarity any51 49 52 54 permission prohibited the commercial be Furthermore,any 5.1.e. Presence ofConsequently, CP4 EPSPSwithout protein in commercial food and feed crops

Herbicide-tolerant crops, primarily those with the Roundup Ready trait, were planted on 49.7 million hectares globally in 2003 (James, 2003b). Roundup Ready soybean is a significant example of the growth of these crops since its introduction in 1996. In the first year of introduction, Roundup Ready soybean was planted on 400,000 hectares in the U.S. (1% of the U.S. soybean area) and has grown to 55% of the 76 million hectares of soybean planted worldwide in 2003. Globally in 2003, Roundup Ready soybean containing the CP4 EPSPS protein was produced on approximately 41.4 million hectares, including almost 13 million hectares in Argentina and half a million hectares in Canada.

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The amino acid sequence of the CP4 EPSPS protein produced in MON 88017 is identical to, or shares greater than 99% sequence identity with, the amino acid sequence of the CP4 EPSPS protein produced in a number of other Roundup Ready crops that have already completed the FDA consultation process and are produced commercially, including soybean, NK603 corn, cotton, and canola. As described above, humans and animals have consumed these crops and/or their processed products, since 1996 (James, 2003b). This demonstrates a history of safe consumption of the CP4 EPSPS proteins present in Roundup Ready crops.

5.2. Safety assessment of the Cry3Bb1 protein

5.2.a. Structural similarity of the Cry3Bb1 to known proteins and toxins

Using the same procedures as described above, potential structural similarities between the Cry3Bb1 protein and other proteins in the ALLPEPTIDES database wereand evaluated and regime. using the FASTA sequence alignment tool. IdentifiedAG proteins were ranked according to their degree of similarity. The best similarity observed was to the Bacillus thuringiensis pesticidal crystal protein Cry3Bb (Accession No.Bayer Q06117 property), an protection isoformpublishing of the Cry3Bb1 of contents protein, demonstrating an overlap of 651 aa, with 99.2% identity andits an E-score of zero, parties.data therefore which was not unexpected. The remaining alignments with significantand/oror E-scores were affiliates. also Cry protein homologues. The Cry3Bb1property proteinintellectualthird produced in MONmay 88017 is a its as member of the Cry3Bb class of proteinthe of variantsand that shares >99% amino acid sequence is regulatory owner. a document identity with the wild type Cry3Bb1any prot suchein contained in the topicallyits applied reproductiondocument commercial microbial product Raven Oil ownerFlowable Bioinsecticide.this of under of rightsthe this documentand/or of rights Potential structural similarities sharedto of betwfall een theuse Cry3Bb1 protein and proteins in the the TOXIN5 database wereThis also evaluatedmay us ingdistribution, theand FASTA sequence alignment tool. rights owner Identified proteins were rankedsubject according to their degree of similarity. The greatest be the violate similarity observed was to thecopy Bacillus thuringiensisof pesticidal crystal protein Cry3Bb and (Accession No. Q06117may ), andocument isoformpublication, of the Cry3Bb1 protein. In this alignment, the It exploitation overlap of 651 aa containedthis noany gaps, showed 99.2% identity, and had an E-score of zero. This alignment is not surprising, becausepermission the Cry3Bb protein is listed within GenBank as prohibited an insecticidal toxin and as suchthe was be in cluded in the TOXIN5 database during its construction. commercial Furthermore,any Consequently,without Results of the FASTA sequence alignments demonstrated the lack of structurally relevant similarity between the Cry3Bb1 protein and any known toxic or pharmacologically active proteins relevant to human or animal health. These data demonstrate the lack of structurally relevant similarities between Cry3Bb1 and toxins or other pharmacologically active proteins that may adversely impact human or animal health.

5.2.b. Acute oral toxicity study with Cry3Bb1 protein

An acute oral toxicity assessment was conducted to evaluate potential adverse clinical signs or detrimental effects on mice from exposure to E. coli-produced Cry3Bb1 protein.

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The results of this study were consistent with the study conducted for the Cry3Bb1 protein variant produced in YieldGard Rootworm, which completed the consultation process with FDA in 2002.

The Cry3Bb1 protein produced in E. coli was shown to be identical to the plant-produced Cry3Bb1 as expressed in MON 88017 (Part VI). Two groups of ten male and female CD1 mice received an acute high dose of the Cry3Bb1 protein by gavage. The target dose of 2,442 mg/kg was based on the maximum attainable Cry3Bb1 concentration of the dosing solution (estimated at 37 mg/mL) and a total dose volume of 66.6 mL/kg body weight. The limited solubility of theCry3Bb1 protein precluded its administration as a single dose. Therefore, dosing was subdivided into two doses of 33.3 mL/kg body weight (66.6 mL/kg total) to achieve the target dose of 2,442 mg/kg. On the day of dosing (Day 0) the two individual doses of 33.3 mg/mL body weight were separated by approximately four hours. A separate group of ten male and ten female protein control animals received bovine serum albumin (BSA) at a dose of 1900 mg/kg. Analysisand of the and regime. dosing solutions revealed that the concentration of Cry3Bb1AG protein was 79% of target and, therefore, the achieved dose was 1930mg/kg. Bayer property protectionpublishing of contents No significant differences were observed in food consumption duringits the study. No parties.data therefore gross pathological findings related to consumption of Cry3Bb1and/or proteinor were observed at affiliates. necropsy. Under the conditions of thisproperty test, nointellectual toxicitythird was observedmay in any of the its as groups. Therefore, the acute oral LDthe 50of of E. coliand-produced Cry3Bb1 protein in mice is is regulatory owner. a document greater than 1930 mg/kg body weight.any suchThis dose was determinedits to be the NOEL. reproductiondocument owner this of under of For comparison, the results of rodentrights acutethe oral toxicity thistests conducted with other Cry3 documentand/or of rights proteins, including the Cry3Bb1to variant of produfall ceduse in YieldGard Rootworm corn (MON the This may distribution,and 863), are summarized in Table 11.rights In each acuteowner test the highest achievable dose level failed to produce evidencesubject of treatment-rela ted adverse effects and was, therefore, be the violate considerate to be a no obsercopyvable effect levelof (NOEL). and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table 11. Acute oral NOELs for various B.t. and plant-produced Cry3 proteins

NOEL Cry3 Protein (mg/kg) a Cry3Bb1 – MON 88017 ≥ 1930

Cry3Bb1 – YieldGard Rootworm corn ≥ 3200

Cry3Bb1 – Wild type present in Raven Oil Flowable Bioinsecticide b ≥ 30

Cry3Aa4 - NewLeaf® potato ≥ 5000

a - NOEL in rodent acute gavage study. In all instances, the highest dose tested was the NOEL. b - Raven contains a mixture of Cry3Bb1, Cry3Aa4 and CrylAc proteins. In the batch tested, and Cry3 proteins constituted 40% (w/w) active ingredient; Cry3Bbland protein representsregime. 66-75% of the Cry3 proteins present in Raven. The highest doseAG tested was 108 CFU/rat, which is approximately a 100 mg/kg body weight dose of total active ingredients. Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. 5.2.c. Safety of the donor organism:property Bacillus intellectual thuringiensisthird may its as the of and is regulatory owner. a document The safety of B.t. as a donor organismany hassuch been previously reviewedits in Monsanto reproductiondocument consultations with FDA for genetically modiownerfied cotton,this corn andof potato products. under of Bacillus thuringiensis subspecies kumamotoensisrightsthe is a spore-forming,this gram-positive documentand/or rights bacterium that is found naturallyto in soil.of fall B.t. strainsuse haveof been used commercially in the the U.S. since 1958 to produceThis microbial-derivedmay distribution,and pr oducts with insecticidal activity (EPA, rights owner 1988). Many strains of B.t.subject have been shown to produce protein crystals or inclusion be the violate bodies that are selectively toxiccopy to certain ordersof and species of insect pests. The current and nomenclature, basedmay on aminodocument acidpublication, identity, allows closely related proteins to be ranked It exploitation together. Each protein is thisassigned any a unique name incorporating four hierarchical ranks consisting (in order) of an Arabic numeral,permission upper case letter, lower case letter, and an Arabic numeral (e.g., Cry3Bb1 protein).prohibited Thus, proteins with identity of the be commercial a) <45% differFurthermore, in aany primary rank (e.g., Cry2, Cry3, etc.), Consequently,without b) >45% but <78% differ in secondary rank (e.g., Cry3A, Cry3B), c) >78% but <95% differ in tertiary rank (e.g., Cry3Ba, Cry3Bb), and d) >95% differ in quaternary rank (e.g., Cry3Bb1, Cry3Bb2) and are considered allelic variants.

In general, the primary rank of the B.t. proteins denotes its specific insecticidal activity; for example, Cry1, Cry2, Cry3, and Cry4 proteins are toxic to Lepidopteran, Lepidopteran/Dipteran, Coleopteran, and Dipteran pests, respectively (Bravo, 1997; Höfte and Whitely, 1989).

® NewLeaf is a registered trademark of Monsanto Technology LLC.

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An exemption from the requirement of a tolerance for the first microbial B.t. product was granted in 1960 by the FDA after an extensive toxicity and infectivity evaluation program. The testing program consisted of acute, subchronic, and chronic studies, which resembled the testing required for conventional chemical pesticides. Registration was granted by the USDA later that same year. In 1971, EPA assumed responsibility for all pesticide tolerance exemptions. Since then, a variety of naturally occurring and genetically modified microbial B.t. products have been registered and included under this tolerance exemption. EPA has established separate tolerance exemptions by amendment for various Cry proteins (e.g., Cry1Ab, Cry1Ac, Cry2Ab2 and Cry3Aa) expressed in genetically-modified food crops (EPA 1995a, 1995b, 1995c, 1996, 1997 and 2001). The conclusion of reasonable certainty of no harm and the resultant tolerance exemptions for this wide array of B.t. mixtures and Cry proteins in food or feed were based on the lack of adverse effects to mammals in numerous toxicological studies. This conclusion is supported by a history of safe use in agriculture for over 40 years (McClintock et al., 1995). There are no adverse effects known to have occurred in humans duringand this and regime. prolonged period of use (EPA, 1998). AG

Bayer property protectionpublishing of contents 5.2.d. Human and animal safety of B.t. Cry proteins its parties.data therefore and/oror affiliates. property intellectualthird may FDA’s statement of policy (FDA, 1992)its and the proposed rule on Plant-Pesticides as Subject to the Federal Insecticide, theFungicide of andand Rodenticide Act (Federal Register is regulatory owner. a document November 23, 1994) stipulate that anyEPA such will exercise regulatory oversightits of plant- reproductiondocument expressed pesticidal proteins and the markersowner used in thethis selectionof of transformed plants. under of rightsthe this On May 11, 2001, a time-limiteddocumentand/or exemption from the requirementrights for a tolerance was to of fall use of established by EPA for the Cry3Bb1 protein and the geneticthe material necessary for its This 1 may distribution,and production in corn (EPA, 2001arights). As part of theowner decision to grant this tolerance exemption, EPA stated: “Thesubject lack of mammalian toxicity at high levels of exposure to be the violate the Cry3Bb1 ... proteins demonstratecopy the safeof ty of the product at levels well above and may documentpublication, maximum exposureIt levels anticipatedexploitation in the crop .” The EPA further stated: “There is a reasonable certainty that nothis harmany will result from aggregate exposure to the U.S. population, including infants and children,permission to the Cry3Bb1 ... proteins and the genetic prohibited material necessary for their production.the This includes all anticipated dietary exposures commercial be and other exposures for which there is reliable information.” Furthermore, no receptors Furthermore,any for Cry proteins haveConsequently, beenwithout identified on intestinal cells of mammals (Noteborn, 1994; Sacchi et al., 1986; Van Mellaert et al., 1988).

5.3. Conclusions

Numerous factors have been considered in the safety assessment of the CP4 EPSPS and Cry3Bb1 proteins that are expressed in MON 88017. An assessment of safety of the CP4 EPSPS leads to the following conclusions, which are consistent with the conclusions reached for the CP4 EPSPS protein produced in a number of Roundup Ready crops:

1 An application to amend the current time-limited tolerance exemption to a permanent tolerance is currently under review by the EPA. A notice of filing published in October 2003 (EPA 2003b).

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a) The donor organism, Agrobacterium sp. strain CP4, is not known for human or animal pathogenicity and is not commonly allergenic. Additionally, Agrobacterium sp. strain CP4 and the CP4 EPSPS protein it produces have been reviewed previously as a part of the safety assessment for other Roundup Ready crops. b) A history of the safe use of CP4 EPSPS protein has been demonstrated, based on the similarity of the CP4 EPSPS protein in MON 88017 to EPSPSs naturally present in food crops (e.g., soybean and corn) and in microbial food sources such as Baker’s yeast (Saccharomyces cerevisiae), and to the CP4 EPSPS protein produced in a number of other Roundup Ready crops that have already completed the FDA consultation process, including soybean, NK603 corn, cotton and canola. c) The CP4 EPSPS protein purified from E. coli was found to be physicochemically and functionally equivalent to the protein produced in MON 88017. d) Large margins of exposure for CP4 EPSPS protein indicate that thereand is virtually and regime. no risk to human and animal health associatedAG with dietary exposure to food and feed products derived from MON 88017. e) Digestibility studies demonstrate that theBayer CP4 EPSPSproperty proteinprotectionpublishing is rapidly degraded of contents in simulated digestive fluids and would be unlikely to elicit potentialits toxic effects. parties.data therefore f) No biologically relevant structural similarities were observedand/oror between the CP4 affiliates. EPSPS protein and pharmacologicallyproperty intellectual activthirde proteins that aremay known to cause its as adverse health effects in humansthe of or animals.and is regulatory owner. a document g) Results from the acute oral anytoxicity such study demonstrate thatits the CP4 EPSPS protein reproductiondocument is not toxic and does not cause anyowner adverse effects.this of under of rightsthe this documentand/or of rights Similarly, analysis of the Cry3Bb1to proteinof fall leads useto the following conclusions, which are the This may distribution,and consistent with the conclusions rightsreached for the Cry3Bb1owner protein produced in YieldGard Rootworm corn: subject be the violate copy of and a) The donor organism,may documentBacilluspublication, thuringiensis, has been used commercially in the It exploitation U.S. since 1958 tothis produce any microbial-derived products with insecticidal activity. The extremely low mammalianpermission toxicity of B.t.-based insecticide products has prohibited been demonstrated in numerousthe be safety studies, and there are no confirmed cases of allergic reactionscommercial to Cry proteins in applicators of microbial-derived B.t. Furthermore,any products duringConsequently, 40without years of use. b) A history of safe use of Cry3Bb1 protein has been demonstrated, based on similarity to the Cry3Bb1 protein produced in YieldGard Rootworm corn (MON 863) and to the wild type Cry3Bb1 protein present in Raven bioinsecticide. The safety and nutritional assessment of the YieldGard Rootworm corn has been previously reviewed by FDA. The conclusion that “corn derived from these new varieties is not materially different in composition, safety and other relevant parameters from corn currently on the market and that genetically modified corn does not raise issues that would require pre-market review or approval by FDA” was reached on December 31, 2001. c) The Cry3Bb1 protein purified from E. coli was found to be physicochemically and functionally equivalent to the protein produced in MON 88017.

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d) Large margins of exposure for the Cry3Bb1 protein indicate that there is virtually no risk to human and animal health associated with dietary exposure to food and feed products derived from MON 88017. e) Digestibility studies demonstrate that the Cry3Bb1 protein is rapidly degraded in simulated digestive fluids and would be unlikely to elicit potential toxic effects. f) No biologically relevant structural similarities were observed between the Cry3Bb1 protein and pharmacologically active proteins that are known to cause adverse health effects in humans or animals. g) Results from the acute oral toxicity study demonstrate that the Cry3Bb1 protein is not toxic and does not cause any adverse effects.

Using the guidance provided by the FDA, a conclusion of “no concern” is reached for the donor organisms and the CP4 EPSPS and Cry3Bb1 proteins. One of the decision trees from the FDA Food Policy (FDA, 1992) is reproduced in Figure 33 and identifies the considerations to be used in evaluating the safety of the donor organism. Theand and regime. information provided in this section and summarizedAG above leads to a finding of “no concerns” for the source of the donor organism. The pathway leading to no concerns for MON 88017 is highlighted with bold arrows. BayerConsiderations property usedprotection publishingin evaluating the of contents safety of the proteins introduced from the donor are identified in Figureits 34, another parties.data therefore decision tree reproduced from the FDA Food Policy (FDA, 1992).and/oror As with the donor, the affiliates. information provided in this section andproperty summarized intellectualthird above leads tomay a finding of “no its as concerns” for the CP4 EPSPS and theCry3Bb1 of proteinsand in MON 88017. The pathway is regulatory owner. a document leading to no concerns for MON 88017any such is highli ghted with bold arrows.its It is therefore reproductiondocument concluded that the data and information prownerovided in Sectionthis 5, ofand supported by other under of data and information in this Part VI,rights demonstratethe that thethis CP4 EPSPS and Cry3Bb1 documentand/or of rights proteins in MON 88017 are safeto fo r ofhuman fall and animaluse consumption. 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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Figure 33. Safety assessment of new varieties: the donor

Is food from the donor Yes commonly allergenic?

No Can it be demonstrated that the allergenic determinant has not been transferred to the new variety of host?

Do characteristics of the donor species, Consult FDA on related species or protocols for and progenitor lines allergenicityand regime. warrant analytical or AG testing and/or toxicological tests? labelling

Bayer property protectionpublishing Yes of contents its parties.data therefore and/oror Do test results affiliates. No property intellectualthird may provide evidence its as that toxicant levels the of and New variety not is No regulatory owner. in the new variety do a documentacceptable any such its not present a safety reproductiondocument concern? owner this of under of rightsthe this documentand/or rights Yes to of fall use of the This may distribution,and rights owner No concernssubject 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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Figure 34. Safety assessment of new varieties: proteins introduced from donor.

Is the newly introduced No protein present in food derived from the plant?

Yes

Is the protein derived from Yes a food source, or No substantially similar to an edible protein?

Is food from the and and regime. donor commonly No AG allergenic? Is the introduced protein Yes Consult Yes reported to be toxic? Bayer property protectionpublishing of FDAcontents its parties.data therefore Can it be No and/oror Yes affiliates. demonstrated property intellectualthird may its that the as Will thethe intake of of the and Does the biological allergenic is regulatory owner. donor protein in new a document function of the determinant has any such its variety be generally reproductiondocument introduced protein not been owner this of comparable to the raise any safety transferred to under ofNo this intake ofrights thethe same or the new variety documentand/or of rights concern, or is the similarto ofprotein fall in use of host? introduced protein donor or other food? the This may distribution,and reported to be toxic? rights owner No subject Yes be the violate No copy of Yes Consult and may documentpublication, FDA It exploitation this anyIs the introduced protein likely to be a permission Ye s Consult macroconstituentprohibited in No concerns No FDA the commercialthe human be or animal diet? Furthermore,any Consequently,without

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PART VII. FOOD/FEED SAFETY AND NUTRITIONAL ASSESSMENT OF MON 88017 CORN

SECTION 1. Corn as the comparable food and feed

Corn, Zea mays L., the host plant, has been a staple of the human diet for centuries. Corn grain and its processed fractions are consumed in a multitude of food and animal feed products. Corn forage is extensively consumed as an animal feed by ruminants. Corn does not contain any known allergens or produce significant quantities of toxins or anti- nutritional factors warranting analytical or toxicological tests (Watson, 1982; White and Pollak, 1995).

and Corn is a highly productive crop, yielding an average of 130 bushelsand perregime. acre in the U.S. during 2002 (NCGA, 2003). Its high yield makes itAG one of the most economical sources of metabolizable energy for feeds, and of starch and sugar for food and industrial Bayer property protectionpublishing products. Approximately two-thirds of the cornof produced in the U.S. contentsis fed to livestock. its Therefore, indirect consumption is much greater than parties.directdata consumption therefore for humans. and/oror affiliates. property intellectualthird may its as the of and SECTION 2. Historical uses ofis corn regulatory owner. a document any such its reproductiondocument owner this of Corn (Zea mays L.) originated in Mexico andunder was grownof as a food crop as early as 2700 rightsthe this documentand/or rights B.C. (Salvador, 1997). It is nowto grown of onfall more usethan of296 million acres globally. The history of corn has been studied extensively and multiplethe hypotheses for its origin and This may distribution,and rights owner parentage have been advancedsubject (Mangelsdor f, 1974). The preponderance of evidence supports the hypothesis bethat corn descended frtheom violateteosinte (Galinat, 1988). Corn is the copy of largest crop grown in the U.S. in terms of acreageand planted and net value. In 2002, its may documentpublication, production coveredIt 79.1 million acresexploitation that yielded 9 billion bushels and had a net value this of $21.2 billion (NCGA, 2003).any permission prohibited the 2.1. History and utilizationcommercial of corn be Furthermore,any The history and developmentConsequently,without of corn has been discussed previously in Part IV, Section 1.3. Corn has been a staple of the human diet for centuries. Corn grain and its processed fractions are consumed in a multitude of food and animal feed products. Corn forage is extensively consumed as an animal feed by ruminants.

2.2. Corn as a food source

In spite of its great value as a source of energy, little whole kernel corn is consumed by humans when compared to corn-based food ingredients (Hodge, 1982; Watson, 1988). The low price and ready availability of processed corn products has resulted in the development of large volume food and industrial uses. Corn is an excellent raw material for the manufacture of starch (Anderson and Watson, 1982). Nearly one quarter of corn

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starch produced is sold as starch products, whereas three quarters of the starch is converted to a variety of sweetener and fermentation products including high fructose corn syrup and ethanol (Watson, 1988; NCGA, 2003; Anderson and Watson, 1982; White and Pollack, 1995). Additionally, corn oil is commercially processed from the germ and accounts for approximately 9% of domestic vegetable oil production (Orthoefer and Sinram, 1987). Each of these materials is a component of many foods including bakery and dairy goods, beverages, confections, and meat products.

2.3. Corn as a feed source

Animal feeding represents the largest use of corn in the U.S. with approximately two- thirds of annual production being fed to cattle, chicken and swine (Hodge, 1982; Perry, 1988; Watson, 1988). Approximately 100 million metric tons of grain is fed to livestock directly as grain. Another 1.5 to 2 million metric tons of wet and dry milling by-products (primarily corn gluten meal and feed) are fed directly or in formulated feedsand (Perry, and regime. 1988). AG

Bayer property protectionpublishing of contents its parties.data therefore SECTION 3. Comparison of the composition and characteristicsand/oror of MON 88017 to affiliates. conventional corn property intellectualthird may its as the of and is regulatory owner. 3.1. Levels of significant nutrients, anti-nutrients,a anddocument secondary metabolites any such its reproductiondocument owner this of under of The composition of forage and grainrights producedthe by MONthis 88017 was evaluated and documentand/or of rights compared to a conventional controlto cornof fallwith similaruse genetic background, as well as the This may distribution,and other commercially available cornrights hybrid s. MONowner 88017 and the conventional control corn were grown at three replicatedsubject field sites in major corn-growing areas of the U.S. be the violate (Iowa, Illinois and Nebraska)copy during the 2002of field season. Four commercially available and corn hybrids were grownmay alsodocument atpublication, each of th e same field sites to provide a total of 12 It exploitation different reference substances.this any At each field site, the test, control and reference seed was planted in a randomized complete blockpermission design with three replicates per block. All the prohibited plants were grown under normalthe agronomic be field conditions for their respective geographic regions. All testcommercial plots received an application of Roundup UltraMAX Furthermore,any herbicide accordingConsequently, to labelwithout directions. Bulk forage samples were harvested at the late dough/early dent stage, and bulk grain samples were harvested when the grain was mature. The materials and methods for the compositional analyses are described in the Appendix IV.

Compositional analyses of the forage samples included proximates (protein, fat, ash, and moisture), acid detergent fiber (ADF), neutral detergent fiber (NDF), minerals (calcium, phosphorous), and carbohydrates by calculation. Compositional analyses of the grain samples included proximates (protein, fat, ash, and moisture), ADF, NDF, total dietary fiber (TDF), amino acids, fatty acids (C8-C22), minerals (calcium, copper, iron, magnesium, manganese, phosphorous, potassium, sodium, and zinc), vitamins (B1, B2, B6, E, niacin, and folic acid), anti-nutrients (phytic acid and raffinose), secondary

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metabolites (furfural, ferulic acid, and p-coumaric acid), and carbohydrates by calculation. In all, 77 different analytical components (nine in forage and 68 in grain) were analyzed. The following 15 compositional analytes with >50% of observations below the limit of quantitation (LOQ) of the assay were excluded from statistical analysis: sodium, 2-furaldehyde, 8:0 caprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:0 myristic acid, 14:1 myristoleic acid, 15:0 pentadecanoic acid, 15:1 pentadecenoic acid, 17:0 heptadecanoic acid, 17:1 heptadecenoic acid, 18:3 gamma linolenic acid, 20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, and 20:4 arachidonic acid. Therefore, 62 components were statistically assessed (nine in forage and 53 in grain) for the compositional equivalence of MON 88017.

Statistical analyses of the compositional data were conducted using a mixed model analysis of variance method. Four sets of analyses were made based on data from each of the three replicated field sites plus data from a combination of all three field sites. Statistical evaluation of the composition data involved comparison of the forageand and and regime. grain from the test substance to the control. StatisticallyAG significant differences were determined at the 5% level of significance (p<0.05). There were 248 comparisons (four sets of analyses x 62 components assessed) conductedBayer propertyon the testprotection material.publishing Using the of contents data for each component obtained from the 12 different commercialits hybrids, a 99% parties.data therefore tolerance interval was calculated to contain, with 95% confidence,and/oror 99% of the values affiliates. contained in the population of commercialproperty corn intellectual hybrids.third For those maycomparisons in which its as the test was statistically different fromthe of the control,and the test range was compared to the is regulatory owner. a document 99% tolerance interval in order to determineany such if the test range was itswithin the interval and reproductiondocument therefore considered to be part of the populationowner of thethis commercial of corn. under of rightsthe this documentand/or of rights Results of the forage and grain sampleto of analysfall is showeduse that there were no statistically the This may distribution,and significant differences (p<0.05)rights between MON 88017owner and the conventional control for 232 of the 248 comparisonssubject conducted (Tables 12-19). There were no statistically be the violate significant differences foundcopy in forage (Tableof 12). For grain, statistically significant and differences were observedmay fordocument thepublication, content of: 16:0 palmitic acid, 18:1 oleic acid, 18:3 It exploitation linolenic acid, 20:0 arachidicthis any acid, copper, methionine, moisture, niacin, and serine (one comparison each); 18:2 linoleic acidpermission (three comparisons); and vitamin B1 (four prohibited comparisons) (Table 19). Five percent,the or approximately 12 (0.05 × 248) comparisons, commercial be were expected to be statistically significant based upon chance alone. Except for vitamin Furthermore,any B1, none of the statisticallyConsequently,without significant differences were in all four analyses (each individual site and the combination of all sites). The vitamin B1 values in grain were statistically lower in MON 88017 compared to conventional corn for all four analyses, but they were similar to literature and historical values of vitamin B1 in corn grain. All test values were also within the 99% tolerance interval for the 16 comparisons observed to be statistically different between MON 88017 and the conventional control. Therefore it is unlikely that these differences are biologically meaningful. The summary of the results is presented in Tables 12 through 19. Literature values and historic ranges for components of corn forage and grain are presented in Table 20. In addition, Tables 1-21 in Appendix IV provide the results of the analysis for each independent site. Supplemental summary information presented in Appendix IV provides calculations of fatty acid composition as percent of dry weight (Table 22), percent of total fat (Table 23),

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and amino acid content calculated as percent of dry weight (Table 24) and percent of total protein (Table 25).

Based on these data, it is concluded that the forage and grain produced from MON 88017 are compositionally equivalent to the forage and grain produced from corn currently on the market.

and and regime. AG

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Table 12. Comparison of proximates, fiber, and mineral content in forage from MON 88017 and conventional corn for combined field sites

Difference (MON 88017 Minus Control) MON 88017 Control Commercial Mean ± S.E. Mean ± S.E. 95% C.I. (Range) a Mean ± S.E. b Component (Units) (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.] and and regime. Ash (% dwt) 3.99 ± 0.24 4.04 ± 0.24 -0.051AG ± 0.28 -0.74,0.64 0.861 (2.62 - 6.78) (3.30 - 5.53) (3.59 - 4.67) (-1.37 – 1.55) [0.72,7.42] Carbohydrates (% dwt) 86.19 ± 0.62 86.48 ± 0.62 Bayer-0.29 ± 0.40property -1.11,0.54publishing 0.478 (81.86 - 89.90) protectioncontents (83.54 - 87.88) (84.43 - 87.71) of (-2.58 – 1.73) [78.70,93.43] its parties.data therefore Fat, total (% dwt) 1.61 ± 0.29 1.65 ± 0.29 -0.039 ± 0.25 and/oror-0.56,0.48 0.878 (0.69 - 2.92) affiliates. (0.80 - 3.13) (0.83 - 2.97)property intellectual(-1.47third – 1.99) may [0.80,2.95] its as the of and Moisture (% fw) 70.86 ± 0.66 70.66is ± 0.66 0.20regulatory ± 0.39 -0.61,1.01owner. 0.615 (65.20 - 78.60) a document (68.50 - 72.70) (69.10 - any72.70) such (-1.40 – 1.90) its [59.37,80.83] reproductiondocument owner this of Protein (% dwt) 8.20 ± 0.31 7.82 ± 0.31 under0.38 ±of 0.25 -0.13,0.88 0.137 (6.31 - 9.96) rightsthe this documentand/or rights (7.44 - 8.97) (6.79to - 8.54)of fall (-0.99use – 1.65)of [4.17,11.81] the ADF (% dwt) 26.54 ± 1.25This 25.45 ± 1.25may distribution,and1.10 ± 1.76 -2.97,5.16 0.549 (19.16 - 35.55) rights owner (24.29 - 29.97) subject(23.34 - 28.13) (-2.58 – 4.08) [13.95,38.96] be the violate NDF (% dwt) 37.34 ± 1.22 copy38.33 ± 1.22 of -0.99 ± 1.42 -3.90,1.91 0.490 (30.27 - 57.93) and (33.44 - 45.05)may (35.86documentpublication, - 41.18) (-4.63 – 6.97) [23.80,54.73] It exploitation Calcium (% dwt) 0.22 ± 0.014 this 0.23any ± 0.014 -0.0092 ± 0.014 -0.044,0.026 0.542 (0.13 - 0.32) (0.19 - 0.26) (0.18 - permission0.31) (-0.054 – 0.024) [0.11,0.32] prohibited the Phosphorus (% dwt) 0.25 ± 0.011 commercial0.25 ± 0.011 be 0.0017 ± 0.013 -0.029,0.032 0.899 (0.16 - 0.31) (0.21 - 0.30) (0.20 - 0.30) (-0.060 - 0.079) [0.095,0.38] Furthermore,any Consequently,without adwt=dry weight; ADF=acid detergent fiber; NDF=neutral detergent fiber; S.E.= standard error of the mean; C.I.= confidence interval; T.I.= tolerance interval bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

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Table 13. Comparison of the amino acid content in grain from MON 88017 and conventional corn for combined field sites

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) a b Component (Units) (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.] Alanine (% total AA) 7.55 ± 0.084 7.55 ± 0.084 -0.0026 ± 0.039 -0.097,0.092 0.949 (7.24 - 8.16) (7.29 - 7.70) (7.34 - 7.79) (-0.19 - 0.18) and [6.66,8.49] and regime. Arginine (% total AA) 4.42 ± 0.11 4.29 ± 0.11 0.13AG ± 0.060 -0.013,0.28 0.066 (3.72 - 5.08) (4.10 - 4.74) (4.01 - 4.63) (-0.12 - 0.36) [3.34,5.67] Bayer property protectionpublishing Aspartic acid (% total AA) 6.22 ± 0.050 6.25 ± 0.050 of -0.032 ± 0.067 -0.20,0.13contents 0.648 (6.18 - 6.81) its parties.data therefore (6.09 - 6.34) (6.04 - 6.45) (-0.34 - 0.18) and/oror [5.77,7.16] affiliates. Cystine (% total AA) 2.14 ± 0.054 2.15 ± 0.054property intellectual-0.013third ± 0.042 -0.098,0.073may 0.766 (1.82 - 2.58) its as (1.93 - 2.26) (1.93 - the2.30) of and(-0.20 - 0.17) [1.46,2.89] is regulatory owner. a document Glutamic acid (% total AA) 20.40 ± 0.18 20.44 ±any 0.18 such -0.036 ± 0.086 its -0.25,0.17 0.686 (19.46 - 21.57) reproductiondocument of (19.80 - 20.87) (19.91 - 20.84) owner (-0.52 - this0.48) [18.01,22.15] under of rightsthe this documentand/or rights Glycine (% total AA) 3.45 ± 0.063 3.45 to± 0.063of fall 0.0061use ±of 0.031 -0.058,0.070 0.844 (3.29 - 4.03) (3.32 - 3.62) (3.18 - 3.61) (-0.081 - 0.19)the [2.81,4.54] This may distribution,and rights owner Histidine (% total AA) 2.99 ± 0.049 subject2.95 ± 0.049 0.032 ± 0.022 -0.023,0.087 0.200 (2.50 - 3.12) be the violate (2.90 - 3.10) copy(2.83 - 3.14) of (-0.056 - 0.10) [2.16,3.60] and may documentpublication, Isoleucine (% total AA) 3.59 ± 0.037It 3.57 ± 0.037exploitation 0.025 ± 0.044 -0.065,0.11 0.577 (3.39 - 3.79) (3.43 - 3.71) this(3.45 any - 3.76) (-0.15 - 0.25) [3.30,3.84] permission Leucine (% total AA) 13.28 ± 0.20 13.31 ± 0.20prohibited -0.037 ± 0.098 -0.28,0.20 0.717 (12.11 - 14.35) the (12.69 - 13.62) commercial(12.76 - 14.11) be (-0.69 - 0.56) [10.72,15.18] a Furthermore,any AA=amino acids; S.E.= standard error of theConsequently, mean; withoutC.I.= confidence interval; T.I.= tolerance interval cWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

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Table 13 (cont). Comparison of the amino acid content in grain from MON 88017 and conventional corn for combined field sites MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. 95% C.I. (Range) a Mean ± S.E. b Component (Units) (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.] Lysine (% total AA) 2.69 ± 0.058 2.66 ± 0.058 0.024 ± 0.047 -0.074,0.12 0.614 (2.44 - 3.27) and (2.42 - 2.87) (2.49 - 2.82) (-0.072 - 0.11) and regime. [2.06,3.73] AG Methionine (% total AA) 1.98 ± 0.059 2.01 ± 0.059 -0.030 ± 0.043 -0.14,0.076 0.515 (1.70 - 2.47) (1.85 - 2.05) (1.83 - 2.20) (-0.15 - 0.12) [1.37,2.60] Bayer property protectionpublishing of contents Phenylalanine (% total AA) 5.18 ± 0.059 5.14 ± 0.059 0.035 ± 0.055 -0.10,0.17its 0.545 (4.82 - 5.39) parties.data therefore (4.97 - 5.31) (5.01 - 5.32) (-0.13 - 0.25) and/oror [4.57,5.71] affiliates. property intellectualthird may Proline (% total AA) 9.39 ± 0.094 9.34 ± 0.094 its 0.046 ± 0.11 -0.18,0.27 0.676 (8.35 - 9.72) as (9.02 - 9.69) (8.85 - the9.80) of (-0.61and - 0.71) [7.60,10.37] is regulatory owner. a document any such its Serine (% total AA) 4.83 ± 0.049 4.91 ± 0.049 -0.081reproduction ± 0.068 document -0.22,0.059 0.244 (4.81 - 5.23) owner this of (4.65 - 5.04) (4.63 - 5.13) under(-0.47 -of 0.42) [4.60,5.43] rightsthe this documentand/or rights Threonine (% total AA) 3.22 ± 0.040 3.25 to± 0.040of fall -0.026use ± 0.045of -0.12,0.067 0.572 (2.96 - 3.55) the (3.10 - 3.38)This (3.06 - 3.37)may distribution,and(-0.25 - 0.24) [2.89,3.84] rights owner Tryptophan (% total AA) 0.54 ± 0.027 subject0.55 ± 0.027 -0.0090 ± 0.018 -0.046,0.028 0.627 (0.44 - 0.83) be the violate (0.48 - 0.60) copy(0.41 - 0.68) of (-0.17 - 0.096) [0.36,0.77] and may documentpublication, Tyrosine (% total AA) 3.35 ± 0.16It 3.43 ± 0.16exploitation -0.079 ± 0.23 -0.61,0.46 0.743 (2.26 - 3.80) (2.35 - 3.66) this(2.58 any - 3.66) (-1.18 - 0.98) [2.62,4.26] permission Valine (% total AA) 4.79 ± 0.039 4.74 ± 0.039prohibited 0.043 ± 0.052 -0.064,0.15 0.414 (4.44 - 5.04) the (4.60 - 4.92) commercial(4.60 - 4.94) be (-0.25 - 0.26) [4.22,5.27]

Furthermore,any aAA=amino acids; S.E.= standard error of theConsequently, mean; withoutC.I.= confidence interval; T.I.= tolerance interval b With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

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Table 14. Comparison of the fatty acid content in grain from MON 88017 and conventional corn for combined field sites

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) a b Component (Units) (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.] 16:0 palmitic (% total 10.24 ± 0.43 11.27 ± 0.43 -1.03 ± 0.60 -2.42,0.37 0.128 (9.29 - 17.81) FA) (10.07 - 10.52) (10.14 - 14.57) (-4.35 - 0.36) and [6.51,16.50] and regime. 16:1 pamitoleic (% total 0.18 ± 0.010 0.18 ± 0.010 -0.0030 AG± 0.0064 -0.019,0.013 0.655 (0.054 - 0.21) FA) (0.16 - 0.21) (0.16 - 0.22) (-0.029 - 0.025) [0.0017,0.28] Bayer property protectionpublishing 18:0 stearic (% total FA) 2.01 ± 0.073 2.07 ± 0.073 of-0.052 ± 0.046 -0.15,0.042contents 0.266 (1.68 - 2.30) its parties.data therefore (1.80 - 2.19) (1.76 - 2.23) (-0.28 - 0.25) and/oror [1.41,2.53] affiliates. 18:1 oleic (% total FA) 22.74 ± 0.23 22.87 ± 0.23property intellectual-0.13third ± 0.24 -0.71,0.46may 0.613 (19.79 - 34.46) its as (22.20 - 23.53) (21.43 - 23.51)the of (-0.94and - 1.13) [9.25,44.14] is regulatory owner. a document 18:2 linoleic (% total FA) 62.85 ± 0.39 61.52 ± 0.39any such 1.34 ± 0.53 its0.093,2.58 0.038 (51.64 - 64.12) reproductiondocument of (61.86 - 63.72) (59.10 - 63.18) owner(-0.64 - 4.19)this [41.22,74.09] under of rightsthe this documentand/or rights 18:3 linolenic (% total 1.21 ± 0.062 1.32 ± to0.062 of fall -0.11use ± 0.077of -0.30,0.079 0.205 (0.84 - 1.91) FA) (1.15 - 1.26) (1.19 - 1.77) (-0.53 - 0.043)the [0.42,1.95] This may distribution,and rights owner 20:0 arachidic (% total 0.37 ± 0.010 subject0.38 ± 0.010 -0.0085 ± 0.0032 -0.015,-0.0019 0.012 (0.36 - 0.45) be the violate FA) (0.35 - 0.39) (0.35copy - 0.41) of(-0.028 - 0.0088) [0.31,0.49] and may documentpublication, 20:1 eicosenoic (% total 0.24 ± 0.0056It 0.25 ± 0.0056exploitation -0.0034 ± 0.0034 -0.010,0.0036 0.323 (0.24 - 0.36) FA) (0.23 - 0.26) this(0.24 any - 0.26) (-0.019 - 0.019) [0.18,0.40] permission 22:0 behenic (% total FA) 0.15 ± 0.0027 0.15 ± 0.0027prohibited -0.0062 ± 0.0038 -0.014,0.0016 0.116 (0.074 - 0.24) the (0.14 - 0.16) (0.14commercial - 0.17) be (-0.018 - 0.014) [0.071,0.25] a Furthermore,any FA=fatty acids; S.E.= standard error of theConsequently, mean; C.I.=without confidence interval; T.I.= tolerance interval bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

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Table 15. Comparison of the mineral content in grain from MON 88017 and conventional corn for combined field sites

MON 88017 Control Difference (MON 88017 Minus Control) Commercial a Component (Units) Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. p- (Range) b (Range) (Range) (Range) (Lower, upper) Value [99% T.I.] 0.0054 ± 0.00035 Calcium (% dwt) 0.0058 ± 0.00035 -0.00040 ± 0.00025 -0.0010,0.00021 0.159 (0.0032 - 0.0060) (0.0047 - 0.0060) (0.0049 - 0.0069) (-0.0013 - -0.00006) and [0.0017,0.0062] and regime. Copper (mg/kg dwt) 1.73 ± 0.086 1.99 ± 0.086 -0.26AG ± 0.12 -0.54,0.016 0.061 (1.01 - 2.34) (1.48 - 2.05) (1.64 - 2.63) (-0.95 - 0.41) [0.17,3.00] Bayer property protectionpublishing Iron (mg/kg dwt) 21.51 ± 0.59 21.84 ± 0.59 of -0.33 ± 0.62 contents-1.60,0.93 0.595 (16.42 - 26.03) its parties.data therefore (20.07 - 22.92) (20.31 - 23.93) (-2.16 - 2.12)and/or or [12.60,31.26] affiliates. Magnesium (% dwt) 0.14 ± 0.0034 0.14 ± 0.0034property intellectual-0.0022third ± 0.0044 may-0.011,0.0069 0.618 (0.10 - 0.14) its as (0.13 - 0.15) (0.13 the- 0.16) of and(-0.024 - 0.018) [0.088,0.16] is regulatory owner. a document Manganese (mg/kg dwt) 9.72 ± 0.38 9.37 ±any 0.38 such 0.35 ± 0.38 its -0.57,1.27 0.384 (4.96 - 9.81) reproductiondocument of (9.01 - 10.76) (7.55 - 10.44) owner (-0.39this - 1.56) [2.45,10.60] under of rightsthe this documentand/or rights Phosphorus (% dwt) 0.39 ± 0.010 0.39to ± 0.010of fall use-0.0042 of ± 0.013 -0.032,0.023 0.754 (0.28 - 0.41) (0.37 - 0.41) (0.36 - 0.43) (-0.052the - 0.042) [0.24,0.44] This may distribution,and rights owner Potassium (% dwt) 0.41 ± 0.012 subject0.42 ± 0.012 -0.0063 ± 0.012 -0.030,0.018 0.592 (0.29 - 0.43) be the violate (0.39 - 0.44) copy(0.38 - 0.47) of (-0.052 - 0.037) [0.27,0.48] and may documentpublication, Zinc (mg/kg dwt) 24.53 ± 0.98It 24.92 ±exploitation 0.98 -0.39 ± 0.62 -1.67,0.89 0.534 (17.15 - 26.18) (22.31 - 27.27) this(22.02 any - 27.18) (-3.87 - 1.90) [13.42,31.37]

permission prohibited adwt=dry weight; S.E.= standard error of the mean; C.I.= confidence interval; T.I.= tolerance interval the be bWith 95% confidence, interval contains 99% of the valuescommercial expr essed in the population of commercial lines. Negative limits were set to zero. Furthermore,any Consequently,without

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Table 16. Comparison of the proximates and fiber content in grain from MON 88017 and conventional corn for combined field sites

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) a b Component (Units) (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.] and Ash (% dwt) 1.54 ± 0.077 1.59 ± 0.077 -0.049 ± 0.087 and -0.23,0.13regime. 0.573 (1.04 - 1.86) (1.31 - 1.68) (1.23 - 1.97) (-0.45 AG- 0.43) [0.94,1.73] Carbohydrates (% dwt) 82.32 ± 0.40 82.33 ± 0.40 -0.019 ± 0.25 -0.62,0.58 0.940 (81.46 - 86.68) Bayer property protectionpublishing (81.61 - 83.39) (80.67 - 83.62) of(-1.39 - 0.94) contents [79.39,89.67] its parties.data therefore Fat, total (% dwt) 3.64 ± 0.13 3.79 ± 0.13 -0.16 ± 0.080 and/oror-0.35,0.041 0.100 (2.38 - 4.43) affiliates. (3.44 - 3.96) (3.53 - 4.36)property (-0.63intellectualthird - 0.15) may [0.74,6.01] its as Moisture (% fw) 11.10 ± 0.99 11.60 ± 0.99the of -0.49and ± 0.35 -1.36,0.37 0.212 (9.15 - 14.90) is regulatory owner. a document (9.03 - 13.20) (9.73 - 14.20)any such (-1.10 - -0.10) its [4.67,17.56] reproductiondocument owner this of Protein (% dwt) 12.51 ± 0.35 12.28 ± 0.35 under0.23 ±of 0.24 -0.36,0.82 0.379 (9.26 - 13.37) rightsthe this (11.63 - 13.00) document(11.22and/or - 13.82) (-0.82 - 1.37) rights [6.20,15.35] to of fall use of ADF (% dwt) 3.77 ± 0.16 3.54 ± 0.16 0.23 ± 0.18the -0.13,0.59 0.203 (2.39 - 4.89) This may distribution,and rights owner (3.31 - 4.40) subject(2.97 - 4.69) (-0.62 - 1.16) [1.89,5.23] be the violate NDF (% dwt) 12.44 ± 0.62 11.87copy ± 0.62 of 0.57 ± 0.50 -0.66,1.79 0.299 (8.41 - 16.54) and (10.99 - 13.58)may (10.38document -publication, 14.29) (-1.21 - 2.64) [3.51,21.65] It exploitation TDF (% dwt) 16.24 ± 0.71 this15.40 any ± 0.71 0.84 ± 0.96 -1.51,3.20 0.414 (11.80 - 23.04) (13.57 - 18.64) (13.18 - 17.84)permission (-2.39 - 4.19) [5.72,27.10] prohibited the commercial be aADF=acid detergent fiber; NDF=neutral detergent fiber; TDF=total dietary fiber; S.E.= standard error of the mean; C.I.= confidence interval; T.I.= tolerance interval Furthermore,any Consequently,without bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

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Table 17. Comparison of the vitamin content in grain from MON 88017 and conventional corn for combined field sites

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) a b Component (Units) (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.] and Folic acid (mg/kg dwt) 0.48 ± 0.021 0.48 ± 0.021 0.0012 ± 0.030 and -0.072,0.075regime. 0.969 (0.28 - 0.61) (0.38 - 0.60) (0.42 - 0.59) (-0.074AG - 0.11) [0.12,0.77] Niacin (mg/kg dwt) 20.94 ± 1.20 21.75 ± 1.20 -0.81 ± 0.42 -1.67,0.050 0.063 (14.11 - 27.77) Bayer property protectionpublishing (17.04 - 24.14) (19.08 - 23.92) of (-2.04 - 0.23) contents [3.19,34.49] its parties.data therefore Vitamin B1 (mg/kg dwt) 2.47 ± 0.14 3.24 ± 0.14 -0.77 ± 0.12 and/oror-1.06,-0.48 <0.001 (2.69 - 3.73) affiliates. (2.30 - 2.69) (2.99 - 3.60)property intellectual(-1.02third - -0.35) may [1.96,4.38] its as Vitamin B2 (mg/kg dwt) 1.10 ± 0.041 1.13 ± the0.041 of -0.025and ± 0.037 -0.12,0.066 0.524 (0.88 - 1.32) is regulatory owner. a document (0.98 - 1.22) (0.99 - any1.33) such (-0.17 - 0.14) its [0.67,1.51] reproductiondocument owner this of Vitamin B6 (mg/kg dwt) 7.16 ± 0.22 7.10 ± 0.22 under0.063 ±of 0.28 -0.59,0.72 0.828 (4.93 - 7.24) rightsthe this (6.57 - 8.06) document(5.65and/or - 8.54) (-1.27 - 2.40) rights [4.29,7.84] to of fall use of Vitamin E (mg/kg dwt) 14.15 ± 1.70 14.07 ± 1.70 0.070 ± 1.46the -2.93,3.07 0.962 (8.09 - 21.97) This may distribution,and rights owner (6.08 - 16.93) subject(1.74 - 17.77) (-11.15 - 14.39) [0,29.69] be the violate a copy of dwt=dry weight; Vitamin B1 =Thiamine; Vitamin B2 =Riboflavin; Vitaminand B6 =Pyridoxine; S.E.= standard error of the mean; C.I.= confidence may documentpublication, interval; T.I.= tolerance interval It exploitation bWith 95% confidence, interval contains 99% of the thisvalues 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 18. Comparison of the secondary metabolites and anti-nutrients content in grain from MON 88017 and conventional corn for combined field sites

Difference (MON 88017 Minus Control) MON 88017 Control 95% CI Commercial a Component (Units) Mean ± S.E. Mean ± S.E. Mean ± S.E. (Lower, (Range) and b (Range) (Range) (Range) and upper)regime. p-Value [99% T.I.] AG Ferulic acid 2175.34 ± 46.31 2121.05 ± 46.31 54.29 ± 49.66 - 0.283 (1717.17 - 2687.57) (1986.75 - 2275.48) (1927.55 - 2339.71) Bayer(-200.92 property - 347.92) protection publishing47.14,155.72 [1415.19,3173.90] (µg/g dwt) of contents its parties.data therefore p-Coumaric acid 169.26 ± 7.26 154.83 ± 7.26 14.43 ± 9.88 and/oror -9.75,38.61 0.194 (152.30 - 319.15) affiliates. (µg/g dwt) (148.45 - 215.25) (141.41 - 173.24)property intellectual(-14.72third - 72.55) may [43.13,384.34] its as Phytic acid (% dwt) 0.95 ± 0.043 0.89 ± the0.043 of and0.058 ± 0.056 -0.058,0.17 0.309 (0.45 - 1.00) is regulatory owner. (0.83 - 1.05) (0.72 - 1.03) a(-0.15 - 0.24)document [0.28,1.12] any such its reproductiondocument of Raffinose (% dwt) 0.17 ± 0.013 0.17 ± 0.013 owner 0.00080this ± 0.0081 -0.019,0.021 0.924 (0.073 - 0.22) under of rightsthe this (0.14 - 0.20) document(0.14and/or - 0.23) (-0.035of - 0.036)rights [0,0.32] to of fall use the a This may distribution,and dwt=dry weight; S.E.= standard error of the mean; C.I.= confidencerights interval;owner T.I.= tolerance interval bWith 95% confidence, interval contains 99% of the valuessubject expressed in the population of commercial lines. Negative limits were set to zero. 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 19. Summary of statistically significant differences in composition of MON 88017 and conventional corn

Tissue/Site/ Mean Mean Mean Diff. (% of Signif. MON 88017 99% Tolerance Component (Units)a MON 88017 Control Control Value) (p-value (Range) Intervalb Grain IA and and regime. 16:0 palmitic (% total FA) 10.16 12.94 -21.50 AG 0.029 (10.11-10.23) [6.51, 16.50] 18:2 linoleic (% total FA) 63.25 60.41 4.70 0.017 (62.73-63.72) [41.22, 74.09] Bayer property protectionpublishing 18:3 linolenic (% total 1.25 1.57 -20.26of 0.036 contents(1.24-1.26) [0.42, 1.95] its parties.data therefore FA) and/oror affiliates. property intellectualthird may Methionine (% total AA) 2.02 2.16 its -6.39 <0.001 (1.96-2.05) [1.37, 2.60] as the of and is regulatory owner. Moisture (% fw) 9.38 9.93 -5.54a document 0.034 (9.03-9.70) [4.67, 17.56] any such its reproductiondocument of Vitamin B1 (mg/kg dwt) 2.54 3.07 owner -17.37 this <0.001 (2.42-2.65) [1.96, 4.38] under of IL rights the this documentand/or rights to of fall use of 18:1 oleic (% total FA) 22.53 23.29 -3.26 the <0.001 (22.50-22.56) [9.25, 44.14] This may distribution,and rights owner 18:2 linoleic (% total FA) 63.11 subject 62.15 1.55 0.003 (62.84-63.29) [41.22, 74.09] be the violate copy of Niacin (mg/kg dwt) 21.10 22.52 -6.30and 0.014 (20.39-21.52) [3.19, 34.49] may documentpublication, It exploitation Vitamin B1 (mg/kg dwt) 2.30 this 3.10 any -25.63 <0.001 (2.30-2.30) [1.96, 4.38] NE permission prohibited the Copper (mg/kg dwt) 1.57 commercial 2.21 be -28.80 0.023 (1.48-1.68) [0.17, 3.00] Serine (% total AA) 4.80Furthermore, any 4.97 -3.37 0.042 (4.80-4.81) [4.60, 5.43] Consequently,without Vitamin B1 (mg/kg dwt) 2.58 3.56 -27.53 <0.001 (2.47-2.69) [1.96, 4.38] adwt=dry weight; fw=fresh weight; AA=amino acids; FA=fatty acids; bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

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Table 19 (cont). Summary of statistically significant differences between MON 88017 and conventional corn

Tissue/Site/ Mean Mean Mean Diff. (% of Signif. MON 88017 99% Tolerance Component (Units)a MON 88017 Control Control Value) (p-value) (Range) Intervalb Grain and and regime. Combination of all sites AG

18:2 linoleic (% total FA) 62.85 61.52 2.17Bayer property 0.038protectionpublishing (61.86-63.72) [41.22, 74.09] of contents its 20:0 arachidic (% total 0.37 0.38 -2.24 parties.data 0.012 therefore(0.35-0.39) [0.31, 0.49] FA) and/oror affiliates. property intellectualthird may its Vitamin B1 (mg/kg dwt) 2.47 3.24 as -23.72 <0.001 (2.30-2.69) [1.96, 4.38] the of and is regulatory owner. a documentits a any such reproduction dwt=dry weight; fw=fresh weight; AA=amino acids; FA=fatty acids; this documentof b owner With 95% confidence, interval contains 99% of the values expressed inunder the population of of commercial lines. Negative limits were set to zero. 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 20. Literature and historical ranges of components of corn forage and grain

Tissue/Component1 Literature Range2 Historical Range3 Forage Proximates (% dwt) Ash 2.43-9.64a; 2-6.6b 2.03-8.23 Carbohydrates 83.2-91.6b; 76.5-87.3a 80.6-90.8 Fat, total 0.35-3.62b; 1.42-4.57a 0.61-4.02 Moisture (% fw) 56.5-80.4a;55.3-75.3b 42-78.8 Protein 4.98-11.56a 3.86-11.0

Fiber (% dwt) Acid detergent fiber (ADF) 18.3-41.0b; 17.5-38.3a 17.6-36.7 b a and Neutral detergent fiber 26.4-54.5 ; 27.9-54.8 and 29.6-55.2regime. (NDF) AG

Bayer property protectionpublishing Minerals (% dwt) of contents b its Calcium 0.0969-0.3184 parties.data 0.0866-0.2754therefore b and/oror Phosphorous 0.1367-0.2914affiliates. 0.1602-0.2914 property intellectualthird may its as the of and Grain is regulatory owner. a document Proximates (% dwt) any such its reproductiondocument of Ash 1.1-3.9downer; 0.89-6.28 b this 0.81-3.09 under of this and/orrights the b a Carbohydrates document 77.4-87.2to of fall; 82.2-88.1 of rights 79.8-89.6 d use b Fat, total 3.1-5.7 ; 2.48-4.81 the 1.74-4.83 This mayd distribution,and b Moisture (% fw) rights7-23 ; 8.18-26.2owner 6.07-24.7 subject d c Protein be 6-12 ; 9.7-16.1the violate 6.15-14.8 copy of and may documentpublication, Fiber (% dwt) It exploitation this Acid detergent fiber (ADF) any 3.3-4.3d; 2.46-11.34a,b 2.3-9.33 permissiond b Neutral detergent fiber 8.3-11.9prohibited; 7.58-15.91 6.88-18.1 the (NDF) commercial be h Total dietary fiberFurthermore, (TDF)any 10.99-11.41 - Consequently,without 1 fw=fresh weight; dw=dry weight; Vitamin B1 =Thiamine; Vitamin B2 =Riboflavin; Vitamin B6 =Pyridoxine 2Literature range references: aRidley et al., 2002. bSidhu et al., 2000. cJugenheimer, 1976. dWatson, 1987. eWatson, 1982. fClassen et al., 1990. gDowd and Vega, 1996. hChoi et al., 1999. 3 Historical range is from control samples analyzed in previous Monsanto Company studies. Conversions: % dw x 104 = µg/g dw; mg/g dw x 103 = mg/kg dw; mg/100g dw x 10 = mg/kg dw

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Table 20 (cont). Literature and historical ranges of components of corn forage and grain

Tissue/Component1 Literature Range2 Historical Range3 Grain Minerals Calcium (% dwt) 0.01-0.1d 0.0024-0.0089 Copper (mg/kg dwt) 0.9-10d 0.98-3.43 Iron (mg/kg dwt) 1-100d 10.4-30.7 Magnesium (% dwt) 0.09-1d 0.082-0.16 Manganese (mg/kg dwt) 0.7-54d 3.2-9.89 Phosphorous (% dwt) 0.26-0.75d 0.24-0.46 Potassium (% dwt) 0.32-0.72d 0.29-0.53 d and Zinc (mg/kg dwt) 12-30 and 14.1-37.2regime. AG Amino Acids (% total protein) (% total amino acid) Bayere property protectionpublishing Alanine 6.4-9.9of 7.06-8.19contents e its Arginine 2.9-5.9 parties.data 3.49-5.48therefore e and/oror Aspartic acid 5.8-7.2affiliates. 5.97-7.36 property intellectuale third may Cystine 1.2-1.6its 1.61-2.94 as the of ande Glutamic acid is 12.4-19.6 regulatory 17.3-20.4owner. e a document Glycine any such2.6-4.7 3.22-4.91its reproductiondocument of Histidine 2.0-2.8owner e this 2.46-3.35 under of this and/orrights the e Isoleucine document to of 2.6-4.0fall of rights 2.95-4.08 e use Leucine 7.8-15.2 the 11.2-14.6 This may distribution,ande Lysine rights 2.0-3.8 owner 2.35-4.18 subject e Methionine be 1.0-2.1the violate 1.61-2.89 copy of e Phenylalanine 2.9-5.7and 4.6-5.76 may documentpublication, e Proline It exploitation6.6-10.3 8.03-9.9 this Serine any 4.2-5.5e 3.45-5.63 permission e Threonine prohibited2.9-3.9 2.87-4.01 the e Tryptophan commercial be 0.5-1.2 0.39-1.04 e Tyrosine Furthermore,any 2.9-4.7 1.93-4.32 Valine Consequently,without 2.1-5.2e 3.94-5.46

1 fw=fresh weight; dw=dry weight; Vitamin B1 =Thiamine; Vitamin B2 =Riboflavin; Vitamin B6 =Pyridoxine 2Literature range references: aRidley et al., 2002. bSidhu et al., 2000. cJugenheimer, 1976. dWatson, 1987. eWatson, 1982. fClassen et al., 1990. gDowd and Vega, 1996. hChoi et al., 1999. 3 Historical range is from control samples analyzed in previous Monsanto Company studies.

Conversions: % dw x 104 = µg/g dw; mg/g dw x 103 = mg/kg dw; mg/100g dw x 10 = mg/kg dw

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Table 20 (cont). Literature and historical ranges of components of corn forage and grain

Tissue/Component1 Literature Range2 Historical Range3 Grain Fatty Acids (% total fat) (% total fatty acid) 16:0 Palmitic 7-19e 8.41-12.5 16:1 Palmitoleic 1e 0.05-0.18 18:0 Stearic 1-3e 1.33-2.61 18:1 Oleic 20-46e 20.1-37.7 18:2 Linoleic 35-70e 48.0-66.1 18:3 Linolenic 0.8-2e 0.74-1.45 20:0 Arachidic 0.1-2e 0.31-0.56 and 20:1 Eicosenoic - and 0.15-0.44regime. 22:0 Behenic - AG 0.075-0.3

Bayer property protectionpublishing Vitamins (mg/kg dwt)of contents d its Folic acid 0.3 parties.data 0.33-0.75 µthereforeg/g dwt d and/oror Niacin 9.3-70affiliates. - property eintellectual third may Vitamin B1 its3-8.6 0.2-0.33 mg/100g dwt as the of ande Vitamin B2 is 0.25-5.6 regulatory 0.83-1.74owner. µg/g dwt a document any suchd e its Vitamin B6 5.3 ; 9.6 reproductiondocument of - ownere d this Vitamin E 3-12.1 ; under17-47 of this0.005-0.037 mg/g dwt and/orrights the document to of fall of rights 1 use fw=fresh weight; dw=dry weight; Vitamin B1 =Thiamine; Vitaminthe B2 =Riboflavin; Vitamin B6 This may distribution,and =Pyridoxine rights owner subject 2Literature range references: aRidley et al., 2002. bSidhu et al., 2000. cJugenheimer, 1976. be the violate dWatson, 1987. eWatson, 1982.copy fClassen et al., 1990.of gDowd and Vega, 1996. hChoi et al., 1999. 3 and Historical range is frommay controldocument samplespublication, analy zed in previous Monsanto Company studies. It exploitation this any 4 3 Conversions: % dw x 10 = µg/g dw; permissionmg/g dw x 10 = mg/kg dw; mg/100g dw x 10 = mg/kg dw prohibited the commercial be Furthermore,any Consequently,without

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3.2. Levels of naturally occurring toxicants and anti-nutrients

According to OECD, “in considering the anti-nutrients and natural toxins in maize, only phytic acid is significant to the animal feed” (OECD, 2002). Levels of anti-nutrients (phytic acid and raffinose) and secondary metabolites (ferulic acid and p-coumaric acid) were measured in grain of MON 88017 and compared to conventional corn (Table 18). No statistically significant differences were observed. Therefore, it is concluded that the levels of anti-nutrients and secondary metabolites in MON 88017 are comparable to those found in conventional corn.

3.3. Any intended changes to the composition of food and feed

There have been no intended changes to the composition of food and feed derived from and MON 88017 compared to conventional corn. Introduction ofand the cp4 epspsregime. and cry3Bb1 coding sequences and the expressed CP4 EPSPS andAG Cry3Bb1 proteins did not alter the compositional profile of MON 88017 compared to conventional corn. No changes other Bayer property protectionpublishing than intended traits that confer tolerance to Roundupof agricultural herbicidescontents and corn rootworm protection have been observed. its parties.data therefore and/oror affiliates. property intellectualthird may its as SECTION 4. Other informationthe relevant of toand the safety and nutritional assessment of is regulatory owner. a document MON 88017 any such its reproductiondocument of owner this under of rightsthe this The long history of safe documentconsumptionand/or of corn as food and feed,rights combined with the to of fall use of demonstration of the compositional equivalence of MONthe 88017 to conventional corn, is This may distribution,and considered sufficient to supportrights the safety and nutritionalowner assessment of MON 88017. subject be the violate copy of and may documentpublication, SECTION 5. SubstantialIt equivalenceexploitation of MON 88017 to conventional corn this any permission The data and information presented inprohibited Part VII, Section 3 establish that MON 88017 corn the and the food and feed derivedcommercial from beit are compositionally equivalent to conventional corn and the comparable foods/feeds derived from it. Further, the compositional data support Furthermore,any the conclusions derivedConsequently, fromwithout the agronomic/phenotypic studies presented to USDA- APHIS where no biologically meaningful changes were associated with MON 88017, including insect and disease susceptibility, growth, reproduction, and seed dormancy, and insect and disease susceptibility.

A detailed nutritional assessment of forage and grain from MON 88017 statistically compared the levels of 62 components (nine in forage and 53 in grain) to the conventional control and reference corn. Results of the forage and grain analysis showed that there were no statistically significant differences (p<0.05) between MON 88017 and the conventional control for 232 of the 248 comparisons conducted. There were no statistically significant differences found in forage. For grain, statistically significant differences were observed for 16 comparisons between MON 88017 and conventional

Monsanto Company 04-CR-109F Page 134 of 223

corn. However, all of the test values were within the 99% tolerance interval. Therefore it is unlikely that these differences are biologically meaningful. It is concluded, based on these data, that the forage and grain produced from MON 88017 are compositionally equivalent to the forage and grain produced from conventional corn currently on the market.

Conclusions

Collectively, these data and a history of safe use of the host organism, corn, as a common source of human food and animal feed, support a conclusion of “no concerns” for every criterion specified in the flowcharts outlined in the FDA’s Food Policy document (Figure 35). Corn MON 88017 is not materially different in composition, safety or agronomic characteristics from conventional corn other than its tolerance to Roundup agricultural herbicides and protection from corn rootworm feeding damage, the intended traits. Sales and consumption of corn grain and processed products derived from MON 88017and would and regime. be fully consistent with the FDA’s Food Policy, theAG Federal Food, Drug and Cosmetic Act, and current practices for the development and introduction of new corn varieties. 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

Monsanto Company 04-CR-109F Page 135 of 223

Figure 35. Safety assessment of new varieties: the host plant

Does the host species have a history of safe use? Yes

Do characteristics of the host species, related species, or No * No progenitor lines warrant analytical or toxicological tests?

Yes and and regime. AG Do test results provide evidence that toxicant Bayer property Newpublishing variety not levels in the new plant No protectioncontents variety do notof present acceptable its a safety concern? parties.data therefore and/oror affiliates. property intellectualthird may its as the of Yes and is regulatory owner. a document any such its reproductiondocument Is the concentrationowner this of and bioavailabilityunder of this and/orrights the documentimportant to fall nutrients inof rights of use Consult No concerns Yes the new variety within theNo This the rangemay distribution, ordinarilyand FDA rightsseen in the hostowner subject species? be the violate copy of document and may publication,exploitation It this any permission prohibited the be commercial Furthermore,any Consequently,without

Monsanto Company 04-CR-109F Page 136 of 223

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Reidell, W.E. 1990. Rootworm and mechanical damage effects on root morphology and water relations in maize. Crop Sci. 30:628-631.

Ridley, W.P., R.S. Sidhu, P.D. Pyla, M.A. Nemeth, M.L. Breeze, and J.D. Astwood. 2002. Comparison of the nutritional profile of glyphosate-tolerant corn event NK603 with that of conventional corn (Zea mays L.). J. Agric. Food Chem. 50(25): 7235-7243.

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References (cont.)

Romano, C.P. 2002. Expression of Cry3B insecticidal proteins in plants. U.S. Patent 6,501,009.

Sacchi, V.F., P. Parenti, G.M. Hanozet, B. Giordana, P. Luthy and M.G. Wolfersberger. 1986. Bacillus thuringiensis toxin inhibits K+ gradient-dependent amino acid transport across the brush border membrane of Pieris brassicae midgut cells. Fed. European Biol. Soc. Lett. 204:213-218.

Salvador, R.J. 1997. Maize. In The Encyclopedia of Mexico: History, Society , and Culture. Fitzroy Dearborn Publishers, Chicago, Illinois.

Sambrook, J., and D. Russell (eds.). 1989. Molecular Cloning: A Laboratoryand Manual. and regime. Cold Spring Harbor Laboratory Press. Cold SpringAG Harbor, New York.

SAS Software Release 8.1 (TS1M0). 1999-2000.Bayer SASproperty Institute protection Inc.,publishing Cary, North of contents Carolina. its parties.data therefore and/oror affiliates. Schnepf, E., N. Crickmore, J. Van Rie,property D. Lereclus, intellectualthird J. Baum, J. Feitelson,may D.R. Zeigler, its as and D. H. Dean. 1998. Bacillus thuringiensisthe of andand its pesticidal crystal proteins. is regulatory owner. a document Microbiol. Molec. Biol. Rev. 62:775-806.any such its reproductiondocument owner this of under of Shull, G.H. 1909. A pure line methodrightsthe of corn breeding.this Am. Breeders Assoc. Rep. 5:51- documentand/or of rights 59 to of fall use the This may distribution,and rights owner Sidhu, R.S., B.G. Hammond,subject R.L. Fuchs, J.-N. Mutz, L.R. Holden, B. George, and T. be the violate Olson. 2002. Glyphosate-tolerantcopy corn: the ofco mposition and feeding value of grain from and glyphosate-tolerant maycorn to thatdocument ofpublication, conventional corn (Zea mays L.). J.Agric Food Chem. It exploitation 48(6): 2305-2312. this any permission prohibited Sjoblad, R.D., J.T. McClintock theand beR. Engler. 1992. Toxicological considerations for protein components of biologicalcommercial pesticide products. Regulatory Toxicol. Pharmacol. Furthermore,any 15:3-9. Consequently,without

Sleisenger, M.H. and J.S. Fordran. 1989. Pp 685-689. In Gastrointestinal disease. Pathophysiology Diagnosis Management. Fourth Ed. Vol 1. Sleisenger, M.H. and J.S. Fordran (Eds). W.B. Saunders Company.

Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J.Mol. Biol. 98:503-517.

Speek, A.J., J. Schijver, and W.H.P. Schreurs. 1985. Vitamin E composition of some seed oils as determined by high-performance liquid chromatography with fluorometric detection. J Food Sci 50(1): 121-124.

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Spike, B.P. and J.J. Tollefson. 1991. Yield response of corn subjected to western corn rootworm (Coleoptera: Chrysomelidae) infestation and lodging. J. Econ. Entomol. 84:1585-1590.

Stalker, D.M., C.M. Thomas, and D.R. Helinski. 1981. Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2. Mol. Gen. Genetics. 181: 8-12.

Steinrucken, H. and N. Amrhein. 1980. The Herbicide Glyphosate is a Potent Inhibitor of 5-Enolypyruvylshikimic acid-3-phosphate Synthase. Biochem. Biophys. Res. Commun. 94(No.4):1207-1212 and and regime. Sutcliffe, J.G. 1978. Complete nucleotide sequenceAG of the Escherichia coli plasmid pBR322. Symposia on Quantitative Biology. 43: 77-90. Bayer property protectionpublishing of contents Thomas, K., M. Aalbers, G.A. Bannon, M. Bartels, R.J. Dearman, D.J.its Esdaile, T.J. Fu, parties.data therefore C.M. Glatt, N. Hadfield, C. Hatzos, S.L. Hefle, J.R. Heylins, R.E.and/oror Goodman, affiliates. B. Henry, C. Herouet, M. Holsapple, propertyG.S. Ladics, intellectual thirdT.D. Landry, S.C.may MacIntosh, E.A. its as Rice, L.S. Privalle, H.Y. Steiner, R.the Teshim of a, R.and van Ree, M. Woolhiser, J. Zawodny. is regulatory owner. a document 2004. A multi-laboratory evaluationany of such a common in vitro pepsinits digestion assay reproductiondocument protocol used in assessing the safety of novelowner proteins. this Regulatory of Toxicology and under of Pharmacology, 39: (in press) rightsthe this documentand/or of rights to of fall use the This may distribution,and Tilch, C. and P.S. Elias. 1984. rightsInvestig ation of theowner mutagenicity of ethylphenyl- glycidate. Mutation Researchsubject 138:1-8. be the violate copy of and USDA Agriculture Handbookmay document No.publication, 379 1970. Forage Fiber Analysis. In Agricultural It exploitation Handbook No. 379. Unitedthis Statesany Department of Agriculture: Washington, D.C. permission prohibited USDA Agriculture Handbook No.the 74be 1973. Energy Value of Foods. In Agricultural Handbook No. 74. Unitedcommercial States Department of Agriculture: Washington, D.C.; pp. 2-11. Furthermore,any Consequently,without USDA-NASS, 2004. http://www.usda.gov/nass/pubs/pubs.htm

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Van Mellaert, H., J. Van Rie, C. Hofmann, and A. Reynaerts. 1988. Conference On Biotechnology, Biological Pesticides and Novel Plant-Pest Resistance for Insect Pest Management, Boyce Thompson Institute, Cornell University, New York.

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Watson, S.A. 1982. Corn: Amazing Maize. General Properties. Pp 3-29. In CRC Handbook of Processing and Utilization in Agriculture, Vol. II: Part 1 Plant Products. Wolff, I.A. (ed.). CRC Press, Boca Raton, Florida.

Watson, S.A. 1987. Structure and composition. In Corn: Chemistry and Technology. Watson, S.A., and Ramstad, P.E., Eds.; American Association of Cereal Chemists: St. Paul, MN; pp. 53-82.

Watson, S.A. 1988. Corn marketing, processing, and utilization. Pp 881-940. In Corn and Corn Improvement. Sprague, G.F., and J.W. Dudley (eds.). American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, Madison, Wisconsin. and and regime. Wauchope, R.D., T.L. Estes, R. Allen, J.L. Baker, A.G.AG Hornsby, R.L. Jones, R.P. Richards, and D.I. Gustafson. 2001. Predicted impact of transgenic herbicide-tolerant corn on drinking water quality in vulnerable watershedsBayer property of mid-western protectionpublishing USA. Pest of contents Management Sci. 58:146-160. its parties.data therefore and/oror affiliates. WHO. 1994. Glyphosate. World Healthproperty Or ganizationintellectualthird (WHO), Intemayrnational Programme its as of Chemical Safety (IPCS), Geneva.the Environmentalof and Health Criteria No. 159. is regulatory owner. a document any such its reproductiondocument White, P.J. and L.M. Pollack. 1995. Cornowner as a food sourcethis in theof United States: Part II. under of Processes, Products, Composition,rights andthe Nutrit ive Values.this Cereal Foods World 40:756- documentand/or of rights 762. to of fall use the This may distribution,and rights owner Williams, K., M. LoPresti,subject and K. Stone. 1997. Internal protein sequencing of SDS- be the violate PAGE-separated proteins: Optimizationcopy of anof in-gel digest protocol. Tech in Protein and Chem 8:79-98. may documentpublication, It exploitation this any Yagami, T., Y. Haishima, A. Nakamura,permission H. Osuna, and Z. Ikezawa. 2000. Digestibility prohibited of allergens extracted from naturalthe rubberbe latex and vegetable foods. J. Allergy Clin. Immunol. 106:752-762. commercial Furthermore,any Consequently,without

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APPENDIX I

Materials and Methods for Molecular Analysis of MON 88017

Molecular analysis was performed using genomic DNA isolated from MON 88017 in order to characterize the integrated DNA. MON 88017 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 to determine 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. and and regime. A. Test Substance AG

Bayer property protectionpublishing The test substance was MON 88017. Genomicof DNA was isolated fromcontents grain of its MON 88017 generation [LH198BC0F1 x LH59] F2 harvestedparties.data in productiontherefore plan 00-01- and/oror 39-05 generated from seed lot TRI-0005-10343-I.affiliates. Additional test substances from the property intellectualthird may MON 88017 breeding tree (Figure 15) wereits used in the insert stability analysis. For this as the of and analysis, DNA was isolated fromis the following seedregulatory lots: GLP-0203-12151-S,owner. GLP- a document any such its 0203-12152-S, GLP-0203-12153-S, GLP-0203-12154-S,reproduction GLP-0203-12155-S,document and owner this of GLP-0203-12156-S. under of rightsthe this documentand/or rights to of fall use of B. Control Substance the This may distribution,and rights owner subject The control substance wasbe a conventional cornthe with violate similar genetic background as the copy of test substance. DNA was isolated from grain harvestedand in production plan 00-01-39-05 may documentpublication, generated from seedIt lot TPC-0005-10351-I.exploitation An additional control substance, the conventional corn LH198,this was any used in the insert stability analysis. For this analysis, permission DNA was isolated from seed lot GLP-0203-12165-S.prohibited the commercial be C. Reference Substances Furthermore,any Consequently,without The primary reference substance was plasmid PV-ZMIR39 that was used in the transformation process to produce MON 88017. For Southern blot analyses of corn genomic DNA, digested plasmid PV-ZMIR39 (approximately 0.5 and 1 genome copy equivalents) was mixed with digested DNA from the control substance and separated by electrophoresis on agarose gels. Additional reference standards included the 1 kb DNA Extension Ladder and 500 bp DNA Ladder from Life Technologies, which were used for size estimations on Southern blots and PCR agarose gels, respectively.

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D. Characterization of Test, Control and Reference Substances

Event-specific PCR assays were used to confirm the identity of the test and control substances prior to use in the study. The stability of the DNA isolated from the test and control substances was determined in each Southern analysis by observation of the digested DNA sample on an ethidium bromide stained agarose gel. The identity of the test and control substances used in the insert stability analysis was confirmed by the molecular fingerprints generated from the Southern blot analyses.

E. Genomic DNA Isolation for Southern Blot Analyses

Genomic DNA from the test and control substances was extracted from corn grain by first processing the grain to a fine powder. Approximately 6 g of the processed grain were transferred to a 50 ml conical tube, then ~16 ml of CTAB extraction buffer [1.5% (w/v) CTAB, 75 mM Tris-HCl pH 8.0, 100 mM EDTA pH 8.0, 1.05 M NaCl,and and 0.75% and regime. (w/v) PVP (MW 40,000)] and 8 µl of RNase (10 mg/ml,AG Roche) were added to the processed grain. The samples were incubated at 65°C for 30-60 minutes with intermittent mixing and then allowed to cool toBayer room temperature.property protection publishing Approximately 15 ml of contents of chloroform:isoamyl alcohol (24:1 (v/v)) were added to the samples.its The suspension parties.data therefore was mixed for 5 minutes and the two phases separated by centrifugationand/oror at ~16,000 x g affiliates. property intellectualthird may for 5 minutes at room temperature. Theits a queous (upper) layer was transferred to a clean as 50 ml conical tube. Approximatelythe 1/10 of volumeand (~1.5 ml) of 10% CTAB buffer is regulatory owner. a document [10% (w/v) CTAB and 0.7 M NaCl]any and such an equal volume of chloroform:isoamylits alcohol reproductiondocument [24:1 (v/v)] were added to the aqueous phase,owner which wasthis then mixedof for 5 minutes. The under of rightsthe this samples were centrifugeddocument at ~16,000and/or x g for 5 minutes to separaterights the phases. The to of fall use of aqueous (upper) layer was removed, mixed with an equalthe volume (~15 ml) of CTAB This may distribution,and precipitation buffer [1% (w/v) CTAB,rights 50 mM Trowneris pH 8.0, and 10 mM EDTA pH 8.0] and allowed to stand at roomsubject temperature for 1-2 hours. The samples were centrifuged at be the violate ~10,000 x g for 10 minutescopy at room temperatureof to pellet the DNA. The supernatant was and may documentpublication, discarded, and the Itpellet was dissolvedexploitation in approximately 2 ml of high salt TE buffer (10mM Tris-HCl pH 8.0, this10 anymM EDTA pH 8.0, and 1 M NaCl). Gentle swirling at 37°C permission was performed to aid in dissolution ofprohibited the pellet. If necessary, samples were centrifuged the at ~23,000 x g at room temperaturecommercial befor 2 minutes to pellet and remove debris. Approximately 1/10 volume (~150 µl) of 3 M NaOAc (pH 5.2) and 2 volumes (~4 ml Furthermore,any relative to the supernatant)Consequently,without of chilled 100% ethanol were added to precipitate the DNA. The precipitated DNA was spooled into a microcentrifuge tube containing 70% ethanol. The DNA was pelleted in a microcentrifuge at maximum speed (~14,000 rpm) for ~5 minutes, vacuum-dried, and re-dissolved in TE buffer (pH 8.0). The DNA was then stored in a 4°C refrigerator.

F. Quantitation of Genomic DNA

Quantity of the DNA in the samples was determined by using a Hoefer DyNA Quant 200 Fluorometer with Roche molecular size marker IX as a DNA calibration standard.

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G. Restriction Enzyme Digestion of Genomic DNA

Approximately 20 µg of genomic DNA from the test substance and 10 µg of genomic DNA from the control substance were used for restriction enzyme digestions. For the insert stability analysis, approximately 10 µg of genomic DNA from the test substance were used. Overnight digests were performed at 37°C in a total volume of ~500 µl using 100 units of the appropriate restriction enzyme. After digestion, the samples were precipitated by adding 1/10 volume (50 µl) of 3 M NaOAc (pH 5.2) and 2 volumes (1 ml relative to the original digest volume) of 100% ethanol, followed by incubation in a -20°C freezer for at least 30 minutes. The digested DNA was pelleted at maximum speed in a microcentrifuge, washed with 70% ethanol, dried, and re-dissolved in TE buffer.

H. DNA Probe Preparation for Southern Blot Analyses

and DNA probe templates outlined in Figure 4a and 4b were preparedand by PCRregime. amplification of plasmid PV-ZMIR39. Approximately 25 ng of eachAG probe template (except the NOS 3’ and tahsp17 3’ polyadenylation sequences) were labeled with 32P-dCTP (6000 Bayer property protectionpublishing Ci/mmol) by a random priming method (RadPrimeof DNA Labeling System,contents Life its Technologies). The NOS 3’ and tahsp17 3’ polyadenylationparties.data sequences therefore were labeled by and/oror PCR using 25 ng of DNA probe template inaffiliates. the following manner: sense and antisense property intellectualthird may primers specific to the template (0.25 µMits each); 1.5 mM MgCl ; 3 µM each of dATP, as 2 32 the of and dGTP and dTTP; ~100 µCi of P-dCTPis (6000 Ci/mmol);regulatory and 2.5 Unitsowner. of Taq DNA a document any such its polymerase in a final volume of 20 µl. The cyclingreproduction conditionsdocument ofwere as follows: 1 cycle at o o owner thiso o 94 C for 3 minutes; 2 cycles at 94 C for 45 underseconds, of 52 thisC for 30 seconds, 72 C for 2 o rightsthe documentand/or rights minutes; and 1 cycle at 72 C forto 10 minutes.of fall Alluse radiolabeled of probes were purified using a Sephadex G-50 column (Roche). the This may distribution,and rights owner subject I. Southern Blot Analysesbe of Genomic DNAthe violate copy of and may documentpublication, Southern blot analysesIt were performedexploitation based on the method described by Southern this (Southern, 1975). The samplesany of DNA digested with restriction enzymes were permission separated, based on size, using 0.8% agarosprohibitede gel electrophoresis. A ‘long run’ and ‘short the run’ were performed duringcommercial this gel be electrophoresis. The ~20 µg samples of digested test substance DNAFurthermore, were dividedany in half for loading ~10 µg on the long run and ~10 µg on the short run. TheConsequently, long runwithout enabled greater separation of higher molecular weight DNAs while the short run allowed smaller molecular weight DNAs to be retained on the gel. The long run samples were loaded onto the gel and typically separated by electrophoresis for ~3-5 hours at 60-70 volts. The short run samples were then loaded in adjacent lanes on the same gel and separated by electrophoresis for ~14-17.5 additional hours at 30-35 volts. In some cases, the long run samples were loaded onto the gel and separated by electrophoresis for 15-17 hours at 30-35 volts. The short run samples in these cases were then loaded in adjacent lanes on the same gel and separated by electrophoresis for an additional 3.5-4.5 hours at 75-85 volts. For the insert stability analysis, a long run and short run were not performed. In this case, all samples were loaded onto the gel and separated by electrophoresis for 14.5 hours at 35 V and then for 3 hours at 60 V. After electrophoresis, the gels were stained in 1.5 µg/ml ethidium bromide for 10-15 minutes

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and photographed. After photographing, the gels were placed in a depurination solution (0.125 N HCl) for 10-15 minutes followed by a denaturing solution (0.5 M NaOH, 1.5 M NaCl) for 30-40 minutes and then a neutralizing solution (0.5 M Tris-HCl pH 7.0, 1.5 M NaCl) for 30-40 minutes. The gels were then placed in a 20X SSC buffer for 5-15 minutes. The DNA from the agarose gels was transferred to Hybond-N nylon membranes (Amersham) using a Turboblotter™ (Schleicher & Schuell). The DNA was allowed to transfer overnight (using 20X SSC as the transfer buffer) and covalently cross- linked to the membrane with a UV Stratalinker® 1800 (Stratagene) using the auto- crosslink setting. The blots were prehybridized for 0.5-7 hours at 60-65°C in an aqueous solution of 250 mM Na2HPO4•7H2O, 7% SDS, and 0.1 mg/ml tRNA. Hybridization with the radiolabeled probe was performed in fresh prehybridization solution for 15-28 hours at 60°C (NOS 3’ and tahsp17 3’ polyadenylation sequence probes) or 65°C (all other probes). Membranes were washed in an aqueous solution of 0.1% SDS and 0.1X SSC for four 15-20 minute periods at the hybridization temperature using fresh solution for each and of the four washes. Multiple exposures of the blots were thenand generated regime. using Kodak Biomax™ MS-2 film in conjunction with one KodakAG Biomax MS intensifying screen at approximately -80°C. Bayer property protectionpublishing of contents its J. PCR Analyses parties.data therefore and/oror affiliates. property intellectualthird may Overlapping PCR products were generatedits that span the insert in MON 88017 (Products as the of and A-G, Figures 17 and 18). These isproducts were sequencedregulatory to verifyowner. the sequence of the a document any such its insert in MON 88017 and to verify the sequence of reproductionthe genomicdocument DNA flanking the 5’ and owner this of 3’ ends of the insert. The PCR analyses wereunder conducted of using 50 ng of genomic DNA or rightsthe this documentand/or rights 0.5 ng of plasmid PV-ZMIR39 positto ofive controlfall DNAuse templateof in a 50 µl reaction volume containing a final concentration of 1.5 mM MgCl , 0.4 theµM of each primer, 200 µM each This may distribution,and 2 rights owner dNTP, and 2.5 units of Taqsubject DNA polymerase. The specific polymerase used to amplify ™ ™ Products A, B, and D-Gbe was REDTaq DNA thePolymerase violate (Sigma). HotStarTaq DNA copy of Polymerase (Qiagen) was used to amplify Productand C. The amplification of Products A, may documentpublication, It exploitation B, and D-G was performedthis under the following cycling conditions: 94°C for 3 minutes; 35 cycles at 94°C for 30 seconds,any 55°C for 30 seconds, 72°C for 2 minutes; 1 cycle at permission 72°C for 10 minutes. The amplificationprohibited of Product C was performed under the following the be cycling conditions: 94°C forcommercial 15 minutes; 35 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C Furthermore,for 2 minutes;any 1 cycle at 72°C for 10 minutes. The PCR products were Consequently,without separated on 1.2 % agarose gels and visualized by ethidium bromide staining. Following electrophoresis, PCR products generated from MON 88017 were excised from the gel and purified using the QIAquick® Gel Extraction Kit (Qiagen). The purified products were sequenced with the initial PCR primers, as well as primers designed internal to the amplified sequences. All sequencing was performed using dye-terminator chemistry.

™ Turboblotter is a trademark of Schleicher & Schuell, Keene, New Hampshire ® Stratalinker is a registered trademark of Stratagene Cloning Systems, La Jolla, California ™ Biomax is a trademark of Eastman Kodak Co., Rochester, New York ™ REDTaq is a trademark of Sigma Chemical Co., St. Louis, Missouri ™ HotStarTaq is a trademark of Qiagen Inc., Valencia, California ® QIAquick is a registered trademark of Qiagen

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APPENDIX II

Materials and Methods used for Characterization of Introduced Proteins in MON 88017

1. Materials and methods used to characterize the CP4 EPSPS protein

1.1. Plant-Produced CP4 EPSPS Protein

The CP4 EPSPS protein was isolated from MON 88017 grain (produced under production plan 02-01-50-01). The identity of the MON 88017 corn grain sample was confirmed using event-specific PCR analysis. A verification of identity of the grain is and archived under lot 60-100023. The CP4 EPSPS protein was storedand in aregime. –80 °C freezer in a buffer solution [50 mM Tris-HCl, pH 7.5, 50 mMAG KCl, and 2 mM DTT] at a total protein concentration of 0.5 mg/mL. The data for the isolation of the CP4 EPSPS protein Bayer property protectionpublishing from MON 88017 grain is archived under lotof 60-100023. contents its parties.data therefore and/oror 1.2. E. coli-Produced CP4 EPSPS Proteinaffiliates. property intellectualthird may its as the of and The E. coli-produced CP4 EPSPSis protein (APS lotregulatory 20-100015) wasowner. used as a reference a document any such its for the functional activity assays, glycosylation analysis,reproduction anddocument western blot analysis. The owner this of CP4 EPSPS protein was stored in a –80 °C underfreezer inof a buffer solution [50 mM Tris-HCl, rightsthe this documentand/or rights pH 7.5, 50 mM KCl, 2 mM DTT,to 1 mMof fallbenz amidine-HCl,use of and 25% (v/v) glycerol] at a total protein concentration of 3.8 mg/mL. the This may distribution,and rights owner subject 1.3. Assay Controls be the violate copy of and may documentpublication, Protein molecular Itweight standards exploitationwere used to calibrate SDS-polyacrylamide gels and this verify protein transfer to PVDFany membranes. A peptide mixture was used to calibrate the permission MALDI-TOF mass spectrometer for trypticprohibited ma ss analysis. NIST BSA was used for the MALDI-TOF intact mass analysis.commercial beTransfer rin was used as the positive control in glycosylation analysis.Furthermore, any An analytical reference protein (10 pmole β-lactoglobulin) was analyzed before andConsequently, after withoutthe test proteins to verify that the N-terminal sequencer met acceptable performance criteria for repetitive yield and sequence identity. Phenylthiohydantoin amino acid standards were used during N-terminal sequencing analysis. The following standards and controls were used during amino acid analysis; NIST BSA, NIST AA Standards, and norvaline standard.

1.4. Protein Purification

The CP4 EPSPS protein was purified from extracts of ground MON 88017 grain using a combination of anion exchange chromatography, ammonium sulfate fractionation, hydrophobic interaction chromatography, and affinity chromatography.

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Defatted corn grain was extracted with a 50 mM Tris-HCl buffer, pH 8.0 [1:10 (w/v) grain to buffer ratio]. The extract was clarified by centrifugation and the supernatant was concentrated, dialyzed into 50 mM sodium carbonate/bicarbonate, 1 mM EDTA buffer, and applied to a Q-sepharose Fast Flow anion exchange column. The column was washed with 50 mM sodium carbonate/bicarbonate, 1 mM EDTA buffer and the eluate was collected. The eluate was clarified by centrifugation and the proteins were precipitated with ammonium sulfate [70% (w/v)]. The ammonium sulfate pellet was re- suspended in 50 mM Tris-HCl, pH 7.5, 1.25 M ammonium sulfate buffer and an aliquot was applied to a Source 15PHE hydrophobic interaction column. Proteins bound to the column were eluted with a linear ammonium sulfate gradient (1.25 M to 0 M). Fractions were pooled based on immunoreactivity with anti-CP4 EPSPS antibodies on lateral flow strips, concentrated, and the buffer was exchanged to 25 mM Bis-Tris Propane, pH 6.5, containing protease inhibitors. The sample was applied to a Source 15 Q anion exchange column. The column was washed with 25 mM Bis-Tris propane, 1 mM DTT, 1 mM benzamidine, 0.5 mM PMSF, 10% (v/v) glycerol, pH 6.5 buffer. Proteins boundand to the and regime. column were eluted with a linear sodium chloride gradientAG (0 M to 0.5 M NaCl). Fractions containing CP4 EPSPS protein were pooled based on immunoreactivity with anti-CP4 EPSPS antibodies on lateral flow strips,Bayer concentrated, property protection andpublishing the buffer was of contents exchanged to 50 mM MES, pH 5.85, 2 mM DTT. The sample was itsapplied to a cellulose parties.data therefore phosphate column for affinity chromatography. The column wasand/or orwashed with 50 mM affiliates. MES, pH 5.85, 2 mM DTT. CP4 EPSPSproperty prot intellectualein wasthird eluted from themay column with its as 0.5 mM PEP, 0.5 mM S-3-P, 50 mMthe MESof pH and5.85, 2 mM DTT buffer. Fractions were is regulatory owner. a document pooled based on immunoreactivityany with such CP4 EPSPS antibodies onits lateral flow strips and reproductiondocument SDS-PAGE analysis. The protein was concenowner trated andthis the bufferof was exchanged to 50 under of mM Tris-HCl, pH 7.5, 50 mM KCl,rights 2 themM DTT. this documentand/or of rights to of fall use the This may distribution,and 1.5. Total Protein Concentrationrights Determinationowner subject be the violate The total protein concentrationcopy of the plant-producedof CP4 EPSPS protein sample was and determined using aminomay aciddocument analysis.publication, Aliquot s of the sample were subjected to vapor It exploitation phase acid hydrolysis followedthis any by amino acid analysis on a Hitachi L-8800 amino acid analysis system. Amino acids werepermission detected using post-column ninhydrin derivatization. prohibited Each protein sample was analyzedthe inbe triplicate. Total protein concentration was calculated as an average ofcommercial the triplicate analysis. Furthermore,any Consequently,without 1.6. Western Blot Analysis

Prior to western blot analysis, proteins were subjected to SDS-PAGE and transferred to a PVDF membrane. Aliquots of the plant- and E. coli-produced CP4 EPSPS proteins were diluted and mixed with loading buffer to a final concentration of 1× loading buffer [62.5 mM Tris, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 2.5% (v/v) 2-mercaptoethanol, 0.005% (w/v) bromophenol blue]. Samples were heated at 99-100 °C for 5 min prior to loading onto a Tris-glycine buffered 4-20% gradient polyacrylamide gel. Bio-Rad (Hercules, California) pre-stained broad range molecular weight markers (10 µL/lane) were included on the gel to verify transfer of proteins to the PVDF membrane. Electrophoresis was performed at a constant voltage of 160 V for 80 min.

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Following electrophoresis, samples were transferred to a 0.45 µm PVDF membrane for 90 min at a constant current of 300 mA in transfer buffer [12 mM Tris, 96 mM glycine, 20% (v/v) methanol, pH 8.3].

For western blot analysis, the following steps were performed. The membrane was blocked by incubation in 5% (w/v) NFDM in 1× PBST for 18-20 h. Antibodies (goat anti-CP4 EPSPS serum, lot 6844572) were diluted (1:2000) in 1% (w/v) NFDM in PBST and incubated with the membrane for 1 h. Excess serum was removed by washing with PBST four times for 10 min each. The membrane was then incubated with a 1:10,000 dilution of anti-goat IgG, HRP conjugated antibody in 1% (w/v) NFDM in PBST for 1 h. Excess antibody was removed by washing with PBST four times for 10 min each. All incubations were performed at room temperature. Immunoreactive bands were visualized using the ECL detection system (Amersham Pharmacia, Uppsala, Sweden) and the membrane was exposed (1 min, 30, 15, 10, 5, and 1 sec) to Hyperfilm ECL high performance chemiluminescence film (Amersham Pharmacia). Films were anddeveloped and regime. using a Konica SRX-101A automated film processorAG (Tokyo, Japan).

Image analysis of immunoreactive bands on filmsBayer was property conducted protection usingpublishing a Bio-Rad model of contents GS-710 calibrated imaging densitometer equipped with Quantity Oneits software version parties.data therefore 4.3.0. The level of signal for the principal bands correspondingand/or toor the CP4 EPSPS affiliates. protein detected in each lane was measuredproperty asintellectual a bandthird contour quantitymay (average band OD its 2 as × band area in mm ). the of and is regulatory owner. a document any such its reproductiondocument The percent difference in immunoreactivityowner between thethis plant- ofand E. coli-produced under of rightsthe this proteins was calculated. document The and/orproteins were considered to haverights equivalent to of fall use of immunoreactivity if they differed by ≤ 10%. the This may distribution,and rights owner 1.7. N-terminal Sequencesubject Analysis be the violate copy of and may documentpublication, Prior to N-terminalIt sequence analysis,exploitation the plant-produced CP4 EPSPS protein sample was separated using SDS-PAGEthis any and transferred to a PVDF membrane. An aliquot of permission plant-produced CP4 EPSPS protein wasprohibited diluted and mixed with loading buffer to a final the concentration of 1× loadingcommercial buffer. be The sample was heated to ~102 °C for 4 minutes prior to loading onto a Tris-glycine buffered 4-20% gradient polyacrylamide gel. Bio- Furthermore,any Rad pre-stained broadConsequently, rangewithout molecular weight markers were included on the gel to verify transfer of the proteins to the membrane. Electrophoresis was performed at a constant voltage of 125 V for 100 min. Following electrophoresis, the gel was soaked for 60 minutes in CAPS transfer buffer and the proteins were transferred to a PVDF membrane (0.2 µm pore size) for 90 minutes at a constant current of 300 mA. Protein bands were stained with Coomassie Brilliant Blue G stain (Bio-Rad) and then the blot was de-stained in a solution of 10% (v/v) acetic acid, 40% (v/v) methanol. Protein bands corresponding to CP4 EPSPS protein (~ 45 kDa) and two other protein bands found at ~48 kDa and ~55 kDa were excised from the membrane and sequenced.

N-terminal sequence analysis was performed for 15 cycles using automated Edman degradation chemistry (Hunkapillar et al., 1983). An Applied Biosystems 494 Procise

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HT Protein Sequencing System (Foster City, CA) with 140C Microgradient system and 785 Programmable Absorbance Detector and Procise Control Software (version 1.1a) were used. Chromatographic data were collected using Atlas99 software (version 3.59a). A PTH-amino acid standard mixture 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. A control protein (10 picomole β-lactoglobulin) was analyzed before and after the test substance protein bands to verify that the sequencer met acceptable performance criteria for repetitive yield and sequence identity.

1.8. MALDI-TOF MS Tryptic Mass Map Analysis

1.8.a. In-gel Trypsin Digestion and Sample Preparation and and regime. Prior to the generation of tryptic fragments for MALDI-TAG OF mass spectroscopy, aliquots of the plant-produced CP4 EPSPS protein in loading buffer were separated by electrophoresis on a Tris-glycine buffered 4-20%Bayer gradient property polyacrylamide protectionpublishing gel. Prior to of contents loading, the plant-produced CP4 EPSPS protein samples were heatedits at ~102 °C for 4 parties.data therefore min. Bio-Rad broad range molecular weight markers were includedand/oror on the gel. affiliates. Electrophoresis was performed at a constantproperty voltageintellectualthird of 125 V for 100may min. Following its as electrophoresis, the gel was stainedthe for of ~1 h withand Coomassie Brilliant Blue G stain and is regulatory owner. a document destained for ~3 h in a solution containingany such 25% (v/v) methanol. its reproductiondocument owner this of under of The band that migrated to ~45 kDarights wasthe exci sed, de-stained,this reduced, alkylated, and documentand/or of rights subjected to an in-gel trypsin digestto of(W illiamsfall et useal., 1997). Each gel fragment was the This µ may distribution,and destained by incubation in 100 rightsL of 40% (v/v) methanolowner and 10% (v/v) glacial acetic acid in a microfuge tube. subjectFollowing destaining, gel fragments were incubated in 100 µL be the violate of 100 mM ammonium bicarbonatecopy buffer forof 30 min at room temperature. Proteins were and reduced in 100 µL ofmay 10 mMdocument dithiothreitolpublication, solution for two hours at 37 °C. Proteins were It exploitation then alkylated by the additionthis any of 100 µL of buffer containing 100 mM iodoacetic acid. The alkylation reaction was allowedpermission to procee d at room temperature for 20 min in the prohibited dark. Gel fragments were incubatedthe in 100 µL of 100 mM ammonium bicarbonate buffer commercial be for 30 min at room temperature. Acetonitrile (100 µL) was added to each microfuge tube Furthermore,any and the reactionsConsequently, were incubatedwithout at room temperature for an additional 30 min. This procedure was repeated two additional times to remove the reducing and alkylating agents from the gel fragments. The gel bands were dried in a SpeedVac concentrator, rehydrated, and the protein digested in the gel overnight at 37 °C with 50 µL 25 mM ammonium bicarbonate solution containing 33 µg/mL trypsin. Digested peptides were extracted for one hour each at room temperature with 50 µL 70% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid (TFA). The supernatant from each extraction sample was combined and dried in a SpeedVac concentrator. The extraction, pooling, and drying were repeated two additional times, and the final dried material was stored overnight in a 4 °C refrigerator followed by reconstitution in 5 µL of 0.1% (v/v) TFA.

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A portion of the digested sample was desalted (Bagshaw et al., 2000) using Millipore (Bedford, MA) ZipTipC18 pipette tips. Prior to desalting, the tips were wetted with methanol and equilibrated with 0.1% (v/v) TFA. A sample (4.7 µL) was applied to a ZipTip and eluted with 5 µL of Wash 1 [0.1% (v/v) TFA], 5 µL of Wash 2 [20% (v/v) acetonitrile containing 0.1% (v/v) TFA], 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].

1.8.b. MALDI-TOF Instrumentation and Mass Analysis

Mass spectral analysis was performed using an Applied Biosystems Voyager DE-Pro Biospectrometry Workstation MALDI-TOF instrument with the supplied Data Explorer software (version 4.0). Calibration of the mass spectrometer was performed using an external peptide mixture from a Sequazyme Peptide Mass Standards kit (Applied Biosystems). Samples (0.3 µL) from each desalting step, as well as a sampleand of the and regime. solution prior to desalting, were co-crystallized withAG 0.75 µL of α-cyano-4-hydroxy cinnamic acid on the analysis plate. All samples were analyzed from 500 to 5000 Daltons in reflector mode using 150 laser shotsBayer per massproperty spectrum protection publishingat a laser intensity of contents setting of 3100 (a unit-less MALDI-TOF instrument specific value).its Protonated (MH+) parties.data therefore peptide masses were observed monoisotopically in reflector modeand/oror (Aebersold, 1993; affiliates. Billeci and Stults, 1993). GPMAW32property softwa intellectualre (Appliedthird Biosystems,may version 4.23) was its as used to generate a theoretical trypsinthe digestof of andthe expected protein sequence. The amino is regulatory owner. a document acid sequence of the plant-producedany CP4 such EPSP S protein was deducedits from the coding reproductiondocument region of the cp4 epsps gene present in MONowner 88017. thisMasses wereof calculated for each under of theoretical peptide and the raw massrights datathe was comparedthis to the calculated theoretical documentand/or of rights masses. Experimental masses (MH+)to of werefall assigneduse to peaks when three (or more) the This may distribution,and isotopically resolved ion peaks rightswere observed inowner the raw mass data. Peaks were not assessed if there were lesssubject than three isotopically resolved peaks in the spectra, when be the violate peak heights were less thancopy approximately twof ice the baseline noise, or when a mass could and may documentpublication, not be assigned dueIt to overlap with exploitationa stronger signal ±2 daltons from the mass analyzed. Known autocatalytic fragmentsthis any from trypsin digestion were identified in the raw data. permission prohibited 1.8.c. Molecular Weight Determinationthe using MALDI-TOF MS commercial be

Furthermore,any Prior to analysis, Consequently,the plant-producedwithout CP4 EPSPS protein and NIST BSA reference protein were desalted using drop dialysis (Görisch, 1988). Briefly, 4µL of protein was placed on a Millipore 25 mm microdialysis disk (type VSWP, 0.025 µm pore size) and dialyzed for 60-120 minutes against HPLC-grade water. A portion of each protein sample (0.3 and 0.5 µL) was spotted on an analysis plate, mixed with 0.75 µL sinapinic acid solution, and air-dried. Mass spectral analysis of the plant-produced CP4 EPSPS protein was performed using an Applied Biosystems Voyager DE-Pro Biospectrometry Workstation Matrix Assisted Laser Desorption and Ionization (MALDI) Time of Flight (TOF) instrument with the supplied Data Explorer software (version 4.0). Mass calibration of the instrument was performed using the desalted BSA reference protein. The mass of the plant-produced CP4 EPSPS protein was reported as an average of three separate mass spectral acquisitions. For comparison, the mass of the CP4 EPSPS protein

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was calculated from the expected amino acid sequence of the protein using the Protean module of DNAstar software (version 5.01).

1.9. Purity and Molecular Weight Determination using SDS-PAGE

An aliquot of the plant-produced CP4 EPSPS protein was diluted and mixed with loading buffer to a final concentration of 1× loading buffer. The sample was heated at ~102 °C for 4 min prior to loading onto a Tris-glycine buffered 4-20% gradient polyacrylamide gel. Three amounts (1, 2, and 3 µg) of total protein were loaded into three separate wells. Bio-Rad broad range molecular weight markers (1 µg each protein/lane) were included on the gel to estimate the molecular weight of the test substance. Electrophoresis was performed at a constant voltage of 125 V for 100 min. Proteins were fixed in the gel for 60 min in a solution containing 40% (v/v) methanol and 7% (v/v) glacial acetic acid, then stained for 2 h with Colloidal Brilliant Blue G stain (Sigma, St. Louis, MO). The gel was and destained for 45 sec in a solution containing 10% (v/v) acetic andacid and regime.25% (v/v) methanol, followed by 2 h in a solution containing 25%AG (v/v) methanol.

Bayer property protectionpublishing Analysis of the gel was performed using a Bio-Radof Laboratories GS-710contents densitometer its with the supplied Quantity One software, version 4.3.0.parties. Moleculardata weighttherefore values of the marker proteins were supplied by the manufacturer and used toand/or estimateor the molecular affiliates. property intellectualthird may weight of each observed band in the plant-its produced CP4 EPSPS protein. All visible as bands within each sample lane werethe quantified.of and Purity was estimated as the percent is regulatory owner. a document optical density of the ~45 kDa bandany relative such to all bands detectedits in the lane. Molecular reproductiondocument weight and purity were reported as an averagownere of all threethis lanesof containing the plant- under of rightsthe this produced CP4 EPSPS protein.documentand/or The plant- and E. coli-producedrights proteins were considered to of fall use of to have equivalent molecular weights if the difference inthe the molecular weights was ≤ This may distribution,and 5%. rights owner subject be the violate copy of 1.10. Phosphate Release Assays and may documentpublication, It exploitation Prior to analysis, the plant-prthis anyoduced CP4 EPSPS protein and the E. coli-produced CP4 permission EPSPS reference standard were dilutedprohibited in a buffer solution containing 50 mM HEPES, the pH 7.0. Enzymatic activitycommercial assays for be both the plant- and the E. coli-produced CP4 EPSPS proteins were conducted in duplicate or triplicate and each replicate assay was Furthermore,any subsequently analyzedConsequently, in withoutduplicate spectrophotometrically. This end-point type colorimetric assay measures the release of inorganic phosphate from one of the substrates, phosphoenolpyruvate (PEP), which is released by the action of the EPSPS enzyme in the presence of shikimate-3-phosphate (S-3-P). Reaction mixtures contained the EPSPS enzyme with 2 mM S-3-P and were initiated with 1 mM PEP (final concentration). The final reagent concentrations in the assay were 50 mM HEPES (pH 7.0), 0.1 mM ammonium molybdate and 5 mM potassium fluoride. Reactions were incubated for two minutes at 25 °C to allow for product formation. The reactions were quenched with malachite green reagent and fixed two minutes later with 33% (w/v) sodium citrate. The EPSPS-catalyzed release of inorganic phosphate from PEP was determined at a wavelength of 660 nm using a PowerWave Xi microplate reader, relative to a standard curve of inorganic phosphate treated with the malachite green reagent and

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33% (w/v) sodium citrate. For EPSPS, one unit (U) of enzyme activity was defined as the amount of enzyme that produced 1 µmole of inorganic phosphate from PEP per minute at 25 °C. Calculations of the specific activities were performed using Microsoft Excel 2000 version 9.0.4402 SR-1. Specific activity values were calculated based on the assay concentration of the CP4 EPSPS protein. The plant and E. coli-produced proteins were considered to have equivalent functional activity if their specific activities differed by ≤ two-fold.

1.11. Glycosylation Analysis

Glycosylation analysis was performed to determine whether the plant-produced CP4 EPSPS protein was post-translationally modified with covalently bound carbohydrate moieties. Aliquots of the test substance, the E. coli-produced CP4 EPSPS reference standard (a negative control), and transferrin (a positive control) were each diluted and and mixed with loading buffer to a final concentration of 1× loadingand buffer.regime. The samples were heated at 99-101 °C for 4 min, and two amountsAG (0.5 and 1 µg) of each sample were loaded, along with Bio-Rad prestained broad range molecular weight markers (10 Bayer property protectionpublishing µL/lane), on a Tris-glycine buffered 4-20% grof adient polyacrylamide gel.contents Electrophoresis its was performed at a constant voltage of 170 V for 70 min.parties. dataAfter electrophoresistherefore proteins were electrotransferred to a 0.45 µm PVDF membrane for 90 and/orminor at a constant current of affiliates. property intellectualthird may 300 mA. its as the of and is regulatory owner. a document Carbohydrate detection was performedany such directly on the PVDF membraneits using the ECL reproductiondocument of glycoprotein detection system (Amershamowner Phar macia).this The PVDF membrane was gently under of rightsthe this shaken for 10 min in PBSdocument andand/or transferred to a solution of 100rights mM sodium acetate to of fall use of containing 10 mM sodium metaperiodate. The membranethe was incubated in the dark for This may distribution,and 20 min in the oxidation reagent.rights The oxidation solutionowner was removed from the subject membrane by two brief berinses with PBS followedthe violateby three sequential 10 min washes in copy of PBS. The membrane was transferred to a solutionand of 100 mM sodium acetate containing may documentpublication, 25 nM biotin hydrazideIt and incubatedexploitation for 60 min. The biotin hydrazide solution was removed by rinsing the membranethis any twice in PBS followed by washing the membrane in permission PBS three times for 10 min each. Theprohibited membrane was blocked overnight in a 4 °C the refrigerator in the blockingcommercial agent supplied be with the kit. The blocking solution was removed by rinsing the membrane twice in PBS followed by washing the membrane in Furthermore,any PBS three times forConsequently, 10 minwithout each. The membrane was incubated with streptavidin-HRP conjugate (diluted 1:6000) in PBS for 30 min to detect carbohydrate moieties bound to biotin. Excess streptavidin-HRP was removed by rinsing and washing as previously described. Bands were visualized using the ECL detection system and the membrane was exposed (30 sec, 3 min, and 10 min) to Hyperfilm ECL high performance chemiluminescence film. Films were developed using a Konica SRX-101A automated film processor.

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2.0. Materials and methods used to characterize Cry3Bb1 protein

2.1. Plant-Produced Cry3Bb1 Protein

The plant-produced Cry3Bb1 protein was isolated from MON 88017 grain. The identity of the MON 88017 corn grain sample was confirmed using event-specific PCR. A copy of the verification of identity certificate for the grain is archived under lot 60-100024. The Cry3Bb1 protein was stored in a –80 °C freezer in a buffer solution containing 100 mM Tris-HCl, pH 8.0. The data for the isolation of the Cry3Bb1 protein from MON 88017 grain is archived under lot 60-100024.

2.2. E. coli-Produced Cry3Bb1 Protein

The characterized E. coli-produced Cry3Bb1 protein (lot 30-100002) was usedand as a and regime. reference standard to establish equivalence in selectAG anal yses. The Cry3Bb1 protein was stored in a –80 °C freezer in a buffer solution [50 mM sodium carbonate-bicarbonate, pH 10.1, 1 mM EDTA] at a total protein concentrationBayer of 1.2property mg/mL protection bypublishing amino acid analysis. of contents its parties.data therefore 2.3. Assay Controls and/oror affiliates. property intellectualthird may its as Bovine serum albumin (BSA) proteinthe ofwas usedand to estimate the total protein concentration is regulatory owner. a document in the Bio-Rad protein assays. Proteinany suchmolecular weight markersits were used to calibrate reproductiondocument SDS-polyacrylamide gels and verify proteinowner transfer tothis PVDF membranes.of A peptide under of rightsthe this mixture was used to calibratedocumentand/or the MALD I-TOF mass spectrometerrights for tryptic mass to of fall use of analysis. Transferrin was used as a positive control in glycosylationthe analysis. This may distribution,and rights owner 2.4. Protein Purificationsubject be the violate copy of and may documentpublication, The Cry3Bb1 proteinIt was purified fromexploitation extracts of ground defatted MON 88017 corn grain using a combinationthis of any anion exchange chromatography and affinity chromatography. permission prohibited the commercial be Cry3Bb1 protein was extracted from three batches (1-1.5 kg each) of defatted Furthermore,any MON 88017 grainConsequently, with eitherwithout 50 mM Tris-HCl, pH 8.0 buffer or 50 mM sodium carbonate/bicarbonate, 1 mM EDTA, pH 10 buffer (buffer A) [1:10 (w/v) grain to buffer ratio]. The extracts were clarified by centrifugation and the supernatant was concentrated 3-4 fold. The samples not extracted in buffer A were combined and dialyzed into buffer A. Samples were loaded onto a Q-sepharose Fast Flow anion exchange column that was pre-equilibrated with buffer A. The column was washed with 25% buffer B (50 mM sodium carbonate/bicarbonate, 0.5 M NaCl, 1 mM EDTA, pH 10 (buffer B). The Cry3Bb1 protein was eluted with 45% buffer B. The protein was concentrated and dialyzed into 50 mM Tris-HCl, pH 8.0. The dialyzed sample was loaded onto protein G agarose columns coupled with monoclonal Cry3Bb1 antibodies for affinity chromatography. The sample was recirculated over the antibody column. Cry3Bb1 protein was eluted from the column with 100 mM triethylamine, pH 11.2. Fractions were

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collected into 100 mM Tris-HCl, pH 8.0, to neutralize the solution. The fractions were concentrated and buffer exchanged into 100 mM Tris-HCl, pH 8.0. Fractions containing proteins similar in molecular weight to the Cry3Bb1 protein were pooled based on visual examination of Colloidal Brilliant Blue G stained SDS polyacrylamide gels.

2.5. Total Protein Concentration Determination

The total protein concentration of the plant-produced Cry3Bb1 protein was estimated using a Bio-Rad (Hercules, CA) protein concentration assay. BSA protein (concentrations ranging from 0.05 to 0.6 mg/mL) was used to create a standard curve. The Cry3Bb1 protein concentration was estimated by comparison to the linear standard curve. Data was collected using a Bio-Tek Instruments, Inc. Powerwave Xi microplate scanning spectrophotometer (Winooski, VT) employing the KC4 software version 2.6, revision 3. Readings were taken at a wavelength of 595 nm. and and regime. 2.6. Western Blot Analysis AG

Prior to western blot analysis, proteins were separatedBayer propertyusing SDS-PAGE protectionpublishing and transferred of contents to a PVDF membrane. Aliquots of plant- and E. coli-produced Cry3Bb1its proteins were parties.data therefore diluted and mixed with loading buffer, to a final concentrationand/or of or1 × loading buffer affiliates. [62.5 mM Tris, pH 6.8, 2% (w/v) SDpropertyS, 10% (v/v)intellectual thirdglycerol, 2.5% (v/v)may its as 2-mercaptoethanol, 0.005% (w/v) thebromophenol of and blue]. Samples were heated at 100 °C is regulatory owner. a document for 5 min prior to loading onto a Tris-glyciany such ne buffered 4-20% polyacrylamideits gel. Bio- reproductiondocument Rad (Hercules, CA) prestained broad rangeowner molecular thisweight markersof (1 µg/band) or under of rightsthe this Precision Plus Dual Colordocument molecularand/or weight markers (1 µg/band)rights were included on the to of fall use of gel to verify transfer of proteins to the PVDF membrane.the Electrophoresis was performed This may distribution,and at a constant voltage of 160 V forrights 80 min. Followingowner electrophoresis, samples were subject transferred to a 0.45 µmbe PVDF membrane forthe 90 minviolate at a constant current of 300 mA in copy of transfer buffer [12 mM Tris, 96 mM glycine, 20%and (v/v) methanol, pH 8.3]. may documentpublication, It exploitation For western blot analysis,this the any following steps were performed. The membrane was permission blocked by incubation in 5% (w/v) NFDMprohibited in 1× PBST for 16 h and 30 min. the Characterized antibodies (goatcommercial anti-Cry3B be b1 serum, lot 6844582) were diluted (1:5000) in 1% (w/v) NFDM in PBST and incubated with the membrane for 1 h. Excess serum Furthermore,any was removed by Consequently,washing withoutwith PBST four times for 5-10 min each. The membrane was then incubated with a 1:10,000 dilution of anti-goat IgG, HRP conjugated antibody in 1% (w/v) NFDM in PBST for 1 h. Excess antibody was removed by washing with PBST four times for 10 min each. All incubations were performed at room temperature. Immunoreactive bands were visualized using the ECL detection system (Amersham Pharmacia, Uppsala, Sweden) and the membrane was exposed (1 min, 30, 15, 10, 5, and 1 sec) to Hyperfilm ECL high performance chemiluminescence film (Amersham Pharmacia). Films were developed using a Konica SRX-101A automated film processor (Tokyo, Japan).

Image analysis of immunoreactive bands on films was conducted using a Bio-Rad model GS-710 calibrated imaging densitometer equipped with Quantity One software version

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4.3.0. The level of signal from the principal bands corresponding to the Cry3Bb1 protein detected in each lane was measured as a band contour quantity (average band OD × band area in mm2). The percent difference in immunoreactivity between the plant- and E. coli- produced Cry3Bb1 proteins was calculated using Microsoft Excel 2000. The proteins were considered to have equivalent immunoreactivity if the average percent difference was ≤ 10%.

2.7. MALDI-TOF MS Tryptic Mass Map Analysis

2.7.a. In-gel Trypsin Digestion and Sample Preparation

Prior to the generation of tryptic fragments for MALDI-TOF mass spectroscopy, aliquots of the plant-produced Cry3Bb1 protein in loading buffer were separated by electrophoresis on a Tris-glycine buffered 4-20% gradient polyacrylamide gel. Prior to and loading, the plant-produced Cry3Bb1 protein samples were heatedand at ~104regime. °C for 4 min. Bio-Rad broad range molecular weight markers wereAG included on the gel. Electrophoresis was performed at a constant voltage of 150 V for 85 min. Following electrophoresis, the gel was stained for 4 h andBayer 20 min property with Coomassie protectionpublishing stain (Bio-Rad) of contents and destained overnight with a solution containing 25% (v/v) methanol.its parties.data therefore and/oror affiliates. property intellectualthird may Bands that corresponded to the sizes expectedits for full-length Cry3Bb1 protein (~77 kDa) as and its major fragments (~66 and ~55the ofkDa) wereand excised, destained, reduced, alkylated, is regulatory owner. a document and subjected to an in-gel trypsin digestany such (Williams et al., 1997). Briefly,its each gel reproductiondocument fragment was destained by incubation in 100owner µ L of 40%this (v/v) methanolof and 10% (v/v) under of rightsthe this glacial acetic acid in a microfugedocumentand/or tube. Following destaining,rights gel fragments were to of fall use of incubated in 100 µL of 100 mM ammonium bicarbonatethe buffer for 30 min at room This may distribution,and temperature. Proteins were reducedrights in 100 µL ofowner 10 mM dithiothreitol solution for two subject hours at 37 °C. Proteins were then alkylated by the addition of 100 µL of buffer be the violate containing 100 mM iodoaceticcopy acid. The alkylof ation reaction was allowed to proceed at and may documentpublication, room temperature Itfor 20 min in the exploitationdark. Gel fragments were incubated in 100 µL of 100 mM ammonium bicarbonatethis anybuffer for 30 min at room temperature. Acetonitrile (100 permission µL) was added to each microfuge tubeprohibited and the reactions were incubated at room the temperature for an additionalcommercial 30 min. be This procedure was repeated two additional times to remove the reducing and alkylating agents from the gel fragments. The gel bands were Furthermore,any dried in a SpeedVacConsequently, concenwithouttrator, rehydrated, and digested in the gel with 50 µL 25 mM ammonium bicarbonate solution containing 33 µg/mL trypsin. The digestion was continued overnight at 37 °C. Trypsin-digested proteins were extracted for one hour each at room temperature with 50 µL 70% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid (TFA). The supernatant from each extraction sample was combined and dried in a SpeedVac concentrator. The extraction, pooling, and drying were repeated two additional times, and the final dried material was stored in a 4 °C refrigerator until reconstituted in 5 µL of 0.1% (v/v) TFA.

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ZipTip and eluted with 5 µL of Wash 1 [0.1% (v/v) TFA], 5 µL of Wash 2 [20% (v/v) acetonitrile containing 0.1% (v/v) TFA], 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].

2.7.b. MALDI-TOF Instrumentation and Mass Analysis

Mass spectral analysis was performed using an Applied Biosystems Voyager DE-Pro Biospectrometry Workstation MALDI-TOF instrument (Foster City, CA) with the supplied Data Explorer software (version 4.0). Calibration of the mass spectrometer was performed using an external peptide mixture from a Sequazyme Peptide Mass Standards kit (Applied Biosystems). Samples (0.3 µL) from each desalting step, as well as a sample of the solution prior to desalting, were co-crystallized with 0.75 µL of α-cyano-4- hydroxy cinnamic acid on the analysis plate. All samples were analyzed from 600 to 5000 daltons (mass standards were analyzed from 500-5000 daltons) in reflectorand mode and regime. using 150 laser shots per mass spectrum at a laser intensityAG setting of 3100, 3150, or 3300 (a unit-less MALDI-TOF instrument specific value). Protonated (MH+) peptide masses were observed monoisotopically in reflector modeBayer (Aebersold,property protection 1993;publishing Billeci and Stults, of contents 1993). GPMAW32 software (Applied Biosystems, version 4.23) wasits used to generate a parties.data therefore theoretical trypsin digest of the expected protein sequence. Theand/or sequenceor of the Cry3Bb1 affiliates. protein used was identical to the aminoproperty acid sequenceintellectualthird deduced frommay the coding region of its as the full-length cry3Bb1 gene presentthe inof MON 88017.and Masses were calculated for each is regulatory owner. a document theoretical peptide and the raw massany data such was compared to the calculatedits theoretical reproductiondocument masses. Experimental masses (MH+) wereowner assigned tothis peaks whenof three (or more) under of isotopically resolved ion peaks wererights observethe d in the rawthis mass data. Peaks were not documentand/or of rights assessed if there were less than threeto of isotopi fall callyuse resolved peaks in the spectra, when the This may distribution,and peak heights were less than approximatelyrights twice ownerthe baseline noise, or when a mass could not be assigned due to overlapsubject with a stronger signal ±2 daltons from the mass analyzed. be the violate Known autocatalytic fragmentscopy from trypsinof digestion were identified in the raw data. and may documentpublication, It exploitation 2.8. Purity and Molecularthis Weightany Determination using SDS-PAGE permission prohibited An aliquot of the plant-producedthe Cry3Bb1 protein was diluted and mixed with loading commercial be buffer to a final concentration of 1x loading buffer. The sample was heated at ~105 °C Furthermore,any for 4 min prior toConsequently, loading withoutonto a Tris-glycine buffered 4-20% gradient polyacrylamide gel. Three amounts (1, 2, and 3 µg) of total protein were loaded into three separate wells. A sample of the E. coli-produced protein (1 µg) was included on the gel for molecular weight comparison. Bio-Rad broad range molecular weight markers (1µg/band) were included on the gel to estimate the molecular weight of the test substance. Electrophoresis was performed at a constant voltage of 150 V for 75 min. Proteins were fixed in the gel for 60 min in a solution containing 40% (v/v) methanol and 7% (v/v) glacial acetic acid, then stained for 2 h with Colloidal Brilliant Blue G stain (Sigma, St. Louis, MO). The gel was destained for 30 sec in a solution containing 10% (v/v) acetic acid and 25% (v/v) methanol, followed by 2 h in a solution containing 25% (v/v) methanol.

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Analysis of the gel was performed using a Bio-Rad Laboratories GS-710 densitometer with the supplied Quantity One software, version 4.3.0. Molecular weight values of the marker proteins were supplied by the manufacturer and used to estimate the molecular weight of each observed band in the plant-produced Cry3Bb1 protein. All visible bands within each lane were quantified. Purity was estimated as the sum of the percent optical density of the three bands identified as Cry3Bb1 protein, relative to all bands detected in the lane. Molecular weight and purity were reported as an average of all three lanes containing the plant-produced Cry3Bb1 protein. Calculations were performed using Microsoft Excel 2000. The plant and E. coli-produced proteins were considered to have equivalent molecular weights if their molecular weights differed by ≤ 5%.

2.9. Insect Bioassays

In order to assess functional activity of the Cry3Bb1 protein produced in MON 88017, aliquots of the plant- and E. coli-produced Cry3Bb1 proteins were used to estimateand the and regime. bioactivity (measured as an LC50 value) of the Cry3Bb1AG proteins incorporated into diets fed to Colorado potato beetle (CPB) larvae, a susceptible insect. The plant- and E. coli- produced proteins were considered to have equivalentBayer propertyfunctional protection activitypublishing if the difference of contents in LC values was ≤ four-fold. its 50 parties.data therefore and/oror affiliates. property intellectualthird may 2.9.a. Samples its as the of and is regulatory owner. a document The test samples included the E. coliany- andsuch plant-produced Cry3Bb1its proteins, lot numbers reproductiondocument 30-100002 and 60-100024, respectively, suspendedowner inthis buffer. Theof E. coli-produced under of rightsthe this protein was suspended indocument a 100and/or mM Tris-HCl buffer, pH 8.0,rights and the plant produced to of fall use of Cry3Bb1 was suspended in a 50 mM sodium carbonatethe buffer, pH 10, with 1mM EDTA. This may distribution,and Samples of each of the buffer typesrights were receivedowner for use as control treatments. The test samples and buffers were subjectstored in a -80° C freezer and a 5° C refrigerator, respectively. be the violate copy of and may documentpublication, 2.9.b. Insects It exploitation this any permission Colorado potato beetle eggs were obtaineprohibitedd from the New Jersey Department of the Agriculture, West Trenton,commercial NJ. Insect be eggs were incubated at temperatures ranging from 15°C to 27°C depending on desired hatch times. Furthermore,any Consequently,without 2.9.c. Bioassays

The Colorado potato beetle (CPB), Leptinotarsa decemlineata, was used to measure activity of the plant- and E. coli-produced Cry3Bb1 protein samples. The bioassay was replicated two times on different days, each using a separate batch of insects. Each bioassay replicate consisted of a series of dilutions from each Cry3Bb1 sample yielding a dose series ranging from 0-3 µg Cry3Bb1 protein/ml diet plus a buffer control. Each bioassay also included a purified water control. The Cry3Bb1 protein doses were prepared by diluting the protein with purified water and incorporating the dilution into an agar-based Colorado potato beetle diet (Bio-

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Serv, Frenchtown, NJ). This dose series in diet was expected to elicit a response range from CPB larvae that would allow for an estimate of the LC50 of the Cry3Bb1 protein. The diet mixture was then dispensed in 0.5 ml aliquots into a 128 well tray (#BIO-BA- 128, CD International, Pitman, NJ). Insect larvae were placed on these diets, approximately 16 insects per treatment, using a soft, fine, natural hair paintbrush. The infested wells were covered by a ventilated adhesive cover (#BIO-CV-16, CD International) and the insects were allowed to feed for a period of approximately seven days in an environmental chamber programmed at 27°C, ambient relative humidity and a lighting regime of 14:10 light:dark. The number of survivors was recorded after the incubation period. Control treatments weights were recorded for quality control purposes only and were not used in the analysis.

2.9.d. Statistical analysis

The bioassay data analyzed consisted of the number of surviving CPB larvaeand for each and regime. dose. Statistical analysis was performed with ReleaseAG 8.2 of the SAS statistical program running under Windows 2000 Professional (SAS Institute Inc., 1999-2001). A Probit analysis was performed to test the effect of theBayer E. coli -property and plant-produced protectionpublishing Cry3Bb1 of contents proteins on the observed mortality and to estimate the dose level requiredits for 50% parties.data therefore and/or mortality (LC50) for each replicate with the following model: or affiliates. property intellectualthird may its as p −+= C)F((1C +theeDose of) and k is kk regulatory owner. a document any such its reproductiondocument owner this of pk : Observed proportion of mortunderality underof a given dose; rightsthe this documentand/or rights C: Natural (threshold)to mortalityof fall rate;use of the F: LogisticThis cumulative distributionmay distribution,and function; rights owner Dose : Dose effectsubject in the scale of base 10 logarithm; and k be the violate e : Random residualcopy effect. of k and may documentpublication, It exploitation An integrated Probit analysisthis any was also performed to test the differences between two permission protein sources with the following model:prohibited the commercial be p −+= C)F((1C Treat + Rep + + eDose ) ijk Furthermore,any i j ijkijk Consequently,without

pijk : Observed proportion of mortality; C: Natural (threshold) mortality rate; F: Logistic cumulative distribution function; Treat : Treatment effect; i Rep j : Effect of the replicate; Dose : Dose effect in the scale of base 10 logarithm; and ijk e : Random residual effect. ijk

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The Fisher’s Exact test was used to test for significant mortality in water and buffer control samples. Significance of tests was determined at the 0.05 level.

2.10. Glycosylation Analysis

Glycosylation analysis was performed to determine whether the plant-produced Cry3Bb1 protein was post-translationally modified with covalently bound carbohydrate moieties. Aliquots of the test substance, the E. coli-produced Cry3Bb1 reference standard (a negative control), and transferrin (a positive control) were each diluted and mixed with loading buffer to a final concentration of 1× loading buffer. The samples were heated at ~98 °C for 4 min and two amounts (0.25 and 0.5 µg) of each sample were loaded, along with Bio-Rad prestained broad range molecular weight markers, on a Tris-glycine buffered 4-20% gradient polyacrylamide gel. Electrophoresis was performed at a constant voltage of 160 V for 75 min. After electrophoresis, the gel was soaked in and transfer buffer for 10 min and proteins were transferred to a 0.45and µm PVDFregime. membrane for 90 min at a constant current of 300 mA. AG

Carbohydrate detection was performed directlyBayer on the propertyPVDF membraneprotectionpublishing using the ECL of contents glycoprotein detection system (Amersham Pharmacia). The PVDF itsmembrane was gently parties.data therefore shaken overnight at 4 °C in PBS and transferred to a solution and/orof 100or mM sodium acetate affiliates. property intellectualthird may containing 10 mM sodium metaperiodate.its The membrane was incubated in the dark for as 20 min in the oxidation reagent. Thethe oxidationof and solution was removed from the is regulatory owner. a document membrane by two brief rinses withany PBS such followed by three sequentialits 10 min washes in reproductiondocument PBS. The membrane was transferred to aowner solution of 100this mM sodiof um acetate containing under of rightsthe this 25 nM biotin hydrazide documentand incubatedand/or for 60 min. The biotinrights hydrazide solution was to of fall use of removed by rinsing the membrane twice in PBS followedthe by washing the membrane in This may distribution,and PBS three times for 10 min each.rights The membraneowner was blocked for 60 min in the blocking agent supplied with the kit.subject The blocking solution was removed by rinsing and washing be the violate the membrane as described copyabove. The membof rane was incubated with streptavidin-HRP and may documentpublication, conjugate (diluted It1:6000) in PBS forexploitation 30 min to detect carbohydrate moieties bound to biotin. Excess of streptavidin-HRPthis any was removed by rinsing and washing the membrane permission as described above. Bands were visualizedprohibited using the ECL detection system and the the membrane was exposed (30commercial sec, 3 min, be and 10 min) to Hyperfilm ECL high performance chemiluminescence film (Amersham Pharmacia). Films were developed using a Konica Furthermore,any SRX-101A automatedConsequently, filmwithout processor.

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APPENDIX III

Materials and Methods used for Analysis of Expression of the Introduced Proteins

1. Test, control, and reference substances

1.1. Test substance

The test substance for this study was MON 88017. Corn plants were grown from seed lot # GLP-0203-12154-S and tissue samples analyzed in this study were produced under Production Plan # 02-01-50-01. and and regime. 1.2. Control substance AG

Bayer property protectionpublishing The control substance for this study was the ofconventional corn hybrid contentsH1200902 with its genetic background similar to MON 80017. Corn plantsparties. weredata grown fromtherefore seed lot # and/oror GLP-0203-12164-S and tissue samples fromaffiliates. the control plants were produced under property intellectualthird may its Production Plan # 02-01-50-01. as the of and is regulatory owner. a document any such its 1.3. Characterization of test and control substances.reproduction document owner this of under of rightsthe this documentand/or rights The identity of the test and controlto substaof fallnces wasuse confirmed of by verifying the chain-of- custody documentation prior to analysis. To further confirmthe the identity of the test and This may distribution,and rights owner control substances, event-specificsubject polymerase chain reaction (PCR) analyses were conducted on seed and grainbe samples. The identitythe violate of grain samples harvested from the copy of field was verified by PCR. and may documentpublication, It exploitation this 1.4. Reference substances. any permission prohibited the The E. coli-produced Cry3Bb1commercial protein be standard (lot # 30-100002) was used as the reference substanceFurthermore, forany the analysis of Cry3Bb1 protein levels. The purity-corrected total protein concentrationConsequently, of thwithoute purified standard was 1.2 mg/ml by amino acid composition analysis. The purity was 98% as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric analysis.

The E. coli-produced CP4 EPSPS protein standard (lot # 20-100015) was used as the reference substance for analysis of CP4 EPSPS protein levels. The purity-corrected total protein concentration of the purified standard was 3.7 mg/ml by amino acid composition analysis. The purity was 97% as determined by SDS-PAGE and densitometric analysis. Certificates of analysis for both the Cry3Bb1 and CP4 EPSPS reference substances were archived with the study data.

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2.0. Generation of plant samples

2.1. Summary of field design

A field production plan # 02-01-50-01 was initiated during the 2002 growing season to generate test and control substances. The field sites were located within the major corn- growing region of the U.S. (Benton County, IA; Stark County, IL; and York County, NE) and provided a variety of environmental conditions. At each site, three replicated plots of MON 88017 and the control H1200902 were planted using a randomized complete block field design. Overseason leaf (OSL), overseason whole plant (OSWP), overseason root (OSR), pollen, silk, forage, forage root, grain, stover, and senescent root tissues were collected from each replicated plot at all field sites. Throughout the field production, sample identity was maintained by using unique sample identifiers and proper chain-of- custody documentation. Upon collection, all tissue samples were placed in uniquely labeled bags or containers. All tissue samples, except grain, were stored andand shipped on and regime. dry ice. Grain samples were stored and shipped at ambientAG temperature.

2.2. Description of the collected tissues Bayer property protectionpublishing of contents its parties.data therefore Grain and/oror affiliates. property intellectualthird may its as All hand-pollinated ears in each testthe andof controland plot were collected at the R6 growth is regulatory owner. a document stage (physiological maturity). Theany ears such were shucked and dried itsto moisture content of reproductiondocument 10-15%. All cobs were shelled prior to shipping.owner this of under of rightsthe this documentand/or of rights Forage to of fall use the This may distribution,and rights owner Forage is defined as the entiresubject plant (leaves, ears, tassels, and stalk) with the root ball be the violate removed. Two whole, self-pollinatedcopy plantsof at the early dent growth stage (R4 - R6) and were collected frommay each testdocument andpublication, control plot. The forage tissue samples were pooled It exploitation from each plot during collection.this any permission prohibited Pollen the be commercial Furthermore,any Approximately 5Consequently, g of pollenwithout was collected non-systematically from each test and control replicated plot at each site at the R1 plant growth stage. Any debris in the pollen sample was removed using a fine mesh sieve.

Silk

Silks were collected non-systematically from five primary ears of plants from each test and control plot at each site. The samples were collected at the time of hand pollination (the R1 plant growth stage). The silk tissue samples were pooled from each plot during collection.

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Stover

Stover is defined as the above ground portion of the plant remaining after grain harvest. Two stover samples were collected from each test and control plot, after the harvest of self-pollinated ears. The stover samples were pooled from each plot during collection.

Overseason Leaf

The youngest immature whorl leaf (2 - 4 inches) samples were collected from 15 plants from each of the test and control plots. The first OSL samples (OSL-1) were collected at the V2 - V3 growth stage; OSL-2 samples were collected at the V5 stage; OSL-3 samples were collected at the V8 stage; and OSL-4 samples were collected at the V11 - V17 stage. The leaves corresponding to each growth stage were pooled from each plot during collection. and and regime. Overseason Whole Plant AG

Two whole plants were collected from each ofBayer the test propertyand control protection plots.publishing An OSWP of contents sample consists of shoot tissue (above-ground portion of the plant includingits leaves, parties.data therefore tassels, ears, etc.). The two whole plant samples were pooledand/or fromor each plot during affiliates. collection. At each field site, the OSWPproperty tissu intellectuale samplesthird were collectedmay to coincide with its as the collection of OSL-1 to OSL-4 theand ofOSR-1 toand OSR-4 samples. is regulatory owner. a document any such its reproductiondocument Overseason Root owner this of under of rightsthe this documentand/or of rights The OSR samples were the belowto groundof fall root massuse that had been cut from the the This may distribution,and corresponding OSWP sample. rightsThe two root sampownerles were pooled from each plot during collection. At each field site,subject the OSR tissue samples were collected to coincide with the be the violate collection of OSL-1 to OSL-4copy and OSWP-1of to OSWP-4 samples. and may documentpublication, It exploitation Forage Root this any permission prohibited Forage root is defined as the rootthe ball be removed from the plant sampled for forage. Forage root tissue samples were collectedcommercial at the early dent growth stage (R4 - R6) from each test Furthermore,any and control plot. Consequently, Collectionwithout of the forage root tissue was similar to that used for the overseason root tissue. The forage root tissue samples were pooled from two plants from each plot during collection.

Senescent Root

Senescent root is the below ground root ball removed from plants sampled for stover. Two senescent root samples were collected from each test and control plot and corresponded to the plants that had been collected for stover. Collection of the senescent root tissue was similar to that used for the overseason root tissue. The senescent root tissue samples were pooled from each plot during collection.

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2.3. Tissue processing and protein extraction methods

2.3.a. Processing

All tissue samples produced at the field sites were shipped to Monsanto for processing. During the processing step, dry ice was combined with the samples (except pollen) and then vertical cutters or mixers were used to thoroughly grind and mix the tissues. All processed tissue samples were stored in a –80°C freezer during the study.

2.3.b. Extraction

The Cry3Bb1 and CP4 EPSPS proteins were extracted from corn tissues according to the validation parameters provided in Section 2.5. of this Appendix III. All processed tissues were kept on dry ice during extract preparation. All tissues were extracted using a Harbil mixer and insoluble material was removed from the extracts by using a Serumand Filter and regime. System (Fisher Scientific, Pittsburgh, PA). The extractsAG were aliquoted and stored in a – 80°C freezer until ELISA analyses. Extraction efficiency for each tissue type was determined by successive extraction of three replicates,Bayer property where protectionthe lastpublishing extraction of contents employed a harsh buffer (e.g., 1X Laemmli buffer). its parties.data therefore and/oror affiliates. property intellectualthird may Tissue Type Tissue-to-Bufferits Ratio Extraction Buffer as the of and is regulatory owner. Forage 1:100 a 1X PBSTdocument with 0.1% (w/v) BSA any such its reproductiondocument Leaf 1:100 owner 1Xthis PBST withof 0.1% (w/v) BSA under of Pollen 1:100rightsthe 1Xthis PBST with 0.1% (w/v) BSA documentand/or rights Root to1:50 of fall use 1Xof PBST with 0.1% (w/v) BSA the Grain This 1:100may distribution,and 1X TBA with 10 mM DCA rights owner subject be the violate copy of and 2.4. ELISA reagentsmay and methodsdocumentpublication, It exploitation this any 2.4.a. CP4 EPSPS antibodies permission prohibited the be Mouse monoclonal antibodycommercial clone 39B6 (IgG2a isotype, kappa light chain; lot # Furthermore,any 6199732) specificConsequently, for the withoutCP4 EPSPS protein was purified from mouse ascites fluid using Protein-A Sepharose affinity chromatography.

The concentration of the purified IgG was determined to be 3.2 mg/ml by spectrophotometric methods. Production of the 39B6 monoclonal antibody was performed by TSD Bioservices, Inc. (Newark, DE). The purified antibody was stored in a buffer (pH 7.2) containing 0.02 M Na2HPO4 · 7H2O, 0.15 M NaCl, and 15 ppm ProClin 300 (Sigma, St. Louis, MO). The detection reagent was goat anti-CP4 EPSPS antibody (produced by Sigma, St. Louis, MO) conjugated to HRP.

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2.4.b. CP4 EPSPS ELISA method

The CP4 EPSPS ELISA was performed using an automated robotic workstation. Mouse anti-CP4 EPSPS antibody was diluted in coating buffer containing 0.15 M NaCl to a final concentration of 1.0 µg/ml and immobilized onto 96-well microtiter plates, followed by incubation in a 4°C refrigerator for >8 h. Plates were washed in 1X PBST, CP4 EPSPS protein standard or sample extract was then added (100 µl/well), and plates were incubated at 37°C. Forage sample extracts were diluted with a 1X PBST/0.1% BSA solution and grain samples were diluted with a solution containing 0.1 M tris, 0.1 M Na2B4O7 · 10H2O, 0.01 M MgCl2, 0.05% (v/v) Tween-20 at pH 7.8, and 0.2% (w/v) L- ascorbic acid (TBA). Plates were washed as before followed by the addition of 100 µl per well of anti-CP4 EPSPS peroxidase conjugate and incubated at 37°C. Plates were developed by adding 100 µl per well of TMB. The enzymatic reaction was terminated by the addition of 100 µl per well of 6 M H3PO4. Quantitation of CP4 EPSPS protein levels and was accomplished by interpolation from a CP4 EPSPS proteinand standard regime. curve that ranged in concentration from 0.456 - 14.6 ng/ml. AG

Bayer property protectionpublishing 2.4.c. Cry3Bb1 antibodies of contents its parties.data therefore and/oror Goat polyclonal antibody (lot # 7107417) affiliates.specific for the Cry3Bb1 protein was purified property intellectualthird may its using Protein-G Agarose affinity chromatography.as The concentration of the purified IgG the of and was determined to be 5.16 mg/mlis by spectrophotometricregulatory methods. owner.The purified antibody a document any such its was stored in a phosphate buffered saline (PBS) bufferreproduction (pHdocument 7.4) containing 0.001 M owner this of KH2PO4, 0.01 M Na2HPO4 · 7H2O, 0.137 underM NaCl, of and 0.0027 M KCl, with 0.02% rightsthe this documentand/or rights NaN3 (w/v) added as a preservative.to of fall use of the This may distribution,and rights owner The purified antibody (lot subject# 7107417) was coupled with biotin (Sigma, St. Louis, MO) according to the manufacturer’sbe instructions andthe assignedviolate lot # 7107462. The detection copy of reagent was NeutrAvidin (Pierce, Rockford, IL)and conjugated to horseradish peroxidase may documentpublication, (HRP). It exploitation this any permission 2.4.d. Cry3Bb1 ELISA method prohibited the commercial be The Cry3Bb1 ELISAFurthermore, wasany performed using an automated robotic workstation. Goat anti- Consequently,without Cry3Bb1 antibody was diluted in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and immobilized onto 96-well microtiter plates at a concentration of 5.0 µg/ml, followed by incubation in a 4°C refrigerator for >12 h. Plates were washed with 1X PBS with 0.05% (v/v) Tween-20 (1X PBST), blocked with the addition of 150 µl per well of 1X PBS with 0.25% (w/v) casein buffer and incubated at 37oC. Plates were washed as before followed by the addition of 100 µl per well of the Cry3Bb1 protein standard or sample in 1X PBST with 0.1% (w/v) BSA and incubated at 37°C. The procedure was completed after separate incubations with the addition of 100 µl per well of biotinylated goat anti-Cry3Bb1 antibody and NeutrAvidin-HRP. Plates were developed by adding 100 µl per well of HRP substrate, 3,3',5,5'- tetramethylbenzidine (TMB, Kirkegaard & Perry, Gaithersburg, MD). The enzymatic reaction was terminated by the addition of 100

Monsanto Company 04-CR-109F Page 171 of 223

µl per well of 6 M H3PO4. Quantitation of Cry3Bb1 protein levels was accomplished by interpolation from a Cry3Bb1 protein standard curve that ranged in concentration from 0.35 - 11.2 ng/ml.

2.4.e. Sensitivity of the ELISA

Limit of detection

The limit of detection (LOD) was calculated as the mean value plus three SD using the data generated with control sample (conventional corn) extracts for each tissue type. The LOD value in “ng/ml” was converted to “µg/g fwt” using the respective dilution factor, tissue-to-buffer ratio, and a tissue-specific method bias correction factor. Limits of detection for CP4 and Cry3Bb1 ELISA are summarized in the Table 7 of Section 2, Part VI. and and regime. Limit of quantitation AG

The limit of quantitation (LOQ) was calculatedBayer based onproperty the lowestprotectionpublishing standard of contents its concentration. The “ng/ml” value was converted to “µparties.g/g datafwt” using thetherefore respective dilution factor, tissue-to-buffer ratio, and a tissue-specific methodand/oror bias correction factor. affiliates. property intellectualthird may Limits of quantitation for CP4 and Cry3Bb1its ELISA are summarized in the Table 7 of as Section 2, Part VI. 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 IV

Materials and Methods used for Analysis of MON 88017 Composition

The composition of grain and forage of MON 88107 was analyzed and compared to conventional corn. For this analysis, MON 88017 and conventional corn were grown at replicated field trials at three locations: NE, IA and IL. Analysis of grain included proximates (protein, fat, ash and moisture), acid detergent fiber (ADF), neutral detergent fiber (NDF), total dietary fiber (TDF), amino acids, fatty acids, minerals (calcium, copper, iron, magnesium, manganese, potassium, phosphorus, sodium and zink), vitamins (B1, B2, B6, E, niacin, and folic acid), anti-nutrients (phytic acid and raffinose), secondary metabolites (furfural, ferulic, and p-coumaric acid), and carbohydrates by calculation. Analysis of forage included proximates, ADF, NDF, minerals (calcium and and phosphorus), and carbohydrates by calculation. and regime. AG The results of compositional analysis are discussed in the Section 3, Part VII and the Bayer property protectionpublishing summary of the analysis for combined sites,of summary of statistical differencescontents and the its literature and historical ranges are presented in Tablesparties. 12 –data 20 of Part VII.therefore Additional and/oror results of forage analysis by site are presenteaffiliates.d in Tables 1 – 3, and results of the grain property intellectualthird may analysis by site are presented in Tables 4its – 21 of this Appendix IV. as the of and is regulatory owner. a document any such its 1.0. Test, control and reference substances reproductiondocument owner this of under of rightsthe this MON 88017 (test), conventionaldocumentand/or corn with a similar geneticrights background (control), and to of fall use of commercial corn hybrids (references) were planted andthe grown in replicated field trials This may distribution,and across the U.S. in 2002. rights owner subject be the violate copy of 1.1. Test substance and may documentpublication, It exploitation The test substance was MONthis any 88017 grown from starting corn seed lot GLP-0203-12154- permission S. Forage and grain samples harvestedprohibited from MON 88017 were analyzed. the commercial be 1.2. Control substance Furthermore,any Consequently,without The control substance was LH198xLH59, a conventional corn hybrid grown from starting seed lot GLP-0203-12164-S. Forage and grain samples were harvested from this corn hybrid.

1.3. Reference substances

The following commercial corn forage and grain samples were used as the reference substances:

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Vendor/Hybrid ID Starting Seed Lot No. Field Site Asgrow/ RX708 REF-0203-12304-S IA Dekalb/ DK579 REF-0203-12305-S IA Garst/ 8464 IT REF-0203-12306-S IA Garst/ 8590 IT REF-0203-12307-S IA Asgrow/ RX690 REF-0203-12308-S IL Dekalb/ DKC60-15 REF-0203-12309-S IL Dekalb/ DKC61-24 REF-0203-12310-S IL Northrup King/ N59-Q9 REF-0203-12311-S IL and Mycogen/ 7474 REF-0203-12316-Sand NEregime. AG Mycogen/ 6431 REF-0203-12317-S NE

Bayer property protectionpublishing Northrup King/ N60-N2 REF-0203-12318-Sof NEcontents its parties.data therefore Northrup King/ N67-H6 REF-0203-12319-Sand/or or NE affiliates. property intellectualthird may its as the of and is regulatory owner. 2.0. Test, control and reference substance characterizationa document any such its reproductiondocument owner this of under of The identity of the forage and grainrights sampthe les from each test,this control, and reference documentand/or rights substance was verified by confirmingto of thefall chain-of-custody use of documentation supplied with the the forage and grain Thiscollected from themay plot distribution,s. and The grain from the test, control, and rights owner reference substances was furthersubject characterized by event-specific PCR analysis. be the violate copy of and 3.0. Field trial descriptionmay documentpublication, It exploitation this Corn seed was planted in theany spring of 2002 at three field sites (IA, IL, NE) in the U.S. permission At each field site, seed from the test, controprohibitedl, and reference starting material was planted the in a randomized complete commercialblock design be with three replicates per block. All the plants were grown underFurthermore, normalany agronomic field conditions for their respective geographic regions. All test Consequently,plots receivedwithout an application of Roundup UltraMAX herbicide according to the label instructions. Bulk forage samples were harvested at the late dough/early dent stage and bulk grain samples were harvested when the grain was mature. Forage and grain samples were harvested from all plots and shipped on dry ice (forage) or at ambient (grain) temperature. A sub-sample specific for compositional analysis was obtained from each bulk forage and grain sample harvested. The samples were ground and stored in a −20°C freezer located at Monsanto Company (St. Louis, MO) until shipped to Covance Laboratories Inc. (Madison, WI) on dry ice for analysis. The labels on the samples listed the composition protocol number, a unique sample number, tissue type, and storage conditions.

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4.0. Analytical methods

A total of 107 ground forage and grain samples were shipped for analysis to Covance Laboratories Inc. for analyses. The samples upon receipt were stored in a −20 °C freezer until their use. Forage samples were analyzed for proximates (protein, fat, ash, and moisture), ADF, NDF, and minerals (calcium, phosphorous). Grain samples were analyzed for proximates (protein, fat, ash, and moisture), ADF, NDF, TDF, amino acids, fatty acids, minerals (calcium, copper, iron, magnesium, manganese, phosphorous, potassium, sodium, and zinc), vitamins (B1, B2, B6, E, niacin, and folic acid), anti- nutrients (phytic acid and raffinose), and secondary metabolites (furfural, ferulic acid, and p-coumaric acid). Each analysis was based on published analytical methods that were approved in advance by the Study Director. Carbohydrate values in ground forage and grain were determined by calculation. The methods used for compositional analyses are summarized below. and and regime. 4.1. 2-Furaldehyde (FURF) AG

The method used was based on a literature methodBayer (Albala-Hurtadoproperty protectionpublishing et al., 1997). The of contents grain sample was extracted with 4% trichloroacetic acid in MilliQ water,its centrifuged, and parties.data therefore filtered. The level of 2-furaldehyde (furfural) in the extract wasand/or determinedor by RP-HPLC affiliates. with UV quantitation. The reference propertystandard intellectual wasthird ACROS 2-furaldehyde,may 99%, lot its as number A013867501. The quantitationthe of limit ofand this method was calculated to be is regulatory owner. a document approximately 0.5 ppm using the followingany such equation: its reproductiondocument owner this of under of (conc. of lowest standard) x (vol)rightsthe x (dil) / (sample weight)this = quantitation limit (ppm) documentand/or of rights to of fall use the This may distribution,and 4.2. Acid detergent fiber (ADF)rights owner subject be the violate The method used was basedcopy on an USDA Agof riculture Handbook No. 379 (1970) method. and The sample was placedmay in a fritteddocumentpublication, vessel and washed with an acidic boiling detergent It exploitation solution that dissolved thethis protein, any carbohydrate, and ash. An acetone wash was used to remove the fats and pigments. Thepermission lignocellu lose fraction was collected on the frit and prohibited determined gravimetrically. Thethe limit be of quantitation of this method was 0.1% fw. commercial Furthermore,any 4.3. Amino acidConsequently, compositionwithout (TAAP)

The method used was based on AOAC International (2000) method 982.30 that estimates the levels of 18 amino acids in the sample: alanine, arginine, aspartic acid (including asparagine), cystine (including cysteine), glutamic acid (including glutamine), glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The sample was assayed by three methods to obtain the full profile. Tryptophan required a base hydrolysis using sodium hydroxide. Sulfur-containing amino acids required an oxidation using performic acid prior to hydrolysis with hydrochloric acid. Analysis of the remaining amino acids was accomplished through direct hydrolysis with hydrochloric acid. The individual amino acids were quantitated using an automated amino acid analyzer. The limit of quantitation

Monsanto Company 04-CR-109F Page 175 of 223

of this method was 0.1 mg/g fw. The reference standards were Pierce K18, lot number DJ58806; Sigma-Aldrich L-Tryptophan, 100%, lot number 88HO4391; Sigma-Aldrich L- Cysteic acid monohydrate, 100%, lot number 111K2608; Sigma-Aldrich L-Methionine sulfone, 100%, lot number 12H3349.

4.4. Ash (ASHM)

The method used was based on AOAC International (2000) method 923.03. The sample was placed in an electric furnace at 550 °C and ignited to drive off volatile organic compounds. The nonvolatile matter remaining was quantitated gravimetrically and the percent ash was determined by calculation. The limit of quantitation of this method was 0.1% fw.

4.5. Carbohydrates (CHO) and and regime. The method used was based on an USDA AgricultureAG Handbook No. 74 (1973) method. The limit of quantitation of this method was 1.0% fw. Carbohydrate values were calculated by difference using the fresh weight-derivedBayer property data and protection thepublishing following equation: of contents its parties.data therefore % carbohydrates = 100% − (% protein + % fat + % and/orash or+ % moisture) affiliates. property intellectualthird may its as 4.6. Fat by acid hydrolysis (FAAH)the of and is regulatory owner. a document any such its reproductiondocument The method used was based on AOAC Internaowner tional (2000)this methodsof 922.06 and 954.02. under of The forage sample was hydrolyzedrights withthe hydrochloric acidthis at an elevated temperature. documentand/or of rights The fat was extracted using diethylto etherof fall follo weduse by hexane. Extracts were washed with the This may distribution,and a dilute alkali solution and filteredrights through a sodiumowner sulfate column. The extract was evaporated, dried, and weighed.subject The limit of quantitation of this method was 0.1% fw. be the violate copy of and 4.7. Fat by soxhletmay extraction document publication,(FSOX) It exploitation this any The method used was based on AOACpermission Intern ational (2000) method 960.39. The grain prohibited sample was weighed into a cellulosethe thimble containing sand or sodium sulfate and dried commercial be to remove excess moisture. Pentane was dripped through the sample to remove the fat. Furthermore,any The extract was evaporated,Consequently,without dried, and weighed. This method was used for grain sample analysis. The limit of quantitation of this method was 0.1% fw.

4.8. Fatty acids (FAPM)

The method used was based on AOCS (1997) method Ce 1-62 that estimates the levels of 22 fatty acids in the sample: 8:0 caprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:0 myristic acid, 14:1 myristoleic acid, 15:0 pentadecanoic acid, 15:1 pentadecenoic acid, 16:0 palmitic acid, 16:1 palmitoleic acid, 17:0 heptadecanoic acid, 17:1 heptadecenoic acid, 18:0 stearic acid, 18:1 oleic acid, 18:2 linoleic acid, 18:3 linolenic, 18:3 gamma linolenic acid, 20:0 arachidic acid, 20:1 eicosenoic acid, 20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, 20:4 arachidonic acid, and 22:0 behenic acid. Lipid in grain samples

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was extracted and saponified with 0.5 N sodium hydroxide in methanol. The saponification mixture was methylated with 14% (weight/volume) boron trifluoride:methanol. The resulting methyl esters were extracted with heptane containing an internal standard. The methyl esters of the fatty acids were analyzed by gas chromatography using external standards for quantitation. The limit of quantitation of this method was 0.003% fw. The reference standards were Nu Chek Prep GLC reference standard Hazelton no. 1, used as 100%, lot number O2-M; Nu Chek Prep GLC reference standard Hazelton no. 2, used as 100%, lot number JA10-H; Nu Chek Prep GLC reference standard Hazelton no. 3, used as 100%, lot number M18-L; Nu Chek Prep GLC reference standard Hazelton no. 4, used as 100%, lot number O2-M; and Nu Chek Prep methyl gamma linolenate, used as 100%, lot number U-63M-A26-L.

4.9. Folic acid (FOAN)

The method used was based on AOAC International (2000) methods 960.46and and 992.05 and regime. and Methods of Analysis for Infant Formulas (1973),AG Section C-2. The sample was hydrolyzed in potassium phosphate buffer with the addition of ascorbic acid to protect the folic acid during autoclaving. Following hydrBayerolysis, theproperty sample protection waspublishing treated with a of contents chicken-pancreas enzyme and incubated approximately 18 hours to itsliberate the bound parties.data therefore folic acid. The amount of folic acid was turbidimetrically determinedand/oror by comparing the affiliates. growth response of the bacteria Lactobacillusproperty intellectual caseithird in the sample versusmay the growth its as response in folic acid standard. Thethe limof it of quantitationand of this method was 0.06 µg/g is regulatory owner. a document fw. The reference standard was USP,any folicsuch acid, 100%, lot numberits P. reproductiondocument owner this of under of 4.10. Minerals/ICP emission spectrometryrightsthe (ICPS) this documentand/or of rights to of fall use the This may distribution,and The method used was based on rightsAOAC Internationalowner (2000) methods 984.27 and 985.01 and a literature method (Dahlquistsubject and Knoll, 1978). Samples were dried, precharred, be the violate and ashed overnight at 500°copy ± 50 °C. Ashedof samples were treated with hydrochloric acid, and dried, and dissolvedmay in 5% (v/v)document publication,hydrochlor ic acid. The amount of each element was It exploitation determined at appropriatethis wavelengths any by comparing the emission of the unknown sample, using inductively coupled permissionplasma, with the emission of the standard solutions. prohibited The limits of quantitation of thisthe method and Spex CertiPrep reference standards are commercial be listed in the table below. Furthermore,any Consequently,without

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Limit of Mineral Lot Numbers Concentration (mg/L) Quantitation (ppm) Calcium O8-67CA 10,000 20.0 Copper 9-38CU 10,000 0.500 Iron 9-02FE 1,000 2.00 Magnesium S8-87MG 10,000 20.0 Manganese 9-16MN 10,000 0.300 Phosphorus P8-77P 10,000 20.0 Potassium Q8-108K 10,000 100 Sodium P7-176NA 10,000 100 Zinc 9-34ZN 1,000 0.400 and and regime. AG

4.11. Moisture (M100). Bayer property protectionpublishing of contents its The method used was based on AOAC International (2000)parties.data methods 926.08therefore and 925.09. and/oror Samples were dried in a vacuum oven at 100°Caffiliates. to a constant weight. The moisture loss property intellectualthird may its was determined and converted to percent moisture.as The limit of quantitation of this the of and is regulatory owner. method was 0.1% fw. a document any such its reproductiondocument owner this of 4.12. Neutral detergent fiber, enzyme methodunder (NDFE)of rightsthe this documentand/or rights to of fall use of The method used was based on AACC (1998) method 32.20the and a USDA Agriculture This may distribution,and rights owner Handbook No. 379 (1970)subject method. Samples were placed in a fritted vessel and washed with a neutral boiling detergenbe t solution to dissolvethe violate the protein, carbohydrate, enzyme, copy of and ash. Fats and pigments were removed usingand an acetone wash. The hemicellulose, may documentpublication, cellulose, and ligninIt fractions were collectedexploitation on a frit and determined gravimetrically. this The limit of quantitation of thisany method was 0.1% fw. permission prohibited the 4.13. Niacin (NIAP) commercial be Furthermore,any The method usedConsequently, was basedwithout on AOAC International (2000) method 944.13. The sample was hydrolyzed with sulfuric acid and the pH was adjusted to remove interferences. The amount of niacin was turbidimetrically determined by comparing the growth response of the bacteria Lactobacillus plantarun in the samples versus the growth response in niacin standard. The limit of quantitation of this method was 0.3 µg/g fw. The reference standard was USP, niacin, 100% lot number H-1.

4.14. p-Coumaric and ferulic acids (ACID)

The method was based on a literature method (Hagerman and Nicholson, 1982). The samples were extracted with methanol using ultrasonication, and the extracts then hydrolyzed using 4 N sodium hydroxide, buffered using acetic acid/sodium hydroxide,

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acidified with 3 N hydrochloric acid, and filtered. The levels of p-coumaric and ferulic acids in the extracts were determined by RP-HPLC with UV quantitation. The reference standards were ACROS p-Hydroxycinnamic acid (p-coumaric acid), 98.8%, lot number A014307101 and ACROS 4-Hydroxy-3-methoxycinnamic acid (ferulic acid), 100%, lot number A11176601. The limit of quantitation for both analytes was calculated to be approximately 50.0 ppm using the following equation:

(conc. of lowest standard) x (vol) x (dil) / (sample weight) = quantitation limit (ppm)

4.15. Phytic acid (PHYT)

The method used was based on two literature methods (Lehrfeld 1989, 1994). Samples were extracted using 0.5 M HCl with ultrasonication. Purification and concentration was performed using a silica-based anion exchange (SAX) column. Sample analysis was conducted using a macroporous polymer HPLC column [PRP-1, 5µm (150 andx 4.1 mm)] and regime. connected to a refractive index detector. The limit AGof quantitation of this method was approximately 0.133% fw. The reference standard was Aldrich phytic acid, dodecasodium salt hydrate, 94%, lot number 14403DO.Bayer property protectionpublishing of contents its parties.data therefore 4.16. Protein (PGEN) and/oror affiliates. property intellectualthird may its as The method used was based on AOACthe of Internationaland (2000) methods 955.04 and 979.09 is regulatory owner. a document and two literature methods (Bradstreet,any such 1965; Kalthoff and Sandell,its 1948). Protein and reproductiondocument other nitrogenous compounds in the sampleowner we re reducedthis to ammoniof a by digestion of the under of sample with sulfuric acid containingrights a the merc ury catalyst thismixture. The acid digest was documentand/or of rights made alkaline, and the ammoniato was of distille fall d anduse titrated with a standard acid. The the This may distribution,and percent nitrogen was determinedrights and converted toowner percent protein by multiplication with 6.25. The limit of quantitationsubject of this method was 0.1% fw. be the violate copy of and 4.17. Pyridoxine/vitaminmay Bdocument (B6A)publication, It 6 exploitation this any The method used was based on AOACpermission Interna tional (2000) method 961.15. The sample prohibited was hydrolyzed with dilute sulfuricthe beacid. The amount of pyridoxine was turbidimetrically determinedcommercial by comparing the growth response of the yeast Furthermore,any Saccharromyces Consequently,carlsbergenesiswithout in the sample with the growth response in pyridoxine standard. The limit of quantitation of this method was 0.07 µg/g fw. The reference standard was USP pyridoxine, 100%.

4.18. Raffinose (SUGT)

This method was based on two literature methods (Mason and Slover, 1971; Brobst, 1972). The grain samples were extracted with deionized water and the extracts treated with a hydroxylamine hydrochloride solution in pyridine containing phenyl-β-D- glucoside as an internal standard. The resulting oximes were converted to silyl derivatives by treatment with hexamethyldisilane and trifluoracetic acid and analyzed by gas chromatography using a flame ionization detector. The reference standard was

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Sigma, D(+)-raffinose, 99%, lot number 091K12741. The quantitation limit of this method was calculated to be 0.0358% fw using the following equation:

(conc. of lowest standard) x (vol) x (dil) / (sample size) = quantitation limit (%)

4.19. Riboflavin/vitamin B2 (B2FV)

The method used was based on AOAC International (2000) method 940.33. The sample was hydrolyzed with dilute HCl. The amount of riboflavin was determined by comparing the growth response of the bacteria, Lactobacillus casei, in the sample hydrolysate with the bacterial growth response in varying amounts of riboflavin standard. The bacterial growth response was measured turbidimetrically. The limit of quantitation of this method was 0.2 µg/g fw. The reference standard was USP riboflavin, 100%.

4.20. Thiamin/vitamin B1 (BIDE). and and regime. AG The method used was based on AOAC International (2000) methods 942.23, 953.17, and 957.17. The sample was autoclaved under weakBayer acid conditionsproperty protection topublishing extract the thiamin. of contents The resulting solution was incubated with a buffered enzyme solutionits to release any parties.data therefore bound thiamin. The solution was purified on an ion-exchangeand/or column.or An aliquot was affiliates. taken and reacted with potassium ferricyanidproperty eintellectual to convertthird thiamin tomay thiochrome. The its as thiochrome was extracted into isobutylthe of alcoholand and read on a fluorometer against a is regulatory owner. a document known standard. The limit of quantitationany such of this method was 0.01mg/100gits fw. The reproductiondocument reference standard was USP, thiamin, 100%.owner this of under of rightsthe this documentand/or of rights 4.21. Total dietary fiber (TDF)to of fall use the This may distribution,and rights owner The method used was basedsubject on AOAC International (2000) method 985.29. Duplicate be the violate samples were gelatinized withcopy alpha-amylase of and digested with enzymes to break down and starch and protein. mayEthanol wasdocument publication,added to each sample to precipitate the soluble fiber. It exploitation The samples were filteredthis and any the residue was rinsed with ethanol and acetone to remove starch and protein degradation productspermission and moisture. Protein content was determined prohibited for one of the duplicates; ash contentthe be was determined for the other. The total dietary fiber in the sample was calculatedcommercial using the protein and ash values. The limit of quantitation ofFurthermore, this methodany was approximately 1.0% fw. Consequently,without

4.22. Vitamin E (EFD2)

The method used was based on three literature methods (Cort et al., 1983; McMurray et al., 1980; Speek et al., 1985). Samples were saponified to break down fat and release vitamin E. The saponified mixture was extracted with ethyl ether and quantitated directly by HPLC on a silica column. The limit of quantitation of this method was approximately 0.003 mg/g fw. The reference standard was USP alpha tocopherol, 100%.

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5.0. Control of bias

The test, control, and reference substances from each respective plot within the field sites were produced under similar agronomic conditions. To control and/or minimize bias, the samples were analyzed in the order specified by a computer-generated randomized sample list. The Study Director generated the randomized sample list and forwarded it to Covance Laboratories Inc. prior to analysis.

6.0. Statistical analysis

6.1. Data processing

The data were statistically analyzed by Certus International, Inc. The following formulas were used for re-expression of the data for statistical analysis: and and regime. Component FromAG (X) To Formula Proximates (excluding moisture), % FW % DW X/d* Fiber, Phytic Acid, Raffinose Bayer property protectionpublishing Calcium, Magnesium, Phosphorus, of ppm FW % DW contents (X/d) X 10-4 its parties.data therefore Potassium, Sodium and/oror affiliates. Copper, Iron, Manganese, Zincproperty intellectual ppmthird FW mg/kg DWmay X/d its Ferulic Acid, Furfural as ppm FW µg/g DW X/d the of and is regulatory owner. Niacin, Folic Acid, Vitamin B2, µg/ga FW document mg/kg DW X/d any such its Vitamin B6 reproductiondocument owner this of Vitamin B1 undermg/100g of FW mg/kg DW 10 (X/d) rightsthe this 3 Vitamin E documentand/or mg/g FW rights mg/kg DW 10 (X/d) to of fall use of Amino Acids (AA) mg/g FW the % Total AA (100)Xj/Σ Xj, This may distribution,and rights owner for each AA j subject Fatty Acids (FA)be g/100gthe violate FW % Total FA (100)Xj/Σ Xj, copy of and for each FA j * may documentpublication, d is the fractionIt of the sampleexploitation that is dry matter. this any permission The following 15 compositional analytesprohibited with >50% of observations below the LOQ of the the assay were excluded fromcommercial statistical be analysis: sodium, 2-furaldehyde, 8:0 caprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:0 myristic acid, 14:1 myristoleic acid, 15:0 Furthermore,any pentadecanoic acid,Consequently, 15:1 pentadecenoicwithout acid, 17:0 heptadecanoic acid, 17:1 heptadecenoic acid, 18:3 gamma linolenic acid, 20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, and 20:4 arachidonic acid.

There were observations below the LOQ observed for 16:1 palmitoleic acid (five values), 22:0 behenic acid (one value) and vitamin E (one value). To include a complete data set for these analytes in the statistical analysis, a value equal to 50% of the LOQ was assigned for these seven data points. The data was also assessed for potential outliers using a studentized PRESS residuals calculation and no outliers were identified in this evaluation.

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6.2. Statistical methodology

At the field sites, the test, control, and reference substances were grown in single plots randomly assigned within each of three replication blocks. The compositional components for the test and control substances were statistically analyzed using a mixed model analysis of variance. The three replicated sites were analyzed separately and combined. 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: and and regime. Yijk = U + Ti + Lj + B(L)jk + LTij + eAGijk ,

where Yijk = unique individual observation, UBayer = overall property mean, protection Ti =publishing hybrid effect, Lj = of contents random location effect, B(L)jk = random block within location effect,its LTij = random parties.data therefore location by hybrid interaction effect, and eijk = residual error. and/or Foror each compositional affiliates. component, the forage and grain fromproperty the test intellectual substancethird was comparedmay to the control. its as the of and is regulatory owner. a document A range of observed values from theany reference such substances were determinedits for each reproductiondocument analytical component. Additionally, the refereowner nce substancesthis dataof were used to develop under of population tolerance intervals. A tolerancerightsthe interval is anthis inte rval that one can claim, with documentand/or of rights a specified degree of confidence,to containsof fall at leastuse a specified proportion, p, of an entire the This may distribution,and sampled population for the parameterrights measured. owner For each compositional component, 99% tolerance intervals weresubject calculated that are expected to contain, with 95% be the violate confidence, 99% of the quanticopyties expressedof in the population of conventional hybrids. and Each tolerance intervalmay estimatedocument publication,was based upon one observation per reference. It exploitation Individual hybrids with multiplethis any observations were summarized across sites to obtain a single estimate for inclusion in toleranpermissionce interval calculations. Because negative prohibited quantities are not possible, calculatedthe be negative lower tolerance bounds were set to zero. commercial ® Furthermore,any SAS software wasConsequently, used towithout generate all summary statistics and perform all analyses. Report tables present p-values from SAS® as either <0.001 or the actual value truncated to three decimal places.

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Table 1. Comparison of the proximates, fiber, and mineral content in forage collected at the IA field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b

and Ash (% dw) 3.62 ± 0.24 4.06 ± 0.24 -0.44 ± 0.35 and -1.28,0.41regime. 0.254 (2.62 - 6.78) (3.30 - 3.90) (3.74 - 4.67) (-1.37AG - 0.16) [0.72,7.42] Carbohydrates (% dw) 87.27 ± 0.48 87.30 ± 0.48 -0.029 ± 0.57 -1.42,1.36 0.960 (81.86 - 89.90) Bayer property protectionpublishing (86.19 - 87.88) (86.78 - 87.71) of (-1.22 - 1.10) contents [78.70,93.43] its parties.data therefore Fat, total (% dw) 1.35 ± 0.24 1.16 ± 0.24 0.19 ± 0.31 and/or-0.57,0.95or 0.560 (0.69 - 2.92) affiliates. (1.02 - 1.97) (0.83 - 1.63)property (-0.0062intellectualthird - 0.34) may [0.80,2.95] its as the of and Moisture (% fw) 69.70 ± 0.51 69.67is ± 0.51 0.033regulatory ± 0.65 -1.56,1.62owner. 0.960 (65.20 - 78.60) a document (68.50 - 70.70) (69.40 - any69.80) such (-1.30 - 0.90) its [59.37,80.83] reproductiondocument owner this of Protein (% dw) 7.75 ± 0.39 7.48 ± 0.39 under0.27 ±of 0.55 -1.07,1.62 0.633 (6.31 - 9.96) rightsthe this documentand/or rights (7.44 - 7.94) (6.79to - 8.43)of fall (-0.99use - 1.10)of [4.17,11.81] the ADF (% dw) 26.05 ± 0.80This 25.92 ± 0.80may distribution,and0.14 ± 1.01 -2.33,2.60 0.897 (19.16 - 35.55) rights owner (24.32 - 28.23) subject(24.17 - 26.90) (-2.58 - 1.54) [13.95,38.96] be the violate copy of NDF (% dw) 39.48 ± 2.39 40.11 ± 2.39 and-0.63 ± 3.38 -8.89,7.64 0.858 (30.27 - 57.93) may documentpublication, (36.51 - 45.05)It (38.08 - 41.18)exploitation (-4.55 - 6.97) [23.80,54.73] this Calcium (% dw) 0.24 ± 0.021 0.27any ± 0.021 -0.027 ± 0.019 -0.073,0.018 0.190 (0.13 - 0.32) permission (0.22 - 0.26) (0.20 - 0.31)prohibited (-0.049 - 0.011) [0.11,0.32] the be Phosphorus (% dw) 0.23 ± 0.011 commercial0.26 ± 0.011 -0.034 ± 0.015 -0.071,0.0030 0.065 (0.16 - 0.31) (0.21 Furthermore,- 0.25) any (0.25 - 0.27) (-0.060 - -0.018) [0.095,0.38] Consequently,without adw=dry weight; fw=fresh weight; ADF=acid detergent fiber; NDF=neutral detergent fiber, S.E. = standard error, C.I.= confidence interval, T.I.= tolerance interval cWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

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Table 2. Comparison of the of proximates, fiber, and mineral content in forage collected at the IL field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b

and Ash (% dw) 4.40 ± 0.43 3.86 ± 0.43 0.54 ± 0.48 and -0.63,1.71regime. 0.305 (2.62 - 6.78) (3.60 - 5.53) (3.59 - 4.01) (0.016AG - 1.55) [0.72,7.42] Carbohydrates (% dw) 86.05 ± 0.62 86.56 ± 0.62 -0.51 ± 0.73 -2.29,1.27 0.510 (81.86 - 89.90) Bayer property protectionpublishing (84.13 - 87.21) (85.94 - 87.06) of (-1.80 - 0.15) contents [78.70,93.43] its parties.data therefore Fat, total (% dw) 1.40 ± 0.24 1.61 ± 0.24 -0.21 ± 0.34 and/or-1.05,0.63or 0.558 (0.69 - 2.92) affiliates. (0.80 - 1.75) (1.32 - 1.87)property intellectual(-0.83third - 0.31) may [0.80,2.95] its as Moisture (% fw) 70.77 ± 0.63 70.00 ±the 0.63 of and0.77 ± 0.65 -0.81,2.35 0.280 (65.20 - 78.60) is regulatory owner. a document (70.20 - 71.80) (69.10 - any71.00) such (-0.70 - 1.90) its [59.37,80.83] reproductiondocument owner this of Protein (% dw) 8.16 ± 0.20 7.97 ± 0.20 under0.19 ±of 0.29 -0.52,0.89 0.541 (6.31 - 9.96) rightsthe this (7.52 - 8.58) document(7.72and/or - 8.21) (-0.48 - 0.66) rights [4.17,11.81] to of fall use of the ADF (% dw) 25.53 ± 0.75This 24.10 ± 0.75may distribution,and1.43 ± 0.97 -0.93,3.80 0.188 (19.16 - 35.55) rights owner (24.81 - 26.17) subject(23.34 - 24.72) (0.88 - 1.95) [13.95,38.96] be the violate NDF (% dw) 37.20 ± 1.22 copy36.76 ± 1.22 of 0.44 ± 1.68 -3.67,4.55 0.803 (30.27 - 57.93) and (33.89 - 39.06)may (35.86documentpublication, - 37.22) (-3.32 - 3.20) [23.80,54.73] It exploitation Calcium (% dw) 0.22 ± 0.015 this0.23 any ± 0.015 -0.0045 ± 0.022 -0.058,0.048 0.841 (0.13 - 0.32) permission (0.20 - 0.25) (0.21 - 0.25)prohibited (-0.054 - 0.024) [0.11,0.32] the Phosphorus (% dw) 0.26 ± 0.016 commercial0.23 ± 0.016 be 0.034 ± 0.021 -0.018,0.086 0.163 (0.16 - 0.31) (0.23 Furthermore,- 0.28) any (0.20 - 0.25) (-0.018 - 0.079) [0.095,0.38] Consequently,without adw=dry weight; fw=fresh weight; ADF=acid detergent fiber; NDF=neutral detergent fiber, S.E. = standard error, C.I.= confidence interval, T.I.= tolerance interval bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

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Table 3. Comparison of the proximates, fiber, and mineral content in forage collected at the NE field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b

and Ash (% dw) 3.96 ± 0.20 4.21 ± 0.20 -0.25 ± 0.19 and -0.71,0.20regime. 0.224 (2.62 - 6.78) (3.59 - 4.36) (4.06 - 4.32) (-0.73AG - 0.10) [0.72,7.42] Carbohydrates (% dw) 85.25 ± 0.69 85.58 ± 0.69 -0.32 ± 0.91 -2.56,1.91 0.735 (81.86 - 89.90) Bayer property protectionpublishing (83.54 - 86.16) (84.43 - 86.18) of (-2.58 - 1.73) contents [78.70,93.43] its parties.data therefore Fat, total (% dw) 2.10 ± 0.46 2.19 ± 0.46 -0.095 ± 0.65 and/oror-1.68,1.49 0.888 (0.69 - 2.92) affiliates. (1.49 - 3.13) (1.14 - 2.97)property intellectual(-1.47third - 1.99) may [0.80,2.95] its as Moisture (% fw) 72.10 ± 0.47 72.30 ±the 0.47 of and-0.20 ± 0.66 -1.81,1.41 0.771 (65.20 - 78.60) is regulatory owner. a document (70.90 - 72.70) (71.90 - any72.70) such (-1.40 - 0.80) its [59.37,80.83] reproductiondocument owner this of Protein (% dw) 8.69 ± 0.35 8.02 ± 0.35 under0.67 ±of 0.50 -0.55,1.89 0.226 (6.31 - 9.96) rightsthe this (8.42 - 8.97) document(7.03and/or - 8.54) (-0.12 - 1.65)rights [4.17,11.81] to of fall use of the ADF (% dw) 28.05 ± 1.32This 26.32 ± 1.32may distribution,and1.73 ± 1.83 -2.75,6.20 0.381 (19.16 - 35.55) rights owner (24.29 - 29.97) subject(24.95 - 28.13) (-0.66 - 4.08) [13.95,38.96] be the violate NDF (% dw) 35.35 ± 1.40 copy38.14 ± 1.40 of -2.79 ± 1.98 -7.63,2.05 0.207 (30.27 - 57.93) and (33.44 - 37.73)may (37.36documentpublication, - 39.50) (-4.63 - 0.37) [23.80,54.73] It exploitation Calcium (% dw) 0.21 ± 0.012 this0.20 any ± 0.012 0.0044 ± 0.012 -0.025,0.034 0.727 (0.13 - 0.32) permission (0.19 - 0.22) (0.18 - 0.24)prohibited (-0.024 - 0.023) [0.11,0.32] the Phosphorus (% dw) 0.27 ± 0.019 commercial0.26 ± 0.019 be 0.0055 ± 0.014 -0.028,0.039 0.705 (0.16 - 0.31) (0.23 Furthermore,- 0.30) any (0.24 - 0.30) (-0.0066 - 0.021) [0.095,0.38] Consequently,without adw=dry weight; fw=fresh weight; ADF=acid detergent fiber; NDF=neutral detergent fiber, S.E. = standard error, C.I.= confidence interval, T.I.= tolerance interval bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

Monsanto Company 04-CR-109F Page 185 of 223

Table 4. Comparison of the amino acid content in grain collected at the IA field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b Alanine (% total AA) 7.37 ± 0.031 7.36 ± 0.031 0.0039 ± 0.039 -0.091,0.099 0.923 (7.24 - 8.16) (7.29 – 7.46) (7.34 - 7.40) (-0.055 - 0.12) and [6.66,8.49] and regime. AG Arginine (% total AA) 4.66 ± 0.065 4.50 ± 0.065 0.15 ± 0.093 -0.075,0.38 0.153 (3.72 - 5.08) (4.57 – 4.74) (4.40 - 4.63) (-0.060 - 0.34) [3.34,5.67] Bayer property protectionpublishing of contents Aspartic acid (% total AA) 6.23 ± 0.058 6.18 ± 0.058 0.047 ± 0.065 -0.11,0.21its 0.497 (6.18 - 6.81) parties.data therefore (6.09 – 6.32) (6.04 - 6.28) (0.0070 - 0.087) and/oror [5.77,7.16] affiliates. property intellectualthird may Cystine (% total AA) 2.16 ± 0.037 2.26 ± 0.037 its -0.11 ± 0.053 -0.24,0.022 0.089 (1.82 - 2.58) as (2.10 – 2.26) (2.20 - the2.30) of (-0.19and - -0.028) [1.46,2.89] is regulatory owner. a document any such its Glutamic acid (% total AA) 20.08 ± 0.079 20.02 ± 0.079 0.060reproduction ± 0.11 document -0.20,0.32 0.599 (19.46 - 21.57) owner this of (19.80 - 20.24) (19.91 - 20.10) under(-0.26 -of 0.34) [18.01,22.15] rightsthe this documentand/or of rights Glycine (% total AA) 3.55 ± 0.032 3.56 to± 0.032of fall -0.0069use ± 0.042 -0.11,0.096 0.875 (3.29 - 4.03) the (3.47 – 3.62)This (3.50 - 3.61)may distribution,(-0.058and - 0.064) [2.81,4.54] rights owner subject Histidine (% total AA) 3.07 ± 0.032 3.08 ± 0.032 -0.010the violate ± 0.038 -0.10,0.082 0.796 (2.50 - 3.12) be copy (3.02 – 3.10) (2.99 - 3.14) of(-0.034 - 0.026) [2.16,3.60] document and may publication,exploitation Isoleucine (% total AA) 3.52 ± 0.046It 3.53 ± 0.046 -0.0057 ± 0.062 -0.16,0.15 0.930 (3.39 - 3.79) this any (3.43 – 3.57) (3.51 - 3.54) (-0.099 - 0.048) [3.30,3.84] permission prohibited Leucine (% total AA) 12.93 ± 0.085 12.91the ± 0.085 0.016 ± 0.094 -0.21,0.25 0.867 (12.11 - 14.35) commercial be (12.69 - 13.11) (12.76 - 13.04) (-0.25 - 0.23) [10.72,15.18] Furthermore,any aAA=amino acid, S.E. = standard error, C.I.= confidenceConsequently, interval,without T.I.= tolerance interval bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

Monsanto Company 04-CR-109F Page 186 of 223

Table 4 (cont.) Comparison of the amino acid content in grain collected at the IA field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b

and Lysine (% total AA) 2.77 ± 0.059 2.72 ± 0.059 0.053 ± 0.082 and -0.15,0.25regime. 0.541 (2.44 - 3.27) (2.67 - 2.87) (2.57 - 2.82) (-0.045AG - 0.11) [2.06,3.73] Methionine (% total AA) 2.02 ± 0.029 2.16 ± 0.029 -0.14 ± 0.015 -0.17,-0.10 <0.001 (1.70 - 2.47) Bayer property protectionpublishing (1.96 - 2.05) (2.11 - 2.20) of (-0.15 - -0.12) contents [1.37,2.60] its parties.data therefore Phenylalanine (% total AA) 5.10 ± 0.067 5.08 ± 0.067 0.019 ± 0.080 and/oror-0.18,0.21 0.818 (4.82 - 5.39) affiliates. (4.97 - 5.18) (5.01 - 5.22)property intellectual(-0.037third - 0.13) may [4.57,5.71] its as the of and Proline (% total AA) 9.50 ± 0.15 9.63is ± 0.15 -0.13regulatory ± 0.21 -0.65,0.39owner. 0.572 (8.35 - 9.72) a document (9.32 - 9.69) (9.54 - any9.80) such (-0.22 - -0.050) its [7.60,10.37] reproductiondocument owner this of Serine (% total AA) 4.87 ± 0.093 4.85 ± 0.093 under0.024 of± 0.10 -0.22,0.27 0.815 (4.81 - 5.23) rightsthe this documentand/or rights (4.65 - 5.03) (4.64to - 4.96)of fall (-0.0024use - of0.063) [4.60,5.43] the Threonine (% total AA) 3.29 ± 0.056This 3.26 ± 0.056may distribution,and0.035 ± 0.070 -0.14,0.21 0.637 (2.96 - 3.55) rights owner (3.15 - 3.38) subject(3.15 - 3.33) (0.0034 - 0.084) [2.89,3.84] be the violate copy of Tryptophan (% total AA) 0.56 ± 0.017 0.59 ± 0.017 -0.032and ± 0.024 -0.090,0.026 0.221 (0.44 - 0.83) may documentpublication, (0.53 - 0.58)It (0.58 - 0.60)exploitation (-0.053 - -0.013) [0.36,0.77] this any Tyrosine (% total AA) 3.59 ± 0.20 3.57 ± 0.20 0.026 ± 0.28 -0.66,0.71 0.929 (2.26 - 3.80) permission (3.48 - 3.66) (3.52 - 3.66)prohibited (-0.052 - 0.14) [2.62,4.26] the commercial be Valine (% total AA) 4.73 ± 0.066 4.74 ± 0.066 -0.0077 ± 0.088 -0.22,0.21 0.933 (4.44 - 5.04) (4.60 Furthermore,- 4.82) any (4.67 - 4.77) (-0.069 - 0.045) [4.22,5.27] Consequently,without aAA=amino acid, S.E. = standard error, C.I.= confidence interval, T.I.= tolerance interval bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

Monsanto Company 04-CR-109F Page 187 of 223

Table 5. Comparison of the amino acid content in grain collected at the IL field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b Alanine (% total AA) 7.65 ± 0.059 7.59 ± 0.059 0.057 ± 0.079 -0.14,0.25 0.498 (7.24 - 8.16) (7.56 - 7.70) (7.52 - 7.65) (-0.087 - 0.18) and [6.66,8.49] and regime. AG Arginine (% total AA) 4.24 ± 0.066 4.23 ± 0.066 0.014 ± 0.060 -0.13,0.16 0.827 (3.72 - 5.08) (4.10 - 4.35) (4.20 - 4.27) (-0.12 – 0.15) [3.34,5.67] Bayer property protectionpublishing of contents Aspartic acid (% total AA) 6.16 ± 0.071 6.29 ± 0.071 -0.13 ± 0.10 -0.37,0.12its 0.253 (6.18 - 6.81) parties.data therefore (6.11 - 6.24) (6.06 - 6.45) (-0.34 – 0.18) and/oror [5.77,7.16] affiliates. property intellectualthird may Cystine (% total AA) 2.21 ± 0.045 2.17 ± 0.045its 0.042 ± 0.057 -0.097,0.18 0.488 (1.82 - 2.58) as (2.18 - 2.24) (2.12 the- 2.19) of and(-0.0079 - 0.11) [1.46,2.89] is regulatory owner. a document any such its Glutamic acid (% total AA) 20.66 ± 0.14 20.59 ± 0.14 0.069reproduction ± 0.19document -0.39,0.53 0.728 (19.46 - 21.57) owner this of (20.43 - 20.87) (20.39 - 20.84) under (-0.40of – 0.48) [18.01,22.15] rightsthe this documentand/or of rights Glycine (% total AA) 3.40 ± 0.042 3.44to ± 0.042of fall -0.035use ± 0.055 -0.17,0.10 0.554 (3.29 - 4.03) the (3.32 - 3.46)This (3.31 - 3.54)may distribution,and(-0.081 - 0.0027) [2.81,4.54] rights owner subject Histidine (% total AA) 2.95 ± 0.024 2.89 ± 0.024 the0.063 violate ± 0.034 -0.019,0.14 0.110 (2.50 - 3.12) be copy (2.93 - 2.99) (2.87 - 2.92) of (0.046 - 0.073) [2.16,3.60] document and may publication,exploitation Isoleucine (% total AA) 3.64 ± 0.064It 3.54 ± 0.064 0.11 ± 0.090 -0.11,0.33 0.284 (3.39 - 3.79) this any (3.53 - 3.71) (3.45 - 3.67) (-0.15 - 0.25) [3.30,3.84] permission prohibited Leucine (% total AA) 13.54 ± 0.13 13.35the ± 0.13 0.18 ± 0.16 -0.20,0.57 0.285 (12.11 - 14.35) commercial be (13.46 - 13.62) (13.06 - 13.69) (-0.16 - 0.56) [10.72,15.18] Furthermore,any Consequently,without aAA=amino acid, S.E. = standard error, C.I.= confidence interval, T.I.= tolerance interval bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

Monsanto Company 04-CR-109F Page 188 of 223

Table 5 (cont.) Comparison of the amino acid content in grain collected at the IL field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b

and Lysine (% total AA) 2.64 ± 0.070 2.62 ± 0.070 0.019 ± 0.099 and -0.22,0.26regime. 0.854 (2.44 - 3.27) (2.42 - 2.78) (2.49 - 2.71) (-0.072 -AG 0.070) [2.06,3.73] Methionine (% total AA) 2.01 ± 0.018 1.99 ± 0.018 0.017 ± 0.026 -0.047,0.081 0.534 (1.70 - 2.47) Bayer property protectionpublishing (1.99 - 2.03) (1.96 - 2.02) (-0.0043of - 0.046) contents [1.37,2.60] its parties.data therefore Phenylalanine (% total AA) 5.27 ± 0.071 5.16 ± 0.071 0.11 ± 0.10 and/or-0.14,0.35or 0.330 (4.82 - 5.39) affiliates. (5.19 - 5.31) (5.05 - 5.32)property (-0.13intellectualthird - 0.25) may [4.57,5.71] its as Proline (% total AA) 9.34 ± 0.15 9.17 ± 0.15the of 0.17and ± 0.22 -0.36,0.70 0.470 (8.35 - 9.72) is regulatory owner. a document (9.02 - 9.56) (8.85 - 9.63)any such (-0.61 - 0.71) its [7.60,10.37] reproductiondocument owner this of Serine (% total AA) 4.81 ± 0.11 4.91 ± 0.11 under-0.10 ±of 0.16 -0.48,0.28 0.544 (4.81 - 5.23) rightsthe this (4.66 - 5.04) document(4.63and/or - 5.13) (-0.47 - 0.42) rights [4.60,5.43] to of fall use of the Threonine (% total AA) 3.19 ± 0.080This 3.26 ± 0.080may distribution,-0.071and ± 0.11 -0.35,0.21 0.554 (2.96 - 3.55) rights owner (3.10 - 3.30) subject(3.06 - 3.37) (-0.25 - 0.24) [2.89,3.84] be the violate Tryptophan (% total AA) 0.56 ± 0.033 0.58copy ± 0.033 of-0.023 ± 0.046 -0.14,0.089 0.628 (0.44 - 0.83) and (0.51 - 0.60)may (0.53document -publication, 0.68) (-0.17 - 0.062) [0.36,0.77] It exploitation Tyrosine (% total AA) 2.90 ± 0.24 this3.44 any ± 0.24 -0.53 ± 0.34 -1.36,0.29 0.162 (2.26 - 3.80) permission (2.35 - 3.41) (3.29 - 3.53)prohibited (-1.18 - 0.12) [2.62,4.26] the Valine (% total AA) 4.82 ± 0.084 commercial4.77 ± 0.084 be 0.047 ± 0.11 -0.23,0.32 0.691 (4.44 - 5.04) (4.69 -Furthermore, 4.92) (4.60 - 4.94) (-0.25 - 0.26) [4.22,5.27] any Consequently,without aAA=amino acid, S.E. = standard error, C.I.= confidence interval, T.I.= tolerance interval bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

Monsanto Company 04-CR-109F Page 189 of 223

Table 6. Comparison of the amino acid content in grain collected at the NE field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b Alanine (% total AA) 7.63 ± 0.038 7.70 ± 0.038 -0.068 ± 0.054 -0.20,0.063 0.248 (7.24 - 8.16) (7.59 - 7.69) (7.59 - 7.79) (-0.19 - 0.095) and [6.66,8.49] and regime. Arginine (% total AA) 4.37 ± 0.069 4.13 ± 0.069 0.24 ±AG 0.098 -0.0022,0.48 0.051 (3.72 - 5.08) (4.28 - 4.49) (4.01 - 4.25) (0.085 - 0.36) [3.34,5.67] Bayer property protectionpublishing Aspartic acid (% total AA) 6.27 ± 0.059 6.29 ± 0.059 of-0.017 ± 0.068 -0.18,0.15contents 0.811 (6.18 - 6.81) its parties.data therefore (6.18 - 6.34) (6.11 - 6.40) (-0.10 - 0.064) and/oror [5.77,7.16] affiliates. Cystine (% total AA) 2.06 ± 0.071 2.03 ± 0.071property intellectual0.027third ± 0.10 -0.22,0.27may 0.795 (1.82 - 2.58) its as (1.93 - 2.20) (1.93 - the2.14) of (-0.20and - 0.17) [1.46,2.89] is regulatory owner. a document Glutamic acid (% total AA) 20.47 ± 0.085 20.71 ± any0.085 such -0.24 ± 0.12 -0.53,0.058its 0.096 (19.46 - 21.57) reproductiondocument of (20.33 - 20.62) (20.47 - 20.84) owner(-0.52 - 0.15)this [18.01,22.15] under of rightsthe this documentand/or rights Glycine (% total AA) 3.41 ± 0.052 3.35 to± 0.052of fall 0.060use ± 0.063of -0.094,0.21 0.378 (3.29 - 4.03) (3.37 - 3.45) (3.18 - 3.44) (-0.037 - 0.19)the [2.81,4.54] This may distribution,and rights owner Histidine (% total AA) 2.93 ± 0.029 subject2.88 ± 0.029 0.044 ± 0.041 -0.056,0.14 0.322 (2.50 - 3.12) be the violate (2.90 - 2.95) copy(2.83 - 2.96) of (-0.056 - 0.10) [2.16,3.60] and may documentpublication, Isoleucine (% total AA) 3.61 ± 0.056It 3.63 ± 0.056exploitation -0.027 ± 0.079 -0.22,0.17 0.748 (3.39 - 3.79) (3.56 - 3.64) this(3.45 any - 3.76) (-0.12 - 0.16) [3.30,3.84] permission Leucine (% total AA) 13.36 ± 0.14 13.67 ± 0.14prohibited -0.31 ± 0.20 -0.79,0.17 0.165 (12.11 - 14.35) the (13.21 - 13.46) commercial(13.26 - 14.11) be (-0.69 - 0.20) [10.72,15.18] a Furthermore,any AA=amino acids Consequently,without bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

Monsanto Company 04-CR-109F Page 190 of 223

Table 6 (cont.) Comparison of the amino acid content in grain collected at the NE field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b

and Lysine (% total AA) 2.65 ± 0.061 2.65 ± 0.061 0.00084 ± 0.070 and -0.17,0.17regime. 0.990 (2.44 - 3.27) (2.60 - 2.70) (2.50 - 2.74) (-0.057AG - 0.095) [2.06,3.73] Methionine (% total AA) 1.91 ± 0.042 1.88 ± 0.042 0.031 ± 0.059 -0.11,0.18 0.614 (1.70 - 2.47) Bayer property protectionpublishing (1.85 - 1.95) (1.83 - 1.94) of(-0.085 - 0.12) contents [1.37,2.60] its parties.data therefore Phenylalanine (% total AA) 5.16 ± 0.046 5.18 ± 0.046 -0.020 ± 0.066 and/or-0.18,0.14or 0.776 (4.82 - 5.39) affiliates. (5.13 - 5.22) (5.14 - 5.20)property (-0.077intellectualthird - 0.075) may [4.57,5.71] its as Proline (% total AA) 9.33 ± 0.094 9.23 ± the0.094 of 0.098and ± 0.13 -0.23,0.42 0.490 (8.35 - 9.72) is regulatory owner. a document (9.14 - 9.64) (9.12 - any9.41) such (-0.20 - 0.51) its [7.60,10.37] reproductiondocument owner this of Serine (% total AA) 4.80 ± 0.046 4.97 ± 0.046 under-0.17 ±of 0.065 -0.33,-0.0084 0.042 (4.81 - 5.23) rightsthe this (4.80 - 4.81) document(4.82and/or - 5.09) (-0.29 - -0.026)rights [4.60,5.43] to of fall use of the Threonine (% total AA) 3.19 ± 0.027This 3.23 ± 0.027may distribution,and-0.042 ± 0.038 -0.14,0.052 0.317 (2.96 - 3.55) rights owner (3.14 - 3.24) subject(3.15 - 3.29) (-0.12 - 0.0037) [2.89,3.84] be the violate Tryptophan (% total AA) 0.50 ± 0.018 copy0.47 ± 0.018 of 0.029 ± 0.025 -0.032,0.090 0.292 (0.44 - 0.83) and (0.48 - 0.51)may (0.41documentpublication, - 0.51) (-0.012 - 0.096) [0.36,0.77] It exploitation Tyrosine (% total AA) 3.54 ± 0.25 this 3.27any ± 0.25 0.27 ± 0.35 -0.59,1.14 0.470 (2.26 - 3.80) permission (3.50 - 3.58) (2.58 - 3.62)prohibited (-0.12 - 0.98) [2.62,4.26] the Valine (% total AA) 4.81 ± 0.060 commercial4.72 ± 0.060 be 0.090 ± 0.084 -0.12,0.30 0.326 (4.44 - 5.04) (4.77 Furthermore,- 4.83) any (4.65 - 4.85) (-0.077 - 0.19) [4.22,5.27] Consequently,without aAA=amino acid, S.E. = standard error, C.I.= confidence interval, T.I.= tolerance interval bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

Monsanto Company 04-CR-109F Page 191 of 223

Table 7. Comparison of the fatty acid content in grain collected at the IA field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b 16:0 palmitic (% total FA) 10.16 ± 0.71 12.94 ± 0.71 -2.78 ± 0.98 -5.18,-0.39 0.029 (9.29 - 17.81) (10.11 - 10.23) (10.14 - 14.57) (-4.35 - -0.033) and [6.51,16.50] and regime. 16:1 pamitoleic (% total FA) 0.20 ± 0.0049 0.21 ± 0.0049 -0.0084AG ± 0.0069 -0.025,0.0084 0.265 (0.054 - 0.21) (0.19 - 0.21) (0.20 - 0.22) (-0.021 - -0.0021) [0.0017,0.28] Bayer property protectionpublishing 18:0 stearic (% total FA) 1.84 ± 0.066 2.01 ± 0.066 of -0.17 ± 0.088 -0.38,0.047contents 0.104 (1.68 - 2.30) its parties.data therefore (1.80 - 1.88) (1.76 - 2.15) (-0.28 - 0.041) and/oror [1.41,2.53] affiliates. 18:1 oleic (% total FA) 22.56 ± 0.27 22.11 ± 0.27property intellectual0.45third ± 0.38 may-0.47,1.37 0.278 (19.79 - 34.46) its as (22.20 - 23.07) (21.43 the- 22.98) of and(-0.78 - 1.13) [9.25,44.14] is regulatory owner. a document 18:2 linoleic (% total FA) 63.25 ± 0.66 60.41 any± 0.66 such 2.84 ± 0.87 its 0.72,4.96 0.017 (51.64 - 64.12) reproductiondocument of (62.73 - 63.72) (59.10 - 62.96)owner (0.75 -this 4.19) [41.22,74.09] under of rightsthe this documentand/or rights 18:3 linolenic (% total FA) 1.25 ± 0.087 1.57to ± 0.087of fall -0.32use ±of 0.12 -0.61,-0.029 0.036 (0.84 - 1.91) (1.24 - 1.26) (1.22 - 1.77) (-0.53 - 0.016)the [0.42,1.95] This may distribution,and rights owner 20:0 arachidic (% total FA) 0.35 ± 0.0029 subject0.36 ± 0.0029 -0.0037 ± 0.0039 -0.013,0.0059 0.384 (0.36 - 0.45) be the violate (0.35 - 0.36) copy(0.35 - 0.36) of (-0.012 - 0.0037) [0.31,0.49] and may documentpublication, 20:1 eicosenoic (% total FA) 0.24 ± 0.0015It 0.24 ± 0.0015exploitation 0.00044 ± 0.0019 -0.0043,0.0052 0.827 (0.24 - 0.36) (0.24 - 0.24) this any(0.24 - 0.24) (-0.0013 - 0.0031) [0.18,0.40] permission 22:0 behenic (% total FA) 0.15 ± 0.0061 0.15 ± 0.0061prohibited -0.0090 ± 0.0087 -0.030,0.012 0.341 (0.074 - 0.24) the (0.14 - 0.15) commercial(0.15 - be0.16) (-0.017 - 0.0059) [0.071,0.25] a Furthermore,any FA=fatty acid, S.E. = standard error, C.I.= confidenceConsequently, interval,without T.I. = tolerance interval bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

Monsanto Company 04-CR-109F Page 192 of 223

Table 8. Comparison of the fatty acid content in grain collected at the IL field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b 16:0 palmitic (% total FA) 10.18 ± 0.083 10.34 ± 0.083 -0.16 ± 0.12 -0.45,0.13 0.223 (9.29 - 17.81) (10.07 - 10.36) (10.18 - 10.42) (-0.35 - 0.19) and [6.51,16.50] and regime. 16:1 pamitoleic (% total FA) 0.18 ± 0.0084 0.17 ± 0.0084 0.0069AG ± 0.012 -0.022,0.036 0.581 (0.054 - 0.21) (0.16 - 0.19) (0.16 - 0.19) (-0.029 - 0.025) [0.0017,0.28] Bayer property protectionpublishing 18:0 stearic (% total FA) 2.04 ± 0.032 2.06 ± 0.032 of -0.026 ± 0.045 -0.14,0.085contents 0.589 (1.68 - 2.30) its parties.data therefore (2.00 - 2.08) (1.99 - 2.12) (-0.089 - 0.094) and/oror [1.41,2.53] affiliates. 18:1 oleic (% total FA) 22.53 ± 0.084 23.29 ± 0.084property intellectual-0.76third ± 0.12 -1.05,-0.47may <0.001 (19.79 - 34.46) its as (22.50 - 22.56) (23.08 the- 23.51) of and(-0.94 - -0.58) [9.25,44.14] is regulatory owner. a document 18:2 linoleic (% total FA) 63.11 ± 0.15 62.15 any± 0.15 such 0.96 ± 0.21 its 0.44,1.48 0.003 (51.64 - 64.12) reproductiondocument of (62.84 - 63.29) (62.01 - 62.25)owner (0.66 -this 1.19) [41.22,74.09] under of rightsthe this documentand/or rights 18:3 linolenic (% total FA) 1.20 ± 0.011 1.21to ± 0.011of fall -0.0053use of± 0.016 -0.045,0.034 0.753 (0.84 - 1.91) (1.19 - 1.23) (1.19 - 1.22) (-0.033 - the0.036) [0.42,1.95] This may distribution,and rights owner 20:0 arachidic (% total FA) 0.37 ± 0.0042 subject0.39 ± 0.0042 -0.011 ± 0.0059 -0.026,0.0030 0.100 (0.36 - 0.45) be the violate (0.37 - 0.38) copy(0.38 - 0.39) of (-0.021 - 0.0019) [0.31,0.49] and may documentpublication, 20:1 eicosenoic (% total FA) 0.24 ± 0.0045It 0.25 ± 0.0045exploitation -0.0077 ± 0.0064 -0.023,0.0080 0.274 (0.24 - 0.36) (0.23 - 0.24) this any(0.24 - 0.26) (-0.019 - 0.0024) [0.18,0.40] permission 22:0 behenic (% total FA) 0.15 ± 0.0046 0.15 ± 0.0046prohibited 0.0012 ± 0.0066 -0.015,0.017 0.856 (0.074 - 0.24) the (0.14 - 0.16) commercial(0.14 - be0.15) (-0.014 - 0.014) [0.071,0.25] a Furthermore,any FA=fatty acid, S.E. = standard error, C.I.= confidenceConsequently, interval,without T.I. = tolerance interval bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

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Table 9. Comparison of the fatty acid content in grain collected at the NE field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b 16:0 palmitic (% total FA) 10.38 ± 0.14 10.51 ± 0.14 -0.14 ± 0.20 -0.62,0.35 0.520 (9.29 - 17.81) (10.10 - 10.52) (10.16 - 10.85) (-0.44 - 0.36) and [6.51,16.50] and regime. 16:1 pamitoleic (% total FA) 0.17 ± 0.0039 0.17 ± 0.0039 -0.0074AG ± 0.0055 -0.021,0.0060 0.224 (0.054 - 0.21) (0.16 - 0.17) (0.16 - 0.18) (-0.013 - 0.00074) [0.0017,0.28] Bayer property protectionpublishing 18:0 stearic (% total FA) 2.16 ± 0.065 2.13 ± 0.065 of 0.036 ± 0.092 -0.19,0.26contents 0.711 (1.68 - 2.30) its parties.data therefore (2.12 - 2.19) (1.93 - 2.23) (-0.11 - 0.25) and/oror [1.41,2.53] affiliates. 18:1 oleic (% total FA) 23.12 ± 0.22 23.19 ± 0.22property intellectual-0.068third ± 0.22 -0.61,0.48may 0.768 (19.79 - 34.46) its as (22.56 - 23.53) (22.59 - the23.50) of (-0.22and - 0.048) [9.25,44.14] is regulatory owner. a document 18:2 linoleic (% total FA) 62.19 ± 0.36 61.99 ± any0.36 such 0.20 ± 0.49 its-1.00,1.41 0.692 (51.64 - 64.12) reproductiondocument of (61.86 - 62.54) (61.21 - 63.18) owner(-0.64 - 0.65)this [41.22,74.09] under of rightsthe this documentand/or rights 18:3 linolenic (% total FA) 1.19 ± 0.019 1.20 ±to 0.019of fall -0.0044use ± of0.026 -0.069,0.060 0.871 (0.84 - 1.91) (1.15 - 1.23) (1.19 - 1.21) (-0.039 - 0.043)the [0.42,1.95] This may distribution,and rights owner 20:0 arachidic (% total FA) 0.39 ± 0.0046 subject0.40 ± 0.0046 -0.010 ± 0.0065 -0.026,0.0054 0.158 (0.36 - 0.45) be the violate (0.38 - 0.39) copy(0.38 - 0.41) of(-0.028 - 0.0088) [0.31,0.49] and may documentpublication, 20:1 eicosenoic (% total FA) 0.25 ± 0.0060It 0.25 ± 0.0060exploitation -0.0031 ± 0.0085 -0.024,0.018 0.731 (0.24 - 0.36) (0.24 - 0.26) this(0.24 any - 0.26) (-0.019 - 0.019) [0.18,0.40] permission 22:0 behenic (% total FA) 0.15 ± 0.0042 0.16 ± 0.0042prohibited -0.011 ± 0.0060 -0.025,0.0039 0.122 (0.074 - 0.24) the (0.14 - 0.15) commercial(0.14 - 0.17) be (-0.018 - 0.0034) [0.071,0.25] a Furthermore,any FA=fatty acid, S.E. = standard error, C.I.= confidenceConsequently, interval,without T.I. = tolerance interval bWith 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero.

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Table 10. Comparison of the mineral content in grain collected at the IA field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b Calcium (% dw) 0.0047 ± 0.00017 0.0050 ± 0.00017 -0.00030 ± 0.00024 -0.00090,0.00030 0.267 (0.0032 - 0.0060) (0.0047 - 0.0047) (0.0049 - 0.0051) (-0.00045 - -0.00020) and [0.0017,0.0062] and regime. Copper (mg/kg dw) 1.74 ± 0.10 1.95 ± 0.10 -0.22 AG± 0.099 -0.46,0.025 0.070 (1.01 - 2.34) (1.71 - 1.78) (1.76 - 2.27) (-0.49 - -0.052) [0.17,3.00] Bayer property protectionpublishing Iron (mg/kg dw) 22.18 ± 0.81 22.31 ± 0.81 of -0.13 ± 1.15 -2.95,2.68contents 0.912 (16.42 - 26.03) its parties.data therefore (21.77 - 22.92) (20.80 - 23.93) (-2.16 - 2.12) and/oror [12.60,31.26] affiliates. Magnesium (% dw) 0.14 ± 0.0068 0.14 ± 0.0068property -0.0056intellectualthird ± 0.0096 may-0.029,0.018 0.579 (0.10 - 0.14) its as (0.13 - 0.15) (0.13 - 0.16)the of (-0.024and - 0.018) [0.088,0.16] is regulatory owner. a document Manganese (mg/kg dw) 9.05 ± 0.40 8.67 ± 0.40any such 0.38 ± 0.52 its -0.90,1.66 0.491 (4.96 - 9.81) reproductiondocument of (9.01 - 9.11) (7.55 - 9.41) owner (-0.37 - 1.56)this [2.45,10.60] under of rightsthe this documentand/or rights Phosphorus (% dw) 0.39 ± 0.018 0.40 ± to0.018 of fall -0.010use ± of0.024 -0.070,0.049 0.682 (0.28 - 0.41) (0.37 - 0.40) (0.36 - 0.43) (-0.052 - 0.042)the [0.24,0.44] This may distribution,and rights owner Potassium (% dw) 0.42 ± 0.017 subject0.44 ± 0.017 -0.015 ± 0.024 -0.074,0.044 0.552 (0.29 - 0.43) be the violate (0.41 - 0.44) (0.40copy - 0.47) of (-0.052 - 0.037) [0.27,0.48] and may documentpublication, Zinc (mg/kg dw) 24.17 ± 0.90It 24.05 ± 0.90exploitation 0.11 ± 1.08 -2.53,2.76 0.919 (17.15 - 26.18) (23.92 - 24.50) (22.02this any - 25.81) (-1.74 - 1.90) [13.42,31.37]

permission prohibited adw=dry weight; mg/kg=ppm (parts per million), S.E. = standard error, C.I.= confidence interval, T.I.= tolerance interval the be bWith 95% confidence, interval contains 99% of the valuescommercial expressed in the population of commercial lines. Negative limits were set to zero. Furthermore,any Consequently,without

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Table 11. Comparison of the mineral content in grain collected at the IL field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b Calcium (% dw) 0.0058 ± 0.00015 0.0060 ± 0.00015 -0.00015 ± 0.00022 -0.00068,0.00038 0.510 (0.0032 - 0.0060) (0.0056 - 0.0060) (0.0058 - 0.0061) (-0.00024 - -0.00006) and [0.0017,0.0062] and regime. Copper (mg/kg dw) 1.89 ± 0.076 1.82 ± 0.076 0.069 ±AG 0.11 -0.19,0.33 0.541 (1.01 - 2.34) (1.68 - 2.05) (1.64 - 1.91) (-0.23 - 0.41) [0.17,3.00] Bayer property protectionpublishing Iron (mg/kg dw) 21.91 ± 0.54 22.06 ± 0.54 of-0.15 ± 0.76 -2.01,1.72contents 0.854 (16.42 - 26.03) its parties.data therefore (21.40 - 22.78) (21.45 - 22.54) (-0.64 - 0.24) and/oror [12.60,31.26] affiliates. Magnesium (% dw) 0.14 ± 0.0024 0.14 ± 0.0024 property-0.00076 intellectualthird ± 0.0034 -0.0091,0.0076may 0.830 (0.10 - 0.14) its as (0.13 - 0.14) (0.14 - 0.14)the of (-0.0057and - 0.0028) [0.088,0.16] is regulatory owner. a document Manganese (mg/kg dw) 10.20 ± 0.27 9.81 ± 0.27any such 0.40 ± 0.36 its-0.49,1.29 0.317 (4.96 - 9.81) reproductiondocument of (9.85 - 10.46) (9.46 - 10.29) owner(0.0092 - 1.00)this [2.45,10.60] under of rightsthe this documentand/or rights Phosphorus (% dw) 0.38 ± 0.0067 0.38 ± 0.0067to of fall 0.0010use ± 0.0095of -0.022,0.024 0.918 (0.28 - 0.41) (0.37 - 0.40) (0.38 - 0.38) (-0.011 - 0.016)the [0.24,0.44] This may distribution,and rights owner Potassium (% dw) 0.40 ± 0.0061 0.41subject ± 0.0061 -0.0073 ± 0.0087 -0.029,0.014 0.433 (0.29 - 0.43) be the violate (0.39 - 0.42) (0.41copy - 0.42) (-0.015of - 0.0084) [0.27,0.48] and may documentpublication, Zinc (mg/kg dw) 26.40 ± 0.55 It 26.66 ± 0.55 exploitation -0.26 ± 0.64 -1.84,1.31 0.697 (17.15 - 26.18) (25.43 - 27.27) (25.99this any - 27.18) (-0.68 - 0.45) [13.42,31.37]

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Table 12. Comparison of the mineral content in grain collected at the NE field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b Calcium (% dw) 0.0056 ± 0.00043 0.0063 ± 0.00043 -0.00076 ± 0.00038 -0.0017,0.00017 0.093 (0.0032 - 0.0060) (0.0054 - 0.0057) (0.0056 - 0.0069) (-0.0013 - -0.00029) and [0.0017,0.0062] and regime. Copper (mg/kg dw) 1.57 ± 0.15 2.21 ± 0.15 -0.64 ±AG 0.21 -1.15, -0.12 0.023 (1.01 - 2.34) (1.48 - 1.68) (1.83 - 2.63) (-0.95 - -0.35) [0.17,3.00] Bayer property protectionpublishing Iron (mg/kg dw) 20.44 ± 0.92 21.16 ± 0.92 of-0.72 ± 1.30 -3.89,2.45contents 0.598 (16.42 - 26.03) its parties.data therefore (20.07 - 20.74) (20.31 - 22.02) (-1.51 - 0.43) and/oror [12.60,31.26] affiliates. Magnesium (% dw) 0.13 ± 0.0071 0.13 ± 0.0071property -0.00030 intellectualthird ± 0.0090 -0.022,0.022may 0.974 (0.10 - 0.14) its as (0.13 - 0.14) (0.13 - 0.14)the of (-0.0027and - 0.0032) [0.088,0.16] is regulatory owner. a document Manganese (mg/kg dw) 9.90 ± 0.84 9.63 ± 0.84any such 0.28 ± 0.90 its-1.92,2.47 0.768 (4.96 - 9.81) reproductiondocument of (9.36 - 10.76) (8.69 - 10.44) owner(-0.39 - 0.90)this [2.45,10.60] under of rightsthe this documentand/or rights Phosphorus (% dw) 0.40 ± 0.026 0.40 ± 0.026to of fall -0.0032use ± 0.031of -0.080,0.073 0.923 (0.28 - 0.41) (0.39 - 0.41) (0.38 - 0.42) (-0.013 - 0.0097)the [0.24,0.44] This may distribution,and rights owner Potassium (% dw) 0.41 ± 0.020 0.41subject ± 0.020 0.0036 ± 0.023 -0.052,0.059 0.880 (0.29 - 0.43) be the violate (0.40 - 0.42) (0.38copy - 0.42) of(-0.019 - 0.023) [0.27,0.48] and may documentpublication, Zinc (mg/kg dw) 23.02 ± 0.99 It 24.05 ± 0.99exploitation -1.03 ± 1.40 -4.45,2.40 0.491 (17.15 - 26.18) (22.31 - 23.77) (22.33this any - 26.85) (-3.87 - 1.43) [13.42,31.37]

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Table 13. Comparison of the proximates and fiber content in grain collected at the IA field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b

and Ash (% dw) 1.55 ± 0.078 1.67 ± 0.078 -0.12 ± 0.11 and -0.39,0.15regime. 0.313 (1.04 - 1.86) (1.37 - 1.66) (1.51 - 1.82) (-0.45AG - 0.15) [0.94,1.73] Carbohydrates (% dw) 82.72 ± 0.27 83.23 ± 0.27 -0.50 ± 0.37 -1.40,0.39 0.216 (81.46 - 86.68) Bayer property protectionpublishing (82.23 - 83.39) (82.75 - 83.62) of(-1.39 - 0.074) contents [79.39,89.67] its parties.data therefore Fat, total (% dw) 3.53 ± 0.064 3.58 ± 0.064 -0.044 ± 0.090 and/or-0.26,0.18or 0.645 (2.38 - 4.43) affiliates. (3.44 - 3.59) (3.53 - 3.61)property (-0.16intellectualthird - 0.062) may [0.74,6.01] its as Moisture (% fw) 9.38 ± 0.18 9.93 ±the 0.18 of and-0.55 ± 0.20 -1.05,-0.054 0.034 (9.15 - 14.90) is regulatory owner. a document (9.03 - 9.70) (9.73 - 10.10)any such (-0.70 - -0.40) its [4.67,17.56] reproductiondocument owner this of Protein (% dw) 12.21 ± 0.24 11.51 ± 0.24 under0.70 ±of 0.30 -0.042,1.45 0.060 (9.26 - 13.37) rightsthe this (11.63 - 12.58) document(11.22and/or - 11.96) (0.28 - 1.37) rights [6.20,15.35] to of fall use of the ADF (% dw) 3.99 ± 0.31This 3.78 ± 0.31may distribution,and0.21 ± 0.37 -0.70,1.12 0.594 (2.39 - 4.89) rights owner (3.50 - 4.40) subject(3.24 - 4.69) (-0.62 - 1.16) [1.89,5.23] be the violate NDF (% dw) 13.24 ± 0.39 copy13.80 ± 0.39 of -0.56 ± 0.56 -1.93,0.80 0.353 (8.41 - 16.54) and (13.07 - 13.58)may (12.99documentpublication, - 14.29) (-1.21 - 0.58) [3.51,21.65] It exploitation TDF (% dw) 16.22 ± 0.61 this17.39 any ± 0.61 -1.17 ± 0.87 -3.30,0.95 0.225 (11.80 - 23.04) permission (15.45 - 16.71) (16.88 - 17.84)prohibited (-1.43 - -0.96) [5.72,27.10]

the be aADF=acid detergent fiber; dw=dry weight; fw=fresh weight;commercial NDF=ne utral detergent fiber; TDF=total dietary fiber, S.E. = standard error, C.I.= confidence interval, T.I.= tolerance interval Furthermore,any bWith 95% confidence, interval contains 99% ofConsequently, the valueswithout expre ssed in the population of commercial lines. Negative limits were set to zero.

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Table 14. Comparison of the proximates and fiber content in grain collected at the IL field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b

and Ash (% dw) 1.49 ± 0.10 1.38 ± 0.10 0.11 ± 0.14 and -0.24,0.47regime. 0.458 (1.04 - 1.86) (1.31 - 1.66) (1.23 - 1.59) (-0.27AG - 0.43) [0.94,1.73] Carbohydrates (% dw) 82.27 ± 0.38 82.39 ± 0.38 -0.12 ± 0.45 -1.22,0.98 0.800 (81.46 - 86.68) Bayer property protectionpublishing (82.16 - 82.39) (82.00 - 83.12) of (-0.96 - 0.34) contents [79.39,89.67] its parties.data therefore Fat, total (% dw) 3.82 ± 0.10 4.11 ± 0.10 -0.29 ± 0.14 and/or-0.63,0.057or 0.087 (2.38 - 4.43) affiliates. (3.72 - 3.96) (3.99 - 4.36)property (-0.63intellectualthird - -0.027) may [0.74,6.01] its as Moisture (% fw) 13.13 ± 0.30 13.87 ±the 0.30 of and-0.73 ± 0.38 -1.65,0.19 0.099 (9.15 - 14.90) is regulatory owner. a document (13.10 - 13.20) (13.50 - any14.20) such (-1.10 - -0.40) its [4.67,17.56] reproductiondocument owner this of Protein (% dw) 12.39 ± 0.34 12.11 ± 0.34 under0.28 ±of 0.38 -0.65,1.21 0.489 (9.26 - 13.37) rightsthe this (12.31 - 12.54) document(11.56and/or - 12.43) (-0.10 - 0.98) rights [6.20,15.35] to of fall use of the ADF (% dw) 3.67 ± 0.22This 3.30 ± 0.22may distribution,and0.37 ± 0.27 -0.29,1.03 0.219 (2.39 - 4.89) rights owner (3.31 - 4.34) subject(2.97 - 3.84) (0.27 - 0.50) [1.89,5.23] be the violate NDF (% dw) 12.59 ± 0.61 copy10.70 ± 0.61 of 1.89 ± 0.87 -0.23,4.01 0.072 (8.41 - 16.54) and (11.74 - 13.58)may (10.39documentpublication, - 10.94) (0.97 - 2.64) [3.51,21.65] It exploitation TDF (% dw) 17.11 ± 0.93 this14.01 any ± 0.93 3.10 ± 1.31 -0.10,6.31 0.055 (11.80 - 23.04) permission (15.44 - 18.64) (13.18 - 14.45)prohibited (1.04 - 4.19) [5.72,27.10]

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Table 15. Comparison of the proximates and fiber content in grain collected at the NE field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b

and Ash (% dw) 1.57 ± 0.15 1.72 ± 0.15 -0.14 ± 0.21 and -0.66,0.38regime. 0.528 (1.04 - 1.86) (1.37 - 1.68) (1.54 - 1.97) (-0.28 AG- 0.032) [0.94,1.73] Carbohydrates (% dw) 81.95 ± 0.26 81.39 ± 0.26 0.57 ± 0.34 -0.26,1.39 0.143 (81.46 - 86.68) Bayer property protectionpublishing (81.61 - 82.29) (80.67 - 82.04) of(-0.092 - 0.94) contents [79.39,89.67] its parties.data therefore Fat, total (% dw) 3.55 ± 0.084 3.69 ± 0.084 -0.14 ± 0.12 and/or-0.42,0.15or 0.295 (2.38 - 4.43) affiliates. (3.44 - 3.69) (3.54 - 3.89)property intellectual(-0.45third - 0.15) may [0.74,6.01] its as Moisture (% fw) 10.80 ± 0.060 11.00 ±the 0.060 of -0.20and ± 0.085 -0.41,0.0079 0.056 (9.15 - 14.90) is regulatory owner. a document (10.80 - 10.80) (10.90 - any11.10) such (-0.30 - -0.10) its [4.67,17.56] reproductiondocument owner this of Protein (% dw) 12.93 ± 0.23 13.22 ± 0.23 under-0.29 ±of 0.27 -0.96,0.38 0.329 (9.26 - 13.37) rightsthe this (12.89 - 13.00) document(12.68and/or - 13.82) (-0.82 - 0.21) rights [6.20,15.35] to of fall use of the ADF (% dw) 3.66 ± 0.22This 3.55 ± 0.22may distribution,and0.11 ± 0.31 -0.66,0.88 0.743 (2.39 - 4.89) rights owner (3.58 - 3.80) subject(3.00 - 4.07) (-0.50 - 0.60) [1.89,5.23] be the violate NDF (% dw) 11.48 ± 0.38 copy11.11 ± 0.38 of 0.38 ± 0.34 -0.46,1.22 0.314 (8.41 - 16.54) and (10.99 - 12.33)may (10.38documentpublication, - 11.59) (-0.22 - 0.75) [3.51,21.65] It exploitation TDF (% dw) 15.40 ± 0.89 this14.79 any ± 0.89 0.60 ± 1.21 -2.35,3.55 0.635 (11.80 - 23.04) permission (13.57 - 17.38) (13.36 - 15.96)prohibited (-2.39 - 2.30) [5.72,27.10]

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Table 16. Comparison of the vitamin content in grain collected at the IA field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b Folic acid (mg/kg dw) 0.50 ± 0.026 0.45 ± 0.026 0.050 ± 0.032 -0.029,0.13 0.170 (0.28 - 0.61) (0.42 - 0.55) (0.42 - 0.47) (-0.032 - 0.11) and [0.12,0.77] and regime. Niacin (mg/kg dw) 22.92 ± 0.59 23.13 ± 0.59 -0.21AG ± 0.64 -1.77,1.35 0.749 (14.11 - 27.77) (21.74 - 24.14) (22.43 - 23.92) (-0.69 - 0.23) [3.19,34.49] Bayer property protectionpublishing of contents Vitamin B1 (mg/kg dw) 2.54 ± 0.066 3.07 ± 0.066 -0.53 ± 0.084 -0.74,-0.33 <0.001 (2.69 - 3.73) its parties.data therefore (2.42 - 2.65) (2.99 - 3.23) (-0.68 - -0.35) and/oror [1.96,4.38] affiliates. Vitamin B (mg/kg dw) 1.01 ± 0.021 1.02 ± 0.021property -0.0081intellectualthird ± 0.029 -0.079,0.063may 0.790 (0.88 - 1.32) 2 its as (0.98 - 1.04) (0.99 - the1.07) of (-0.057and - 0.042) [0.67,1.51] is regulatory owner. a document Vitamin B (mg/kg dw) 7.01 ± 0.34 7.56 ± any0.34 such -0.55 ± 0.36 its-1.43,0.32 0.171 (4.93 - 7.24) 6 reproductiondocument of (6.57 - 7.28) (6.79 - 8.54) owner(-1.27 - -0.18)this [4.29,7.84] under of rightsthe this documentand/or rights Vitamin E (mg/kg dw) 14.79 ± 0.45 13.95to ± 0.45of fall 0.83use ± 0.59of -0.61,2.28 0.207 (8.09 - 21.97) (14.62 - 15.01) (13.55 - 14.46) (0.27 - 1.46)the [0,29.69] This may distribution,and rights owner a subject dw=dry weight; Vitamin B1 =Thiamine; Vitamin B2 =Riboflavin;be Vitamin B6 =Pyridoxine,the violate S.E. = standard error, C.I.= confidence interval, T.I.= tolerance interval copy of b document and With 95% confidence, interval contains 99% of themay values expressedpublication, exploitationin the population of commercial lines. Negative limits were set to zero. It this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table 17. Comparison of the vitamin content in grain collected at the IL field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b Folic acid (mg/kg dw) 0.43 ± 0.026 0.45 ± 0.026 -0.020 ± 0.037 -0.11,0.070 0.603 (0.28 - 0.61) (0.38 - 0.50) (0.43 - 0.47) (-0.074 - 0.064) and [0.12,0.77] and regime. Niacin (mg/kg dw) 21.10 ± 0.30 22.52 ± 0.30 -1.42AG ± 0.42 -2.44,-0.39 0.014 (14.11 - 27.77) (20.39 - 21.52) (22.20 - 23.08) (-1.91 - -0.68) [3.19,34.49] Bayer property protectionpublishing of contents Vitamin B1 (mg/kg dw) 2.30 ± 0.061 3.10 ± 0.061 -0.79 ± 0.086 -1.00,-0.58 <0.001 (2.69 - 3.73) its parties.data therefore (2.30 - 2.30) (3.03 - 3.14) (-0.83 - -0.73) and/oror [1.96,4.38] affiliates. Vitamin B (mg/kg dw) 1.15 ± 0.066 1.13 ± 0.066property intellectual0.025third ± 0.076 -0.16,0.21may 0.753 (0.88 - 1.32) 2 its as (1.12 - 1.22) (1.08 - the1.17) of (-0.045and - 0.14) [0.67,1.51] is regulatory owner. a document Vitamin B (mg/kg dw) 7.49 ± 0.44 6.78 ± any0.44 such 0.72 ± 0.58 its-0.70,2.13 0.261 (4.93 - 7.24) 6 reproductiondocument of (6.88 - 8.06) (5.65 - 7.46) owner(-0.58 - 2.40)this [4.29,7.84] under of rightsthe this documentand/or rights Vitamin E (mg/kg dw) 11.47 ± 3.47 11.57to ± 3.47of fall -0.10use ± 4.91of -12.72,12.52 0.984 (8.09 - 21.97) (6.08 - 16.13) (1.74 - 17.23) (-11.15 - 14.39)the [0,29.69] This may distribution,and rights owner a subject dw=dry weight; Vitamin B1 =Thiamine; Vitamin B2 =Riboflavin;be Vitamin B6 =Pyridoxine,the violate S.E. = standard error, C.I.= confidence interval, T.I.= tolerance interval copy of b and With 95% confidence, interval contains 99% of themay values expredocumentssedpublication, in the population of commercial lines. Negative limits were set to zero. It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table 18. Comparison of the vitamin content in grain collected at the NE field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b Folic acid (mg/kg dw) 0.51 ± 0.041 0.54 ± 0.041 -0.027 ± 0.058 -0.17,0.12 0.662 (0.28 - 0.61) (0.44 - 0.60) (0.48 - 0.59) (-0.054 - 0.015) and [0.12,0.77] and regime. Niacin (mg/kg dw) 18.80 ± 0.76 19.59 ± 0.76 -0.79AG ± 1.02 -3.28,1.70 0.465 (14.11 - 27.77) (17.04 - 19.73) (19.08 - 19.89) (-2.04 - -0.16) [3.19,34.49] Bayer property protectionpublishing of contents Vitamin B1 (mg/kg dw) 2.58 ± 0.073 3.56 ± 0.073 -0.98 ± 0.10 -1.23,-0.73 <0.001 (2.69 - 3.73) its parties.data therefore (2.47 - 2.69) (3.49 - 3.60) (-1.02 - -0.90) and/oror [1.96,4.38] affiliates. Vitamin B (mg/kg dw) 1.14 ± 0.061 1.24 ± 0.061property -0.092intellectualthird ± 0.073 -0.27,0.088may 0.256 (0.88 - 1.32) 2 its as (1.10 - 1.18) (1.16 - the1.33) of (-0.17and - 0.021) [0.67,1.51] is regulatory owner. a document Vitamin B (mg/kg dw) 6.99 ± 0.23 6.97 ± any0.23 such 0.026 ± 0.22 its-0.52,0.57 0.912 (4.93 - 7.24) 6 reproductiondocument of (6.67 - 7.20) (6.47 - 7.63) owner(-0.52 - 0.40)this [4.29,7.84] under of rightsthe this documentand/or rights Vitamin E (mg/kg dw) 16.18 ± 0.74 16.71to ± 0.74of fall -0.52use ± 1.04of -3.08,2.03 0.633 (8.09 - 21.97) (15.70 - 16.93) (15.15 - 17.77) (-2.08 - 1.78)the [0,29.69] This may distribution,and rights owner a subject dw=dry weight; S.E. = standard error, C.I.= confidence interval, T.I.= tolerance intervalthe violate be copy Vitamin B1 =Thiamine of document and Vitamin B2 =Riboflavin may publication,exploitation Vitamin B =Pyridoxine It 6 this any

b permission With 95% confidence, interval contains 99% of the values expressed in prohibitedthe population of commercial lines. Negative limits were set to zero. the commercial be Furthermore,any Consequently,without

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Table 19. Comparison of the secondary metabolites and anti-nutrients content in grain collected at the IA field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b and and regime. Ferulic acid (µg/g dw) 2092.58 ± 83.12 2024.32 ± 83.12 68.26AG ± 117.54 -219.36,355.88 0.582 (1717.17 - 2687.57) (1986.75 - 2275.48) (1927.55 - 2187.67) (-200.92 - 347.92) [1415.19,3173.90] Bayer property protectionpublishing p-Coumaric acid (µg/g dw) 162.93 ± 6.63 164.32 ± 6.63 of -1.39 ± 9.37 -24.32,21.55contents 0.887 (152.30 - 319.15) its (153.93 - 168.19) (156.20 - 173.24) (-9.58parties. - 11.99)data therefore [43.13,384.34] and/oror affiliates. Phytic acid (% dw) 0.94 ± 0.093 0.87 ± property0.093 intellectualthird0.069 ± 0.12 may-0.22,0.35 0.575 (0.45 - 1.00) its (0.85 - 1.03) (0.76 - 1.01) as (0.014 - 0.097) [0.28,1.12] the of and is regulatory owner. a document Raffinose (% dw) 0.15 ± 0.0049 0.14 any± 0.0049 such 0.0079 ± 0.0070 its -0.0091,0.025 0.298 (0.073 - 0.22) reproductiondocument (0.14 - 0.16) (0.14 - 0.15)owner (-0.0018this - 0.022)of [0,0.32] under of rightsthe this a documentand/or rights dw=dry weight, S.E. = standard error, C.I.= confidence interval, T.I.=to oftolerance fall intervaluse of bWith 95% confidence, interval contains 99% of the values expressed in the population of commercithe al lines. Negative limits were set to zero. 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 20. Comparison of the secondary metabolites and anti-nutrients content in grain collected at the IL field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b and and regime. Ferulic acid (µg/g dw) 2198.75 ± 52.20 2140.23 ± 52.20 58.52AG ± 73.81 -122.10,239.14 0.458 (1717.17 - 2687.57) (2142.86 - 2266.97) (2046.24 - 2241.58) (-98.72 - 220.73) [1415.19,3173.90] Bayer property protectionpublishing p-Coumaric acid (µg/g dw) 169.60 ± 10.01 155.98 ± 10.01 of 13.63 ± 14.16 -21.02,48.27contents 0.373 (152.30 - 319.15) its (148.45 - 202.53) (149.13 - 163.17) (-14.72parties. - 53.40)data therefore [43.13,384.34] and/oror affiliates. Phytic acid (% dw) 0.92 ± 0.039 0.99 ± property0.039 intellectual-0.066third ± 0.055 may-0.20,0.068 0.272 (0.45 - 1.00) its (0.83 - 1.00) (0.95 - 1.03) as (-0.15 - -0.016) [0.28,1.12] the of and is regulatory owner. a document Raffinose (% dw) 0.18 ± 0.0093 0.17 any± 0.0093 such 0.015 ± 0.013 its -0.017,0.047 0.305 (0.073 - 0.22) reproductiondocument (0.17 - 0.19) (0.16 - 0.18)owner (-0.013this - 0.036)of [0,0.32] under of rightsthe this a documentand/or rights dw=dry weight, S.E. = standard error, C.I.= confidence interval, T.I.=to oftolerance fall intervaluse of bWith 95% confidence, interval contains 99% of the values expressed in the population of commercithe al lines. Negative limits were set to zero. 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 21. Comparison of the secondary metabolites and anti-nutrients content in grain collected at the NE field site from MON 88017 and conventional corn

MON 88017 Control Difference (MON 88017 Minus Control) Commercial Mean ± S.E. Mean ± S.E. Mean ± S.E. 95% C.I. (Range) Component (Units)a (Range) (Range) (Range) (Lower, upper) p-Value [99% T.I.]b

and Ferulic acid (µg/g dw) 2234.68 ± 53.04 2198.59 ± 53.04 36.09 ± 75.01and -147.46,219.64regime. 0.647 (1717.17 - 2687.57) (2186.10 - 2264.57) (2098.77 - 2339.71) (-75.13AG - 154.60) [1415.19,3173.90] p-Coumaric acid (µg/g dw) 175.26 ± 10.40 144.20 ± 10.40 31.06 ± 14.69 -4.88,67.01 0.078 (152.30 - 319.15) Bayer property protectionpublishing (153.59 - 215.25) (141.41 - 148.48)of (8.47 - 72.55) contents [43.13,384.34] its parties.data therefore Phytic acid (% dw) 0.98 ± 0.071 0.81 ± 0.071 0.17 ± 0.10and/or or -0.072,0.42 0.135 (0.45 - 1.00) affiliates. (0.91 - 1.05) (0.72 - property0.89) intellectualthird(0.087 - 0.24) may [0.28,1.12] its as Raffinose (% dw) 0.18 ± 0.010 0.20the ± 0.010of and -0.020 ± 0.014 -0.055,0.015 0.207 (0.073 - 0.22) is regulatory owner. a document (0.16 - 0.20) (0.18any - 0.23)such (-0.035 - -0.012) its [0,0.32] reproductiondocument owner this of a under of dw=dry weight, S.E. = standard error, C.I.= confidence interval, T.I.=rights tolerancethe interval this b documentand/or rights With 95% confidence, interval contains 99% of the values expressedto inof the fallpopulation use of commerciof al lines. Negative limits were set to zero. 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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SUPPLEMENTAL SUMMARY

Fatty and amino acid composition of MON 88017 and conventional corn calculated as percent of dry weight, percent of total fat, and percent of total protein

Data Description

A SAS data set containing MON 88017 and conventional control corn forage and grain compositional analysis data was analyzed. Test and control materials were grown in single plots, randomly assigned within each of three replication blocks, at sites IA, IL and NE.

The calculation of fatty acid and amino acid composition in MON 88017 was done using following formulas: and and regime. AG Component From (X) To Formula Fatty Acid (FA) % FW %Bayer DW property protectionpublishing X/d of contents % Total Fat its X/(ff) parties.data therefore Amino Acid (AA) mg/g FW % Total Proteinand/or or X/(10*fp) affiliates. property intellectualthird may its % DW X/(10*d) as the of and is regulatory owner. a document d is the fraction of the sample that is dryany matter.such its reproductiondocument ff is the total fat fraction of fresh weight obtainedowner by proximatethis analysis;of ff = % total fat / 100. under of fp is the protein fraction of fresh weightrights obtainedthe by proximatethis analysis; fp = % protein /100. documentand/or rights to of fall use of the Statistical ApproachThis may distribution,and rights owner subject the violate Individual site and combinedbe copy site means and ranges were calculated for each test and of ® control fatty acid compositional analysis componentand using SAS software. A range of may documentpublication, It exploitation observed commercial referencethis hybrid values was also determined for each fatty acid compositional analysis component.any permission prohibited the Results commercial be Furthermore,any For each fatty acidConsequently, compositionalwithout analysis component, means and ranges are presented for each of the test and control hybrids of interest. Tables 22-25 presents means and ranges for each site individually and combined with the overall observed commercial reference range. The calculations are presented in the Tables as follows:

Table 22 - fatty acid content calculated as percent of dry weight Table 23 – fatty acids content calculated as percent of total fat Table 24 – amino acid content calculated as percent of dry weight Table 25 – amino acid content calculated as percent of total protein

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Table 22. Fatty acid content in grain from MON88017 and control LH198xLH59 corn calculated as percent of dry weight

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Fatty Acid % DW 16:0 Palmitic All Sites 0.35 0.39 (0.22 - 0.50) (0.32 - 0.38) (0.32 - 0.48) Site IA 0.32 0.41 (0.32 - 0.33) (0.32 - 0.48) Site IL 0.37 0.40 (0.36 - 0.38) (0.39 - 0.42) Site NE 0.34 0.35 and (0.33 - 0.35) (0.33 -and 0.38) regime. 16:1 Palmitoleic All Sites 0.0061 AG 0.0064 (0.0017 - 0.0068) (0.0053 - 0.0071) (0.0052 - 0.0080) Bayer property protectionpublishing Site IA 0.0064of 0.0067 contents its (0.0060 - 0.0071) (0.0061parties.data - 0.0071) therefore and/oror Site IL 0.0064affiliates. 0.0067 property intellectualthird may its (0.0056 - 0.0070)as (0.0060 - 0.0080) the of and is regulatory owner. Site NE 0.0056 a document0.0058 any such its reproductiondocument (0.0053 owner- 0.0057) (0.0052this - 0.0063)of under of 18:0 Stearic All Sites rightsthe 0.068 this 0.072 (0.043 - 0.079) documentand/or rights to (0.058of fall- 0.077) use of(0.055 - 0.083) the ThisSite IA may0.059 distribution, and 0.064 rights owner subject (0.058 - 0.061) (0.055 - 0.071) be the violate Site ILcopy 0.074of 0.080 and may documentpublication,(0.072 - 0.077) (0.078 - 0.083) It exploitation Site thisNE any 0.072 0.071 (0.069permission - 0.074) (0.063 - 0.079) prohibited 18:1 Oleic All Sites the be 0.77 0.80 (0.46 - 1.27) commercial (0.69 - 0.86) (0.69 - 0.98) Furthermore,any Consequently,Site withoutIA 0.72 0.71 (0.69 - 0.76) (0.69 - 0.72) Site IL 0.82 0.91 (0.78 - 0.86) (0.87 - 0.98) Site NE 0.77 0.78 (0.73 - 0.82) (0.74 - 0.83)

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Table 22 (cont.) Fatty acid content in grain from MON 88017 and control LH198xLH59 corn calculated as percent of dry weight

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Fatty Acid % DW 18:2 Linoleic All Sites 2.12 2.14 (1.32 - 2.48) (1.94 - 2.41) (1.87 - 2.59) Site IA 2.02 1.93 (1.95 - 2.06) (1.87 - 1.96) Site IL 2.28 2.42 (2.17 - 2.41) (2.33 - 2.59) Site NE 2.07 2.07 and (1.94 - 2.17) (1.98 -and 2.17) regime. 18:3 Linolenic All Sites 0.041 AG 0.046 (0.024 - 0.058) (0.037 - 0.045) (0.038 - 0.059) Bayer property protectionpublishing Site IA 0.040of 0.050 contents its (0.038 - 0.041) parties.(0.038data - 0.059) therefore and/oror Site IL 0.043affiliates. 0.047 property intellectualthird may its (0.042 - 0.045)as (0.045 - 0.050) the of and is regulatory owner. Site NE 0.040 a document0.040 any such its reproductiondocument (0.037 owner- 0.041) (0.038this - 0.043)of under of 20:0 Arachidic All Sites rightsthe 0.013 this 0.013 (0.0084 - 0.016) documentand/or rights to (0.011of fall - 0.014)use of(0.011 - 0.016) the ThisSite IA may0.011 distribution, and 0.011 rights owner subject (0.011 - 0.012) (0.011 - 0.012) be the violate Site ILcopy 0.014of 0.015 and may documentpublication,(0.013 - 0.014) (0.015 - 0.016) It exploitation Site thisNE any 0.013 0.013 (0.012permission - 0.013) (0.013 - 0.014) prohibited 20:1 Eicosenoic All Sites the be 0.0082 0.0085 (0.0055 - 0.013) commercial(0.0074 - 0.0089) (0.0074 - 0.010) Furthermore,any Consequently,Sitewithout IA 0.0076 0.0075 (0.0074 - 0.0077) (0.0074 - 0.0078) Site IL 0.0086 0.0095 (0.0083 - 0.0089) (0.0091 - 0.010) Site NE 0.0084 0.0085 (0.0080 - 0.0086) (0.0078 - 0.0093)

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Table 22 (cont.) Fatty acid content in grain of MON 88017 and control LH198xLH59 corn calculated as percent of dry weight

MON88017 LH198xLH59 Mean Mean Commercial corn Component Location (Range) (Range) (Range) Fatty Acid % DW 22:0 Behenic All Sites 0.0050 0.0053 (0.0017 - 0.0063) (0.0044 - 0.0059) (0.0045 - 0.0064) Site IA 0.0046 0.0049 (0.0044 - 0.0049) (0.0045 - 0.0053) Site IL 0.0055 0.0058 (0.0049 - 0.0059) (0.0054 - 0.0064) Site NE 0.0049 0.0053 and (0.0047 - 0.0050) (0.0047 - 0.0059)and regime. AG

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Table 23. Fatty acid content in MON88017 and control LH198xLH59 corn calculated as percent of total fat

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Fatty Acid % Total Fat 16:0 Palmitic All Sites 9.50 10.30 (8.61 - 16.44) (8.83 - 10.00) (8.77 - 13.35) Site IA 9.18 11.59 (8.83 - 9.42) (8.77 - 13.35) Site IL 9.63 9.78 (9.57 - 9.71) (9.73 - 9.82) Site NE 9.70 9.52 and (9.51 - 10.00) (9.29and - 9.88)regime. 16:1 Palmitoleic All Sites 0.17 AG 0.17 (0.050 - 0.20) (0.15 - 0.20) (0.14 - 0.20) Bayer property protectionpublishing Site IA of0.18 0.19 contents its (0.17 - 0.20) parties.data (0.17 - 0.20)therefore and/oror Site IL affiliates.0.17 0.16 property intellectualthird may its (0.15as - 0.18) (0.15 - 0.18) the of and is regulatory owner. Site NE 0.16a document0.16 any such its reproductiondocument (0.15owner - 0.16) this (0.14of - 0.17) under of 18:0 Stearic All Sites rightsthe 1.87 this 1.89 (1.45 - 2.09) documentand/or rights to of (1.62fall - 2.10)use of (1.52 - 2.03) the This Site IA may distribution,1.66and 1.80 rights owner subject (1.62 - 1.69) (1.52 - 1.97) be the violate Sitecopy IL of1.93 1.95 and may documentpublication, (1.90 - 1.95) (1.90 - 2.00) It exploitation thisSite any NE 2.02 1.93 permission(1.97 - 2.10) (1.78 - 2.03) prohibited 18:1 Oleic Combinedthe be 21.11 20.92 (18.26 - 31.94) commercialSites Furthermore,any Consequently,without (19.35 - 23.30) (19.63 - 22.48) Site IA 20.39 19.72 (19.35 - 21.11) (19.63 - 19.88) Site IL 21.32 22.05 (20.90 - 21.66) (21.71 - 22.48) Site NE 21.63 21.00 (20.40 - 23.30) (20.71 - 21.42)

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Table 23 (cont.) Fatty acid content in MON88017 and control LH198xLH59 corn calculated as percent of total fat

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Fatty Acid % Total Fat 18:2 Linoleic All Sites 58.34 56.29 (46.22 - 59.73) (54.63 - 61.59) (53.00 - 59.47) Site IA 57.15 53.87 (54.63 - 59.42) (53.00 - 54.46) Site IL 59.71 58.83 (58.20 - 60.76) (58.48 - 59.47) Site NE 58.16 56.17 and (56.35 - 61.59) (54.32and - 58.41) regime. 18:3 Linolenic All Sites 1.13 AG 1.21 (0.77 - 1.76) (1.07 - 1.16) (1.05 - 1.62) Bayer property protectionpublishing Site IA 1.13of 1.40 contents its (1.07 - 1.16) parties.(1.06data - 1.62) therefore and/oror Site IL 1.14affiliates. 1.14 property intellectualthird may its (1.13 - as1.14) (1.14 - 1.15) the of and is regulatory owner. Site NE 1.11 a document1.08 any such its reproductiondocument (1.09owner - 1.14) this(1.05 -of 1.10) under of 20:0 Arachidic All Sites rightsthe 0.35 this 0.35 (0.31 - 0.42) documentand/or rights to (0.31of fall - 0.37) use of (0.30 - 0.37) the ThisSite IA may0.32 distribution, and 0.32 rights owner subject (0.31 - 0.33) (0.30 - 0.33) be the violate Site ILcopy 0.35of 0.37 and may documentpublication,(0.35 - 0.36) (0.36 - 0.37) It exploitation Site NEthis any 0.36 0.36 permission(0.36 - 0.37) (0.36 - 0.37) prohibited 20:1 Eicosenoic All Sites the be 0.22 0.22 (0.21 - 0.33) commercial (0.21 - 0.24) (0.21 - 0.24) Furthermore,any Consequently,Site IAwithout 0.21 0.21 (0.21 - 0.22) (0.21 - 0.22) Site IL 0.22 0.23 (0.22 - 0.23) (0.23 - 0.24) Site NE 0.24 0.23 (0.23 - 0.24) (0.22 - 0.24)

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Table 23 (cont.) Fatty acid content in MON88017 and control LH198xLH59 corn calculated as percent of total fat

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Fatty Acid % Total Fat 22:0 Behenic All Sites 0.14 0.14 (0.068 - 0.22) (0.12 - 0.15) (0.13 - 0.15) Site IA 0.13 0.14 (0.12 - 0.14) (0.13 - 0.15) Site IL 0.14 0.14 (0.13 - 0.15) (0.13 - 0.15) Site NE 0.14 0.14 and (0.13 - 0.14) (0.13and - 0.15) regime. AG

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Table 24. Amino acid content in MON88017 and control LH198xLH59 corn calculated as percent of dry weight

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Amino Acid % DW Alanine Combined 0.93 0.93 (0.67 - 1.06) Sites (0.84 - 1.00) (0.81 - 1.11) Site IA 0.89 0.83 (0.84 - 0.92) (0.81 - 0.84) Site IL 0.91 0.91

(0.90 - 0.93) (0.88 - 0.93) and and regime. Site NE 0.99 AG 1.03 (0.98 - 1.00) (0.96 - 1.11) Arginine All Sites 0.54Bayer property protection0.52 publishing (0.42 - 0.55) of contents (0.50 - 0.58) (0.49 - 0.58)its parties.data therefore Site IA 0.56 and/or0.51or affiliates. property intellectualthird may (0.55its - 0.57) (0.49 - 0.53) as the of and Site IL is 0.51 regulatory 0.51 owner. a document any such its (0.50 - 0.52) reproduction(0.49document - 0.52) owner this of Site NE 0.57under of 0.55 rightsthe this documentand/or rights to of (0.56fall - 0.58)use of (0.54 - 0.58) the Aspartic Acid AllThis Sites may distribution,0.77and 0.77 (0.60 - 0.82) rights owner subject (0.73 - 0.82) (0.68 - 0.88) be the violate Site IA copy 0.75of 0.70 and may documentpublication, It exploitation(0.73 - 0.77) (0.68 - 0.72) Site IL this any 0.74 0.75 permission (0.73prohibited - 0.75) (0.74 - 0.78) the Site NE commercial be 0.81 0.84 Furthermore,any (0.80 - 0.82) (0.81 - 0.88) Cystine Consequently,All Siteswithout 0.26 0.26 (0.20 - 0.29) (0.25 - 0.28) (0.25 - 0.28) Site IA 0.26 0.26 (0.26 - 0.26) (0.25 - 0.26) Site IL 0.26 0.26 (0.26 - 0.27) (0.25 - 0.27) Site NE 0.27 0.27 (0.25 - 0.28) (0.27 - 0.28)

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Table 24 (cont.) Amino acid content in MON88017 and control LH198xLH59 corn calculated as percent of dry weight

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Amino Acid % DW Glutamic Acid All Sites 2.51 2.50 (1.78 - 2.81) (2.28 - 2.68) (2.22 - 3.00) Site IA 2.42 2.26 (2.28 - 2.48) (2.22 - 2.28) Site IL 2.47 2.47 (2.43 - 2.52) (2.38 - 2.52) Site NE 2.66 2.78 and (2.62 - 2.68) (2.58and - 3.00) regime. Glycine All Sites 0.43 AG 0.42 (0.35 - 0.43) (0.40 - 0.45) (0.39 - 0.46) Bayer property protectionpublishing Site IA 0.43of 0.40 contents its (0.42 - 0.44) parties.(0.39data - 0.41) therefore and/oror Site IL 0.41affiliates. 0.41 property intellectualthird may its (0.40 -as 0.41) (0.41 - 0.42) the of and is regulatory owner. Site NE 0.44 a document0.45 any such its reproductiondocument (0.44owner - 0.45) this(0.43 -of 0.46) under of Histidine All Sites rightsthe 0.37 this 0.36 (0.26 - 0.36) documentand/or rights to of(0.35 fall - 0.38)use of (0.34 - 0.41) the ThisSite IA may 0.37distribution, and 0.35 rights owner subject (0.36 - 0.38) (0.34 - 0.36) be the violate Site ILcopy 0.35of 0.35 and may documentpublication, (0.35 - 0.36) (0.34 - 0.35) It exploitation Site NEthis any 0.38 0.39 permission(0.37 - 0.38) (0.36 - 0.41) prohibited Isoleucine All Sites the be 0.44 0.44 (0.32 - 0.48) commercial (0.40 - 0.47) (0.39 - 0.53) Furthermore,any Consequently,Site IAwithout 0.42 0.40 (0.40 - 0.44) (0.39 - 0.40) Site IL 0.44 0.42 (0.43 - 0.44) (0.40 - 0.45) Site NE 0.47 0.49 (0.46 - 0.47) (0.44 - 0.53)

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Table 24 (cont.) Amino acid content in MON88017 and control LH198xLH59 corn calculated as percent of dry weight

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Amino Acid % DW Leucine All Sites 1.64 1.63 (1.10 - 1.87) (1.46 - 1.75) (1.43 - 2.03) Site IA 1.56 1.46 (1.46 - 1.61) (1.43 - 1.48) Site IL 1.62 1.60 (1.60 - 1.64) (1.51 - 1.68) Site NE 1.73 1.84 and (1.70 - 1.75) (1.67 - 2.03)and regime. Lysine All Sites 0.33 AG 0.33 (0.27 - 0.36) (0.29 - 0.35) (0.29 - 0.36) Bayer property protectionpublishing Site IA 0.33of 0.31 contents its (0.33 - 0.34) parties.(0.29data - 0.32) therefore and/oror Site IL 0.32affiliates. 0.31 property intellectualthird may its (0.29 - as0.33) (0.31 - 0.32) the of and is regulatory owner. Site NE 0.34 a document0.36 any such its reproductiondocument (0.34owner - 0.35) this(0.34 - of0.36) under of Methionine All Sites rightsthe 0.24 this 0.24 (0.17 - 0.26) documentand/or rights to of(0.24 fall - 0.25)use of (0.23 - 0.27) the ThisSite IA may 0.24distribution, and 0.24 rights owner subject (0.24 - 0.25) (0.24 - 0.25) be the violate Site ILcopy 0.24of 0.24 and may documentpublication,(0.24 - 0.25) (0.23 - 0.25) It exploitation Sitethis NE any 0.25 0.25 permission(0.24 - 0.25) (0.24 - 0.27) prohibited Phenylalanine All Sites the be 0.64 0.63 (0.44 - 0.69) commercial (0.59 - 0.68) (0.56 - 0.75) Furthermore,any Consequently,Sitewithout IA 0.61 0.57 (0.59 - 0.64) (0.56 - 0.59) Site IL 0.63 0.62 (0.63 - 0.63) (0.59 - 0.66) Site NE 0.67 0.70 (0.66 - 0.68) (0.65 - 0.75)

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Table 24 (cont.) Amino acid content in MON88017 and control LH198xLH59 corn calculated as percent of dry weight

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Amino Acid % DW Proline All Sites 1.16 1.14 (0.80 - 1.20) (1.09 - 1.26) (1.05 - 1.31) Site IA 1.14 1.09 (1.09 - 1.19) (1.06 - 1.11) Site IL 1.12 1.10 (1.09 - 1.14) (1.05 - 1.18) Site NE 1.21 1.24 and (1.19 - 1.26) (1.16 - 1.31)and regime. Serine All Sites 0.59 AG 0.60 (0.46 - 0.66) (0.55 - 0.63) (0.53 - 0.72) Bayer property protectionpublishing Site IA 0.59 of 0.55 contents its (0.57 - 0.61) (0.53parties.data - 0.57) therefore and/oror Site IL 0.58affiliates. 0.59 property intellectualthird may its (0.55 - 0.61)as (0.57 - 0.62) the of and is regulatory owner. Site NE 0.62 a document0.67 any such its reproductiondocument (0.62 -owner 0.63) (0.64this - 0.72)of under of Threonine All Sites rightsthe 0.40 this0.40 (0.32 - 0.42) documentand/or rights to (0.37of fall- 0.42) use (0.36of - 0.45) the ThisSite IA may0.40 distribution, and 0.37 rights owner subject (0.39 - 0.41) (0.36 - 0.38) be the violate Site ILcopy 0.38 of 0.39 and may documentpublication,(0.37 - 0.40) (0.38 - 0.40) It exploitation Site NEthis any 0.41 0.43 permission(0.41 - 0.42) (0.42 - 0.45) prohibited Tryptophan All Sites the be 0.066 0.067 (0.052 - 0.080) commercial(0.061 - 0.072) (0.054 - 0.083) Furthermore,any Consequently,Site IAwithout 0.067 0.067 (0.061 - 0.072) (0.064 - 0.068) Site IL 0.067 0.070 (0.062 - 0.072) (0.062 - 0.083) Site NE 0.065 0.064 (0.063 - 0.067) (0.054 - 0.071)

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Table 24 (cont.) Amino acid content in MON88017 and control LH198xLH59 corn calculated as percent of dry weight

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Amino Acid % DW Tyrosine All Sites 0.41 0.42 (0.23 - 0.48) (0.28 - 0.47) (0.34 - 0.52) Site IA 0.43 0.40 (0.42 - 0.45) (0.39 - 0.41) Site IL 0.35 0.41 (0.28 - 0.41) (0.41 - 0.42) Site NE 0.46 0.44 and (0.46 - 0.47) (0.34and - 0.52) regime. Valine All Sites 0.59 AG 0.58 (0.43 - 0.61) (0.53 - 0.63) (0.52 - 0.67) Bayer property protectionpublishing Site IA 0.57of 0.54 contents its (0.53 - 0.59) parties.(0.52data - 0.55) therefore and/oror Site IL 0.58affiliates. 0.57 property intellectualthird may its (0.57 -as 0.59) (0.55 - 0.61) the of and is regulatory owner. Site NE 0.62 a document0.63 any such its reproductiondocument (0.62owner - 0.63) this(0.59 -of 0.67) 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 25. Amino acid content in MON88017 and control LH198xLH59 corn calculated as percent of total protein

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Amino Acid % Total Protein Alanine All Sites 7.43 7.52 (7.06 - 8.33) (7.18 - 7.75) (7.04 - 8.05) Site IA 7.26 7.23 (7.18 - 7.37) (7.04 - 7.48) Site IL 7.38 7.52 (7.29 - 7.55) (7.45 - 7.62) Site NE 7.66 7.82 and (7.60 - 7.75) (7.55and - 8.05) regime. Arginine All Sites 4.35 AG 4.27 (3.74 - 5.13) (4.02 - 4.70) (4.10 - 4.52) Bayer property protectionpublishing Site IA 4.59of 4.42 contents its (4.51 - 4.70) parties.(4.30data - 4.52) therefore and/oror Site IL affiliates.4.09 4.19 property intellectualthird may its (4.02as - 4.20) (4.10 - 4.28) the of and is regulatory owner. Site NE 4.38 a document4.19 any such its reproductiondocument (4.28owner - 4.50) this (4.17 of- 4.22) under of Aspartic Acid All Sites rightsthe 6.13 this 6.23 (6.04 - 6.95) documentand/or rights to of (5.83fall - 6.35)use of (6.00 - 6.41) the ThisSite IA may distribution,6.14and 6.07 rights owner subject (5.95 - 6.26) (6.02 - 6.10) be the violate Sitecopy IL 5.95of 6.23 and may documentpublication, (5.83 - 6.12) (6.00 - 6.38) It exploitation Sitethis NE any 6.30 6.38 permission(6.19 - 6.35) (6.36 - 6.41) prohibited Cystine All Sites the be 2.11 2.14 (1.85 - 2.51) commercial (1.95 - 2.24) (2.01 - 2.33) Furthermore,any Consequently,Sitewithout IA 2.13 2.22 (2.07 - 2.24) (2.11 - 2.33) Site IL 2.13 2.15 (2.10 - 2.16) (2.08 - 2.20) Site NE 2.06 2.06 (1.95 - 2.20) (2.01 - 2.12)

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Table 25 (cont.) Amino acid content in MON88017 and control LH198xLH59 corn calculated as percent of total protein

MON88017 LH198xLH59 Mean Mean Commercial Component Location (Range) (Range) (Range) Amino Acid % Total Protein Glutamic Acid All Sites 20.09 20.36 (18.68 - 22.02) (19.62 - 20.78) (19.07 - 21.71) Site IA 19.79 19.66 (19.62 - 20.00) (19.07 - 20.30) Site IL 19.94 20.39 (19.63 - 20.47) (20.19 - 20.60) Site NE 20.55 21.03 and (20.35 - 20.78) (20.35and - 21.71)regime. Glycine All Sites 3.40 AG 3.43 (3.18 - 4.23) (3.25 - 3.59) (3.28 - 3.55) Bayer property protectionpublishing Site IA 3.50of 3.49 contents its (3.39 - 3.59) parties.data(3.46 - 3.53) therefore and/oror Site IL 3.28affiliates. 3.41 property intellectualthird may its (3.25 as- 3.32) (3.28 - 3.55) the of and is regulatory owner. Site NE 3.42 a document3.40 any such its reproductiondocument (3.38owner - 3.45) this (3.32of - 3.45) under of Histidine All Sites rightsthe 2.94 this 2.94 (2.46 - 3.16) documentand/or rights to of (2.84fall - 3.08)use of (2.81 - 3.07) the ThisSite IA may distribution,3.03and 3.03 rights owner subject (2.95 - 3.08) (2.99 - 3.07) be the violate Site copyIL 2.85of 2.86 and may documentpublication, (2.84 - 2.87) (2.81 - 2.93) It exploitation Sitethis NE any 2.94 2.93 permission(2.90 - 2.97) (2.85 - 2.98) prohibited Isoleucine All Sites the be 3.53 3.55 (3.33 - 3.79) commercial (3.40 - 3.64) (3.36 - 3.84) Furthermore,any Consequently,Sitewithout IA 3.47 3.46 (3.40 - 3.51) (3.36 - 3.57) Site IL 3.52 3.50 (3.46 - 3.57) (3.38 - 3.64) Site NE 3.62 3.69 (3.57 - 3.64) (3.43 - 3.84)

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Table 25 (cont.) Amino acid content in MON88017 and control LH198xLH59 corn calculated as percent of total protein

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Amino Acid % Total Protein Leucine All Sites 13.07 13.26 (11.85 - 14.65) (12.57 - 13.57) (12.22 - 14.72) Site IA 12.74 12.68 (12.57 - 12.83) (12.22 - 13.17) Site IL 13.07 13.22 (12.94 - 13.27) (13.02 - 13.55) Site NE 13.41 13.89 and (13.22 - 13.57) (13.19and - 14.72) regime. Lysine All Sites 2.65 AG 2.65 (2.35 - 3.28) (2.37 - 2.85) (2.47 - 2.76) Bayer property protectionpublishing Site IA 2.73of 2.67 contents its (2.61 - 2.85) parties.(2.59data - 2.71) therefore and/oror Site IL 2.55affiliates. 2.60 property intellectualthird may its (2.37 -as 2.64) (2.47 - 2.72) the of and is regulatory owner. Site NE 2.66 a document2.69 any such its reproductiondocument (2.60owner - 2.70) this(2.61 -of 2.76) under of Methionine All Sites rightsthe 1.95 this 2.00 (1.73 - 2.41) documentand/or rights to of(1.87 fall - 2.03)use of (1.85 - 2.22) the ThisSite IA may 1.99distribution,and 2.12 rights owner subject (1.94 - 2.03) (2.02 - 2.22) be the violate Site copyIL 1.94of 1.97 and may documentpublication, (1.89 - 1.99) (1.92 - 2.00) It exploitation Sitethis NE any 1.92 1.91 permission(1.87 - 1.95) (1.85 - 1.94) prohibited Phenylalanine All Sites the be 5.10 5.12 (4.62 - 5.53) commercial (4.91 - 5.26) (4.80 - 5.42) Furthermore,any Consequently,Sitewithout IA 5.02 4.99 (4.91 - 5.10) (4.80 - 5.27) Site IL 5.09 5.11 (5.05 - 5.12) (4.94 - 5.27) Site NE 5.18 5.26 (5.13 - 5.26) (5.12 - 5.42)

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Table 25(cont.) Amino acid content in MON88017 and control LH198xLH59 corn calculated as percent of total protein

MON88017 LH198xLH59 Mean Mean Commercial corn Component Location (Range) (Range) (Range) Amino Acid % Total Protein Proline All Sites 9.24 9.30 (8.14 - 9.55) (8.84 - 9.66) (8.65 - 9.90) Site IA 9.36 9.45 (9.20 - 9.47) (9.14 - 9.90) Site IL 9.01 9.08 (8.84 - 9.22) (8.65 - 9.53) Site NE 9.36 9.37 and (9.22 - 9.66) (9.12 and- 9.51) regime. Serine All Sites 4.76 AG 4.89 (4.67 - 5.20) (4.50 - 4.98) (4.58 - 5.20) Bayer property protectionpublishing Site IA 4.80of 4.76 contents its (4.54 - 4.98) parties.(4.68data - 4.85) therefore and/oror Site IL 4.65affiliates. 4.87 property intellectualthird may its (4.50 - as4.94) (4.58 - 5.02) the of and is regulatory owner. Site NE 4.82 a document5.04 any such its reproductiondocument (4.80owner - 4.84) this(4.86 - 5.20)of under of Threonine All Sites rightsthe 3.17 this 3.23 (3.02 - 3.53) documentand/or rights to (2.99of fall - 3.35) use of (3.03 - 3.38) the ThisSite IA may3.25 distribution, and 3.20 rights owner subject (3.08 - 3.35) (3.18 - 3.23) be the violate Site ILcopy 3.08of 3.23 and may documentpublication,(2.99 - 3.23) (3.03 - 3.38) It exploitation Site NEthis any 3.20 3.27 permission(3.15 - 3.24) (3.26 - 3.28) prohibited Tryptophan All Sites the be 0.53 0.55 (0.44 - 0.81) commercial (0.49 - 0.58) (0.41 - 0.67) Furthermore,any Consequently,Site IAwithout 0.55 0.58 (0.52 - 0.58) (0.57 - 0.61) Site IL 0.54 0.58 (0.50 - 0.58) (0.53 - 0.67) Site NE 0.50 0.48 (0.49 - 0.52) (0.41 - 0.52)

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Table 25 (cont.) Amino acid content in MON88017 and control LH198xLH59 corn calculated as percent of total protein

MON88017 LH198xLH59 Commercial Mean Mean corn Component Location (Range) (Range) (Range) Amino Acid % Total Protein Tyrosine All Sites 3.30 3.41 (2.22 - 3.78) (2.23 - 3.63) (2.60 - 3.77) Site IA 3.54 3.50 (3.43 - 3.63) (3.38 - 3.69) Site IL 2.81 3.41 (2.23 - 3.35) (3.26 - 3.54) Site NE 3.56 3.32 and (3.53 - 3.59) (2.60and - 3.77) regime. Valine All Sites 4.71 AG 4.72 (4.33 - 5.26) (4.56 - 4.87) (4.50 - 4.89) Bayer property protectionpublishing Site IA 4.66of 4.65 contents its (4.56 - 4.72) parties.(4.57data - 4.82) therefore and/oror Site IL 4.65affiliates. 4.73 property intellectualthird may its (4.60 - as4.69) (4.50 - 4.89) the of and is regulatory owner. Site NE 4.82 a document4.79 any such its reproductiondocument (4.77owner - 4.87) this(4.62 -of 4.89) 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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