This Document Is the Property of Bayer AG And/Or Any of Its Affiliates. It

Food and Feed Safety and Nutritional Assessment of the Lepidopteran-protected Corn MON 89034 (OECD Unique Identifier MON-89Ø34-3)

regime.and Conclusion Based on Data and Information Evaluated Accordingand to FDA’s Policy on Foods from New PlantAG Varieties

Bayer property protectionpublishing of contents its parties.data therefore and/oror Octoberaffiliates. 13, 2006 property intellectualthird may its as the of and is regulatory owner. FDA BNF No.a 00105 document any such its reproductiondocument of Monsantoowner 06-CR-161Fthis under of rightsthe this documentand/or rights to of fall use of Prepared by: the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any Contributors:

permission prohibited the commercial be Furthermore,any Consequently,without

Submitted by:

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

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 1 of 323 PART I: CERTIFICATION, CONTENTS AND SUMMARY

Table of Contents

PART I: CERTIFICATION, CONTENTS AND SUMMARY ...... 2 Table of Contents ...... 2 List of Tables ...... 7 and List of Figures ...... 8and regime. Certification ...... AG 11 Release of Information...... 12 Bayer property protectionpublishing Abbreviations, Acronyms and Definitions...... 13of contents its Narrative Summary ...... 19parties.data therefore and/oror affiliates. PART II: SYNOPSIS OF CONSULTATIONproperty intellectualthird SUMMARY ...... 25may its as SECTION 1. Name and Addressthe of of the Submitter...... 25and is regulatory owner. a document SECTION 2. The subjectany of this such Summary and the Plant Species fromits which They reproductiondocument were Derived...... 25owner this of under of this SECTION 3. Distinctiveand/orrights Designationsthe Given to the Subject of This Summary...... 25 document to fall of rights SECTION 4. Identity and Sourcesof of the Geneticuse Material Introduced into the This MON 89034 ...... 25may distribution,and rights owner SECTION 5. subjectThe Intended Technical Effect of MON 89034...... 26 be the violate SECTION 6. Thecopy Applications and Usesof of MON 89034 ...... 27 and SECTIONmay 7. Applicationsdocumentpublication, for which MON 89034 is not Suitable...... 30 It exploitation References this...... 31 any permission PART III: STATUS OF SUBMprohibitedISSIONS TO OTHER REGULATORY AGENCIES...... 34the commercial be SECTION 1. Status of Submission to USDA-APHIS ...... 34 Furthermore,any SECTIONConsequently, 2. without Status of Submission to U.S. EPA ...... 34 SECTION 3. Status of Submissions to Foreign Governments...... 34

PART IV: DEVELOPMENT OF MON 89034 ...... 35 SECTION 1. Corn as a Crop...... 35 1.1. Scientific name and taxonomic classification of corn ...... 36 1.2. Growth and Reproductive Characteristics of Corn...... 38

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 2 of 323 1.3. History of corn development ...... 38 SECTION 2. Characterization of the Vector Used in Transformation ...... 40 2.1. Method of transformation ...... 40 2.2. Plasmid PV-ZMIR245 ...... 41 2.2.1. T-DNA I...... 42 2.1.1.1. The cry1A.105 gene and Cry1A.105 protein ...... 42 2.1.1.2. The cry1A.105 regulatory sequences...... 42 2.1.1.3. The cry2Ab2 gene and Cry2Ab2 protein ...... 42 2.1.1.4. The cry2Ab2 regulatory sequences...... 42 2.1.1.5. T-DNA borders...... 43 2.2.2. T-DNA II ...... 43and and regime. 2.2.2.1. nptII gene and NPTII protein...... 43AG 2.2.2.2. nptII regulatory sequences...... 43 2.2.2.3. T-DNA borders...... 43 Bayer property protectionpublishing 2.2.3. Genetic elements outside theof T-DNA borders...... 43contents its SECTION 3. Characteriztion of the Introduced Geneticparties. Material...... 52data therefore and/oror 3.1. Insert and copy number...... 60 affiliates. property intellectualthird may 3.2. Intactness of the cry1A.105its and cry2Ab2 expression cassettes ...... 62 as 3.2.1. e35S promoter/theCab of leader...... 63and is regulatory owner. a document 3.2.2. Ract1 intron...... 63any such its reproductiondocument 3.2.3. cry1A.105 coding sequenceowner ...... 66this of under of 3.2.4. Hsp17 3' endrights sequence...... 66the this documentand/or rights 3.2.5. FMV promoter...... 69to of fall use of the 3.2.6.This Hsp70 intron...... 69may distribution,and rights owner 3.2.7. SSU-CTPsubject targeting sequence/cry2Ab2 coding sequence...... 70 3.2.8.be nos 3' end sequence...... 74the violate copy of 3.2.9. Analysis to confirm the absenceand of plasmid PV-ZMIR245 backbone ...... 76 may documentpublication, 3.3.It Analysis to confirmexploitation the absence of T-DNA II...... 78 3.3.1. Analysisthis any to confirm the absence of the nptII coding sequence...... 78 permission 3.3.2. Analysis to confirmprohibited the absence of the overall T-DNA II region...... 78 3.4. Insert stabilitythe across generations of MON 89034 ...... 82 commercial be 3.4.1. Summary...... 82 Furthermore,3.4.2.any Stability of the T-DNA I insert across generations of MON 89034...... 84 Consequently,without 3.4.3. Confirmation of the absence of T-DNA II in multiple generations of MON 89034...... 86 3.4.4. Confirmation of the absence of plasmid PV-ZMIR245 backbone sequence in multiple generations of MON 89034...... 86 3.5. Organization and sequence of the insert DNA in MON 89034 ...... 90 3.6. Inheritance of the lepidopteran protection trait in MON 89034 ...... 90 3.7. Conclusion ...... 92

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 3 of 323 SECTION 4. Other data and information about the development of MON 89034 ...... 93 References ...... 93

PART V: PRESENCE OF GENES THAT ENCODE RESISTANCE TO ANTIBIOTICS...... 98

PART VI: CHARACTERIZATION OF THE PROTEINS INTRODUCED INTO MON 89034...... 99 SECTION 1. Identity and Characterization of the Cry1A.105 and Cry2Ab2 Proteins Produced in MON 89034...... 99 1.1. Identity and function of the Cry1A.105 protein...... 100 regime.and 1.2. Characterization of the Cry1A.105 protein...... 102and AG 1.2.1. Cry1A.105 protein molecular weight equivalence ...... 102 1.2.2. Cry1A.105 protein immunoreactivity...... 102 Bayer property protectionpublishing 1.2.3. N-terminus intactness of theof Cry1A.105 protein ...... 105contents its 1.2.4. Cry1A.105 tryptic peptide mapping byparties. MALDI-TOFdata MS...... 105therefore and/oror 1.2.5. Lack of glycosylation for Cry1A.105 protein...... 108 affiliates. property intellectualthird may 1.2.6. Cry1A.105 functionalits activity ...... 108 as 1.2.7. Conclusions thefor characterizationof and of the Cry1A.105 protein ...... 112 is regulatory owner. a document 2.1. Identity and functionany ofsuch the Cry2Ab2 protein...... 112its reproductiondocument 2.2. Characterization of the Cry2Ab2owner protein...... 113this of under of 2.2.1. Cry2Ab2 proteinrights moleculathe r weight equivalencethis ...... 113 documentand/or rights 2.2.2. Cry2Ab2 toprotein of immunoreactivity...... 113fall use of the 2.2.3.This Analysis of the N-terminalmay distribution, andsequence ...... 117 rights owner 2.2.4. Cry2Ab2subject tryptic peptide mapping by MALDI-TOF MS...... 117 2.2.5.be Lack of glycosylation of MONthe 89034-producedviolate Cry2Ab2...... 119 copy of 2.2.6. Functional activity of the Cry2Ab2and protein...... 119 may documentpublication, It 2.2.7. Conclusions of exploitationthe characteri zation of the Cry2Ab2 protein...... 123 this SECTION 2. Levelsany of the Cry1A.105 and Cry2Ab2 Proteins Produced in MON 89034permission ...... 124 prohibited SECTION 3. Dietarythe Exposure Assessment...... 129 commercial be 3.1. Human dietary safety assessment ...... 129 Furthermore,any Consequently,3.1.1. Riskwithout assessment of the Cry1A.105 and Cry2Ab2 proteins to mammals ..129 3.1.2. Corn consumption...... 130 3.1.3. Intake of the Cry1A.105 and Cry2Ab2 proteins...... 131 3.1.4. Margins of exposure ...... 131 3.2. Animal dietary safety assessment ...... 132 3.2.1. Corn consumption...... 133 3.2.2. Dietary intake of the Cry1A.105 and Cry2Ab2 proteins ...... 133

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 4 of 323 3.2.3. Margins of exposure ...... 133 SECTION 4. Assessment of the Potential for Allergenicity of the Cry1A.105 and Cry2Ab2 Proteins Produced in MON 89034...... 134 4.1. Approach to the assessment of allergenicity...... 134 4.1.1. Rational for studying structural similarity to known allergens...... 134 4.1.2. Rational for studying stability in simulated digestive fluids ...... 134 4.2. Assessment of the potential for allergenicity of the Cry1A.105 protein ...... 135 4.2.1. Source of the Cry1A.105 protein...... 135 4.2.2. Bioinformatics analyses of sequence similarity of the Cry1A.105 protein produced in MON 89034 to allergens...... 135 regime.and 4.2.3. Digestibility of the Cry1A.105 in simulated gastricand digestive fluid ...... 137 AG 4.2.4. Digestibility of the Cry1A.105 protein in simulated intestinal fluid ...... 140 4.2.5. Proportion of Cry1A.105 protein to the total protein in MON 89034 Bayer property protectionpublishing grain...... 142of contents 4.3. Assessment of the potential for allergenicity of the Cry2Ab2 proteinits ...... 142 parties.data therefore 4.3.1. Source of the Cry2Ab2 protein...... 142and/oror affiliates. 4.3.2. Bioinformatics analysisproperty of sequenintellectualthirdce similarity of the Cry2Ab2may protein its produced in MON 89034as to allergens ...... 142 the of and is regulatory owner. 4.3.3. Digestibility of the Cry2Ab2 prota ein in simulateddocument gastric fluid ...... 143 any such its 4.3.4. Digestibility of the Cry2Ab2 proteireproductionn in Simulateddocument Intestinal Fluid ...... 146 owner this of 4.3.5. Proportion of Cry2Ab2 proteinunder to theof total protein in MON 89034 grain148 rightsthe this 4.4. Conclusions...... 148documentand/or rights to of fall use of SECTION 5. Assessment of the Potential for Toxicitythe of the Cry1A.105 and This may distribution,and Cry2Ab2rights Proteins...... 149 owner subject 5.1. Approach to the assessment of toxicity...... 149 be the violate 5.2. Safety ofcopy the donor organism:of Bacillus thuringiensis...... 149 and 5.3.may Assessment document ofpublication, the potential for toxicity of the Cry1A.105 protein ...... 150 It exploitation 5.4. Similarity of Cry1A.105 to Cry1A proteins with a history of safe use and this any consumption...... 151 permission 5.4.1. Structural similarityprohibited of the Cry1A.105 protein with Cry1A class of Bt the proteinscommercial ...... 151 be 5.4.2. Functional similarity of the Cry1A.105 Protein to the Cry1A class of Bt Furthermore,anyproteins ...... 152 Consequently,without 5.4.3. Structural similarity of Cry1A.105 to known toxins or other biologically active proteins...... 153 5.4.4. Acute oral toxicity study with the Cry1A.105 protein...... 153 5.4.5. Exposure to the Cry1A.105 protein ...... 154 5.5. Assessment of the potential for toxicity of the Cry2Ab2 protein ...... 155 5.5.1. Similarity of the Cry2Ab2 protein with proteins with a history of safe use and consumption ...... 155

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 5 of 323 5.5.2. Structural similarity of the Cry2Ab2 to known toxins or other biologically active proteins ...... 155 5.5.3. Acute oral toxicity study with Cry2Ab2 protein...... 155 5.5.4. Exposure to the Cry2Ab2 protein ...... 156 5.6. Conclusions...... 156 References ...... 157

PART VII. FOOD AND FEED SAFETY AND NUTRITIONAL ASSESSMENT OF MON 89034...... 162 SECTION 1. Corn as the Comparable Food and Feed ...... 162 and SECTION 2. Historical Uses of Corn ...... 162and regime. AG 2.1. History and utilization of corn ...... 162 2.2. Corn as a food source...... 163 Bayer property protectionpublishing 2.3. Corn as a feed source ...... 164of contents its SECTION 3. Comparison of the Composition and Characteristicsparties.data of MONtherefore 89034 to Conventional Corn...... 165and/oror affiliates. 3.1. Assessment of significantproperty nutrie intellectualnts, antinutrients,third and key secondarymay its metabolites in corn forage andas grain ...... 165 the of and is regulatory owner. 3.2. Levels of nutrients in corn forage anda grain ...... 167document any such its 3.3. Levels of key anti-nutrients and secondaryreproduction metabolitesdocument in corn forage and owner this of grain...... 168under of rightsthe this SECTION 4.document Otherand/or Information Relevant to the Safety andrights Nutritional Assessment to of fall use of of MON 89034...... 186the This may distribution,and SECTION 5. Substantialrights Equivalenc e of MONowner 89034 to Conventional Corn ...... 186 subject Referencesbe ...... 188the violate copy of APPENDICES...... 191and may documentpublication, It exploitation APPENDIX A: Materials and Methods for the Molecular Analysis of MON 89034...192 this any APPENDIX B: Materials permissionand Methods for the Characterization of the Cry1A.105 and Cry2Ab2 prohibitedProteins in MON 89034...... 197 the be APPENDIX C:commercial Materials and Methods for the Safety Assessment of the Furthermore,any Cry1A.105 and Cry2Ab2 Proteins ...... 216 APPENDIXConsequently, D:without Summary of The Tryptic Masses of the Cry1A.105 And Cry2Ab2 Proteins Identified Using MALDI-TOF MS...... 244 APPENDIX E: Materials and Methods used for the Estimation of Cry1Ab.105 and Cry2Ab2 Protein Levels in Tissues of MON 89034 ...... 254 APPENDIX F: Materials and Methods used for Compositional Analysis of MON 89034...... 259 APPENDIX G: Compositional Analyses Data for Individual Sites ...... 272

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 6 of 323 APPENDIX H: Supplementary Compositional Analysis Data...... 316

List of Tables

Table IV.1. Taxonomic Classification of Corn and Its Close Relatives...... 37 Table IV.2. Summary of Genetic Elements in Vector PV-ZMIR245...... 47 Table IV.3. Summary of Genetic Elements in MON 89034...... 58 Table IV.4. Segregation Analyses of MON 89034a...... 92 and Table VI.1. Amino Acid Sequence Identity Between the Cry1A.105and and Cry1Ac,regime. Cry1Ab, and Cry1F Proteins...... AG 101

Table VI.2. EC50 Values of E. coli- and MON 89034-produced Cry1A.105 Proteins in a Corn Earworm Diet-incorporationBayer Bioassay...... property protectionpublishing 110 of contents Table VI.3. EC Values of the MON 89034- and E.coli-produced Cry2Ab2 Proteins 50 its parties.data therefore in a Corn Earworm Diet-incorporation Bioassay...... and/oror 121 Table VI.4. Cry1A.105 and Cry2Ab2affiliates. Protein Levels in MON 89034 Tissues...... 126 property intellectualthird may its Table VI.5. Cry1A.105 Proteins Levelsas in Overseason Tissues of MON 89034 ...... 127 the of and Table VI.6. Cry2Ab2 Proteinsis Levels in Overseasonregulatory Tissues of MON owner.89034 ...... 128 a document any such its Table VI.7. Acute Dietary Intake and Margin of Exposurereproduction (MOE)document of for the Cry1A.105 and Cry2Ab2owner Proteins from thisHuman Consumption of MON under of this 89034 inand/or the U.S.rights ...... the 132 document fall of rights Table VI.8. Daily Dietaryto Intakeof and Margin ofuse Exposure (MOE) for the Cry1A.105 the This and Cry2Ab2 Proteinsmay fromdistribution, Consumptionand of MON 89034 by Poultry rights owner andsubject Livestock...... 133 Table VII.1. Comparison of Proximates, Fitheber, violate and Mineral Content in Forage from be copy Test (MON 89034) and Conventionalof Control (LH198 x LH172) Corn and may for Combineddocumentpublication, Sites ...... 170 It exploitation Table VII.2. Comparison of the Amino Acid Content in Grain from Test (MON this any 89034) and Conventional Control (LH198 x LH172) Corn for permission Combined Sites ...... prohibited 171 the Table VII.3. Comparisoncommercial of bethe Fatty Acid Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Furthermore,anyCombined Sites ...... 174 Consequently,without Table VII.4. Comparison of the Mineral Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites... 175 Table VII.5. Comparison of the Proximates and Fiber Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites ...... 176 Table VII.6. Comparison of the Vitamin Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites... 177

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 7 of 323 Table VII.7. Comparison of the Anti-nutrients and Secondary Metabolites Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites...... 178 Table VII.8. Summary of Differences (p<0.05) for the Comparison of Analyte Levels for MON 89034 vs. the Conventional Control (LH198 x LH172) and Commercial References ...... 179 Table VII.9. Literature and ILSI Database Ranges of Components of Corn Forage and Grain...... 183

and and regime. AG List of Figures

Figure IV.1. Plasmid Map of Vector PV-ZMIR245...... Bayer property protectionpublishing 45 of contents Figure IV.2. Process Map for Transformation, Selection, Regeneration itsand parties.data therefore Evaluation of MON 89034 ...... and/oror 46 affiliates. Figure IV.3. Deduced Amino propertyAcid Seque intellectualnce ofthird the Cry1A.105 Proteinmay Produced in its MON 89034...... as 51 the of and Figure IV.4. Deduced Aminois Acid Sequence ofregulatory the Cry2Ab2 Protein Producedowner. in a document MON 89034...... any such its 51 reproductiondocument of Figure IV.5. Description of the Recombinationowner Processthis th at Explains the Modified under of 5’End of the rightsInsert...... the this 54 documentand/or of rights Figure IV.6. Genetic Elementsto of andfall Restri ctionuse Sites of Vector PV-ZMIR245 Used the This in Southern Blotmay Analyses distribution, (Probesand 1-17)...... 55 rights owner Figure IV.7. Geneticsubject Elements and Restriction Sites of Vector PV-ZMIR245 Used bein Southern blot Analyses (Probesthe violate 18 - 23)...... 56 copy of Figure IV.8. Schematic Representation of andthe Insert and Genomic Flanking may Sequencesdocumentpublication, in MON 89034...... 57 It exploitation Figure IV.9. Southernthis any Blot Analysis of MON 89034: Insert and Copy Number ...... 61 Figure IV.10. Southern Blotpermission Analysis of MON 89034: e35S Promoter/Cab Leader ..... 64 prohibited Figure IV.11. Southernthe Blot Analysis of MON 89034: Ract1 Intron...... 65 commercial be Figure IV.12. Southern Blot Analysis of MON 89034: cry1A.105 Coding Sequence .... 67 FigureFurthermore, IV.13.any Southern Blot Analysis of MON 89034: Hsp17 3' End Sequence...... 68 Consequently,without Figure IV.14. Southern Blot Analysis of MON 89034: FMV Promoter...... 71 Figure IV.15. Southern Blot Analysis of MON 89034: Hsp70 Intron...... 72 Figure IV.16. Southern Blot Analysis of MON 89034: SSU-CTP Targeting Sequence/cry2Ab2 Coding Sequence...... 73 Figure IV.17. Southern Blot Analysis of MON 89034: nos 3' end Sequence ...... 75

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 8 of 323 Figure IV.18. Southern Blot Analysis of MON 89034: PV-ZMIR245 Backbone Sequence...... 77 Figure IV.19. Southern Blot Analysis of MON 89034: npt II Coding Sequence...... 80 Figure IV.20. Southern Blot Analysis of MON 89034: T-DNA II...... 81 Figure IV.21. Breeding History of MON 89034...... 83 Figure IV.22. Insert Stability of MON 89034: T-DNA I...... 85 Figure IV.23. Analysis of Multiple Generations of MON 89034: T-DNA II...... 88 Figure IV.24. Backbone Analysis of Multiple Generations of MON 89034: PV- ZMIR245 Backbone Sequence...... 89 Figure VI.1. Schematic Representation of the Origin of Cry1A.105 Protein Domainsand 101 and regime. Figure VI.2. SDS-PAGE of the MON 89034- andAG E. coli-produced Cry1A.105 Proteins...... 103 Figure VI.3. Western Blot Analysis of MON 89034- and E. coli-produced Bayer property protectionpublishing Cry1A.105 Proteins...... of contents 104 its Figure VI.4. N-terminus Intactness by Immunoblot Analysisparties.data Using Anti-N-terminaltherefore Peptide Antibody...... and/oror 106 affiliates. Figure VI.5. Sequence Coverageproperty Map of theintellectual MONthird 89034-produced Cry1A.105may its as Protein by MALDI-TOFthe of MS andAnalysis...... 107 is regulatory owner. Figure VI.6. Glycosylation Analysis of the MONa 89034-produceddocument Cry1A.105 any such its Protein ...... reproductiondocument 109 owner this of Figure VI.7. Functional Equivalence ofunder the MON of89034- and E. coli-produced rightsthe this documentCry1A.105and/or Proteins Against Corn Earworm (CEW)rights ...... 111 to of fall use of Figure VI.8. Comparison of the Putative N-terminal Sequencesthe of the Cry2Ab2 This Proteins Producedmay in MONdistribution, and89034, Recombinant E. coli, and Bacillus rights owner thuringiensissubject ...... 113 the Figure VI.9. be SDS-PAGE of the MON 89034- andviolate E. coli -produced Cry2Ab2 copy of Proteins...... and 115 may documentpublication, FigureIt VI.10. Western Blot Analysisexploitation of MON 89034- and E. coli-produced Cry2Ab2this any Proteins...... 116 Figure VI.11. Peptide Sequencepermission Coverage Map of the MON 89034-Produced prohibited Cry2Ab2 Protein by MALDI-TOF MS Analysis...... 118 the be Figure VI.12. Peptidecommercial Sequence Coverage Map of the MON 89034-derived Furthermore,anyCry2Ab2 Proteolytic Fragment by MALDI-TOF MS Analysis ...... 118 FigureConsequently, VI.13. Glycosylationwithout Analysis of the MON-89034- and E.coli-produced Cry2Ab2 Proteins...... 120 Figure VI.14. Functional Equivalence of the MON 89034- and E. coli-produced Cry2Ab2 Proteins Against Corn Earworm (CEW) ...... 122 Figure VI.15. Colloidal Brilliant Blue G Stained SDS-gels of Cry1A.105 Protein Digestion in SGF...... 138 Figure VI.16. Western blot Analysis of Cry1A.105 Protein Digestion in SGF...... 139

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 9 of 323 Figure VI.17. Western Blot Analysis of Cry1A.105 protein Digestion in SIF...... 141 Figure VI.18. Colloidal Brilliant Blue G Stained SDS-PAGE Gels of Cry2Ab2 Protein Digestion in SGF ...... 144 Figure VI.19. Western Blot Analysis of Cry2Ab2 Protein Digestion in SGF...... 145 Figure VI.20. Western Blot Analysis of Cry2Ab2 Protein Digestion in SIF ...... 147 Figure VI.21. Phylogram Establishing that the Cry1A.105 and Cry1Ac Proteins are Located in the Same Cluster Based on Amino Acid Sequence Identity .. 152 Figure VII.1. Safety Assessment of New Varieties: the Host Plant ...... 187

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 FDA BNF No. 00105 / Monsanto 06-CR-161F Page 10 of 323 and and regime. AG

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 except to the extent it is requested under the Freedom of Information Act (FOIA), 5 U.S.C., § 552; FDA complies with the provisions of FOIA and FDA’s implementation regulations (21 CFR Part 20); and this information is responsive to the specific request. Except in accordance with the Freedom of Information Act, 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 the as of and owner. is regulatory a documentits any such reproductiondocument of owner this 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

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

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 12 of 323 Abbreviations1, Acronyms and Definitions

1× LB Laemmli Buffer [62.5mM Tris-HCl, 5% (v/v) 2-mercaptoethanol, 2% (w/v) sodium dodecyl sulfate, 0.005% (w/v) bromophenol blue, 10% (v/v) glycerol, pH 6.8] 5× LB Five times concentrated 1× LB 2T-DNA Plasmid vector containing two separate T-DNA regions each surrounded by left and right borders of the Ti plasmid 35S Promoter and leader from the Cauliflower mosaic virus (CaMV) 35S RNA and AA or aa Amino Acid and regime. AG AACC American Association of Cereal Chemists aadA Bacterial promoter and coding sequence for an aminoglycoside- Bayer property protectionpublishing modifying enzyme,of 3'(9)-O-nucleotidyltransferasecontents from the transposon Tn7 its parties.data therefore ACB Asian corn borer, Ostrinia funicalis and/oror affiliates. property intellectualthird may AEX Anion Exchangeits Chromatography as AOAC Associationthe of of Officialand Analytical Chemists is regulatory owner. a document AOCS Americanany Oilsuch Chemists Society its reproductiondocument of AD6 Allergen, gliadin,owner and glutenin thisprotein sequence database under of rightsthe this ADF documentand/orAcid Detergent Fiber rights to of fall use of ALLPEPTIDES Protein sequence database comprisedthe of NRAA and SwissProt This databasesmay distribution,and rights owner ALLERGEN- subject be Computer program thefor theviolate search against known allergens SEARCH copy of bp documentbase pair and may publication,exploitation BCWIt Black cutworm, Agrotis ipsilon this any BRS Biotechnologypermission Regulatory Sciences prohibited BSA Bovinethe Serum Albumin commercial be Bt Bacillus thuringiensis Furthermore,bw any body weight Consequently,without Cab 5' untranslated leader of the wheat chlorophyll a/b-binding protein CaMV Cauliflower Mosaic Virus CAPS 3-[cyclohexyl amino]-1-propanesulfonic acid

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

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 13 of 323 CEW Corn earworm, Helicoverpa zea CFR Code of Federal Regulations CHAPS 3[(3-Cholamidopropyl)dimethylammonio]-propanesulfonic acid CI Confidence Interval Cry Crystal proteins from Bacillus thuringiensis CS Coding Sequence Cry1A.105 A chimeric protein comprised of domains from the naturally occurring Cry1Ab, Cry1F, and Cry1Ac proteins of Bacillus thuringiensis and cry1A.105 Coding sequence for the Cry1A.105 proteinand regime. AG Cry2Ab2 A Cry2 class crystal protein from Bacillus thuringiensis subsp. kurstaki. Bayer property protectionpublishing cry2Ab2 Coding sequence offor the Cry2Ab2 protein contents its Cry1Ab A naturally-occurring protein parties.produceddata by Bt spp. kurstakitherefore HD-1 with activity against certain Lepidopteranand/or insectsor affiliates. Cry1Ac A naturally-occurringproperty intellectual proteinthird produced by Bt sppmay. kurstaki HD-73 its with activity againstas certain Lepidopteran insects the of and is regulatory owner. Cry3 A class of Bt crystal aproteins withdocument insecticidal activity against any such its coleopteran insects reproductiondocument owner this of Cry3Bb1 A naturally-occurringunder protein of produced by B.t. spp. kumamotoensis rightsthe this documentand/orwith activity against certain Coleopteranrights insects to of fall use of CTP Chloroplast Transit Peptide the This may distribution,and CTAB Cetrightsyltrimethyla mmoniumowner bromide subject CV Coefficient of Variationthe violate be copy DAP Days After Plantingof and may documentpublication, dCTPIt Deoxycexploitationytidine trip hosphate this DF anyDilution Factor permission dNTP Deoxynucleotideprohibited trip hosphate the DTT commercialDithiot behreit ol dw or DW dry weight Furthermore,any dwtConsequently, without d ry weight of tissue e35S The promoter and leader from cauliflower mosaic virus (CaMV) 35S RNA containing the duplicated enhancer region

EC50 Effective protein concentration to inhibit the growth of the target insect by 50% ECB European corn borer, Ostrinia nubilalis

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 14 of 323 E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid ECL Enhanced Chemiluminescence ELISA Enzyme-Linked Immunosorbent Assay EPPS 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid FA Fatty Acid FAW Fall armyworm, Spodoptera frugiperda FASTA Algorithm used to find local high scoring alignments between a pair of protein or nucleotide sequences and and regime. FAARP Food Allergy Research andAG Resource Program FIFRA Federal Insecticide, Fungicide and Rodenticide Act (U.S.) FMV Figwort mosaic virusBayer 35S promoterproperty publishing protectioncontents FR Federal Register of its parties.data therefore FW or fw Fresh Weight and/oror affiliates. fwt fresh weightproperty of tissue intellectual third may its as gDNA genomicthe DNAof and is regulatory owner. GLP Good Laboratory Practicea document any such its reproductiondocument HEPES N-[2-(hydroxyeownerthyl)p iperazine-N'-(2-ethis thanesof ulfonic acid)] under of this HPLC and/orHighrights Performancethe Liquid Chromatography document fall of rights HRP Horserto adishof Peroxidase use the Hsp17 This 3' nontranslatedmay distribution, regionand of the gene for wheat heat shock protein rights owner subject17.3 which ends transcription and directs polyadenylation the Hsp70 be violate copyMaize heat shockof protein 70 gene I documentIntron and may publication,exploitation I-Ract1It Intron from the rice actin gene this any IgG Immunoglobulin G permission ILSI-CCD Internationalprohibited Life Sciences Institute-Crop Composition Database the be kb commercialkilobase Furthermore,kDa any kilodalton Consequently,without L Leader Left Border DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA LOQ Limit of Quantitation LOD Limit of Detection mA milliampere

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 15 of 323 MAFF Ministry of Agriculture, Forestry and Fisheries (Japan) MALDI-TOF Matix Assisted Laser Desorption Ionization - Time of Flight MH+ Protonated mass ion MHLW Ministry of Health, Labor and Welfare (Japan) MMT Million Metric Tonnes MOE Margin of Exposure MOA Mode of Action MON 810 A Monsanto corn product, producing the insecticidal Bt Cry1Ab protein regime.and MON 89034 A Monsanto corn product, and the subjectand of this application, AG which produces the Bt Cry1A.105 and Cry2Ab2 proteins MS Mass Spectrometry Bayer property protectionpublishing MWCO Molecular weightof cut-o ff contents its MW Molecular Weight parties.data therefore and/oror MWM Molecular Weightaffiliates. Marker property intellectualthird may n Number of Observationsits as the of and n.a. or na notis available regulatory owner. a document any such its NCGA National Corn Grower’s Associationreproduction document owner this of n.d. or nd not detectable under of rightsthe this documentand/or rights NDF Neutralto of Detergent fall Fiberuse of NFDM Non-fat Dry Milk the This may distribution,and rights owner NIST subjectNational Institute of Standards and Technology NMWC be Nominal molecular theweight violate cut-off copy of NOEL documentNo Observable Effectand Level may publication,exploitation NRAAIt A public protein database maintained by the NCBI at the National this anyInstitutes of Health, Bethesda, MD permission nos 3’ transcriptprohibited termination sequence of the nopaline synthase (nos) genethe from Agrobacterium tumefaciens commercial be nptII Coding sequence of neomycin phosphotransferase II gene that Furthermore,any confers resistance to neomycin and kanamycin Consequently,without NPTII Neomycin phosphotransferase II OD Optical Density OECD Organization for Economic Co-operation and Development OR origin of replication ori-PBR322 origin of replication from pBR322 for maintenance of plasmid in E. coli

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 16 of 323 ori V origin of replication for Agrobacterium derived from the broad host range plasmid RK2 OSL Overseason Leaf OSR Overseason Root OSWP Overseason Whole Plant P Promoter PAGE Polyacrylamide Gel Electrophoresis PBS Phosphate Buffered Saline PBST Phosphate Buffered Saline containing Tween-20 regime.and PCR Polymerase Chain Reaction and AG PMSF Phenylmethylsulfonyl Fluoride ppm parts per million Bayer property protectionpublishing PTH Phenylthiohydantoinof contents its parties.data therefore PVDF Polyvinylidene Difluoride and/oror affiliates. PVPP Polyvinylppropertyolypyrrolidone intellectualthird may its PV-ZMIR245 Plasmid vector usedas to develop MON 89034 the of and is regulatory owner. Ract1 the rice actin gene a document any such its reproductiondocument Right Border DNA region fromowner Agrobacterium this tumefaciensof containing the right border sequence underused for transferof of the T-DNA rightsthe this documentand/or rights rop Codingto of sequence fall for repressoruse of o f primer protein for maintenance of plasmid copy number in E. colithe This may distribution,and RP-HPLC Reversedrights Phase-High Performanceowner Liquid Chromatography subject SAS be Statistical Analysisthe System, violate an integrated system of software copyproducts providedof by the SAS Institute, Inc. headquartered in Cary, and may documentNorthpublication, Carolina, USA It exploitation SCB Sugarcane borer, Diatraea saccharalis this any SD Standardpermission Deviation prohibited SDS-PAGE Sodiumthe Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis commercial be SE Standard Error Furthermore,any SGFConsequently, without Simulated Gastric Fluid SOP Standard Operating Procedure sp. species subsp. subspecies SSU-CTP DNA region containing the targeting sequence for the transit peptide region of maize ribulose 1,5-bisphosphate carboxylase small subunit and the first intron

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 17 of 323 SWCB Southwestern corn borer, Diatraea grandiosella SwissProt A public protein database maintained by the Swiss Institute of Bioinformatics, Geneva, Switzerland, and EMBL Taq Thermus aquaticus, a thermophilic bacterium T Terminator TS Targeting Sequence TCA Trichloroacetic Acid T-DNA Transfer DNA

T-DNA I Transferred DNA containing the cry1A.105 and cry2Ab2and expression cassettes in plasmid vector PV-ZMIR245and regime. AG T-DNA II Transferred DNA containing the nptII gene cassette in plasmid vector PV-ZMIR245 Bayer property protectionpublishing TDF Total Dietary Fiberof contents TI its Tolerance Interval parties.data therefore and/oror TFA Trifluoroacetic Acid affiliates. property intellectualthird may TMB 3,3’,5,5’-Tetramits ethylbenzidene as the of and TOXIN5 Toxinis protein sequence databaseregulatory owner. a document Tris Tris(hydroxymethyl)aminomethaneany such its reproductiondocument owner this of Tween-20 Polyoxyethylenesorbitanunder monolaurateof rightsthe this USDA-APHIS and/or United States Department of Agriculture – Animal and Plant document to of fall of rights Health Inspection Serviceuse the This may distribution,and USDA-NASS Unitedrights States Departmentowner of Agriculture – National Agricultural subjectStatistics Service be the violate U.S. EPA copyUnited States Environmentalof Protection Agency and U.S. FASmay documentUnitedpublication, States Foreign Agriculture Service It exploitation U.S. FDA United States Food and Drug Administration this any UV Ultravpermissioniolet prohibited VOI Verificationthe of Identity commercial be v/v volume to volume ratio Furthermore,w/v any weight to volume ratio Consequently,without w/w weight to weight ratio wt weight wt CAB A 5’ untranslated leader of the wheat chlorophyll a/b/-binding protein WBCW Western bean cutworm, Striacosta albicosta

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 18 of 323 Narrative Summary

Food and Feed Safety and Nutritional Assessment of the Lepidopteran-protected Corn MON 89034

Monsanto Company has developed, through the use of recombinant DNA techniques, MON 89034, a corn product that is protected from damage caused by larval feeding of lepidopteran insect pests. MON 89034 produces the Bacillus thuringiensis (Bt) Cry1A.105 and Cry2Ab2 proteins that are active against lepidopteran insects. MON 89034 is a second-generation product that will effectively address a corn grower’s need to control a wide spectrum of lepidopteran pests. The combination of the Cry1A.105and and and regime. Cry2Ab2 insecticidal proteins in a single plant providesAG excellent control of lepidopteran insect pests and offers an effective insect resistance management tool.

Corn (Zea mays L.) is the largest crop grownBayer in the U.S.property in terms protection of acreagepublishing planted and of contents net value. In 2005, 81.8 million acres of corn was planted in the U.S. itsThe average yield parties.data therefore of corn grain was 148 bushels per acre, with a total productionand/or ofor 11 billion bushels valued at $21 billion. The corn cropaffiliates. is susceptible to feeding damage from insect pests property intellectualthird may resulting in significant economic itslosses. In 1997, Monsanto commercialized the first- ® as generation YieldGard Cornthe Borerof corn and(hereafter referred to as MON 810), which is regulatory owner. produces a Cry1Ab protein that provides effectivea protectiondocument against damage caused by any such its lepidopteran insect pests, especially the Europeanreproduction corn borerdocument (ECB,of Ostrinia nubilalis) and the corn earworm (CEW, Helicoverpaowner zea). Thethis benefits of MON 810 and other Bt under of corn products have includedrights morethe effective control ofthis lepidopteran larval pests, lower documentand/or rights levels of harmful mycotoxinsto of in grain,fall and reductionuse of in the use of chemical insecticides. Bt corn products have been widely adopted by growers,the and by 2006, insect-protected This may distribution,and corn was planted in overrights 40% of the U.S corn owneracres. subject be the violate The introduction copyof the second-generationof product MON 89034 is expected to provide enhanced benefits for the control of lepidoptand eran pests of corn compared to existing may documentpublication, products.It MON 89034 will exploitationhave a wider spectrum of activity against lepidopteran pests and strengthenthis insect any resistance management, continue to reduce the potential for mycotoxins in grain, enable more efficient plant breeding of this multi-genic trait into permission superior hybrids compared to prohibitedMON 810, and potentially reduce the refuge acreage required for resistance themanagement purposes. Taken together, adoption of MON 89034 commercial be is likely to enhance economic and other benefits (occupational health, flexibility of use, etc)Furthermore, to farmersany and improve the quality of grain and the safety of the derived food and feedConsequently, products. without

MON 89034 was produced by Agrobacterium-mediated transformation of corn with the PV-ZMIR245 vector, which is a binary vector containing two separate transfer DNA’s (2T-DNA). The first T-DNA, designated as T-DNA I, contains the cry1A.105 and the cry2Ab2 expression cassettes. The second T-DNA, designated as T-DNA II, contains the

® YieldGard is a registered trademark of Monsanto Company LLC.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 19 of 323 nptII (neomycin phosphotransferase II) expression cassette. During transformation, both T-DNAs were inserted into the genome. The nptII selectable marker gene was used for the selection of transformed cells in the presence of neomycin. Once the transgenic cells were identified, the selectable marker gene was no longer needed. Traditional breeding was used to produce plants that only contained the cry1A.105 and cry2Ab2 expression cassettes (T-DNA I) and did not contain the nptII expression cassette (T-DNA II), thereby, producing marker-free corn MON 89034.

The data and information presented in this summary demonstrate that the foods and feeds derived from MON 89034 are as safe and nutritious as the comparable foods and feeds derived from conventional corn. This conclusion is based on several lines of evidence. The first is the detailed molecular characterization of the inserted DNA. Results confirm regime.and the insertion of a single functional copy of the cry1A.105and and cry2Ab2 expression AG cassettes at a single locus within the genome. The second is a detailed biochemical characterization of the Cry1A.105 and Cry2Ab2 proteins produced in MON 89034. Data demonstrate that the two Cry proteins produBayerced in MONproperty 89034 protection are publishingequivalent to their counterparts produced by recombinant strainsof of Escherichia coli for use incontents various safety its assessment studies. The third is a thorough assessmentparties. ofdata the allergenicitytherefore and toxicity potential of the Cry1A.105 and Cry2Ab2 proteins based on extensiveand/oror studies. The results affiliates. demonstrate the safety of theproperty Cry1A.105 intellectual andthird Cry2Ab2 proteins duemay to the lack of its allergenic potential and the lack of acuteas toxicity when ingested, and their similarity to the of and Cry proteins that have a ishistory of safe use. Theregulatory fourth is the estimationowner. of protein levels a document and a dietary safety assessmentany suchbased on anticipated exposure andits the results of acute reproductiondocument toxicology tests. Results show thatowner there are no meaningfulthis risksof to human or animal health from dietary exposure to the Cry1Aunder.105 andof Cry2Ab2 proteins based on the large rightsthe this margins of exposuredocumentand/or (MOEs) obtained. The fifth is the rightscompositional and nutritional to of fall use of assessment of MON 89034 grain and forage, whichthe confirms that MON 89034 is compositionallyThis equivalent to andmay as distribution, safe andas conventional corn. rights owner subject Molecular characterization of MON 89034the by violateSouthern blot analyses was conducted to be copy determine: 1) the number of inserts andof copies in the genome, 2) intactness of the genetic and elementsmay within thedocument insert,publication, 3) absence of the T-DNA II encoding the selectable marker, 4) It exploitation absence of backbonethis sequences, and 5) stability of the inserted DNA across multiple generations. Resultsany demonstrat ed that the DNA inserted into the corn genome is present permission at a single locus and contains prohibitedone functional copy of the cry1A.105 and the cry2Ab2 expression cassettes. All genetic elements were shown to be present in the inserted DNA the be as expected. However,commercial the e35S promoter, which regulates expression of the cry1A.105 gene,Furthermore, was modifiedany and the Right Border sequence present in PV-ZMIR245 was replaced by aConsequently, Left Borderwithout sequence in MON 89034. There were no other elements, either full length or partial, present other than those associated with the intended insert, and no backbone plasmid DNA or nptII sequences were detected. PCR and DNA sequence analyses provided the complete DNA sequence of the insert and confirmed the organization of the elements within the insert. The stability of the integrated DNA and absence of the T-DNA II and backbone sequences in multiple generations of MON 89034 was also confirmed. The heritability of the cry1A.105 and cry2Ab2 genes was confirmed by segregation analysis of several generations of MON 89034. These results are

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 20 of 323 consistent with the conclusion of a single active site of insertion that segregates according to the Mendelian laws of genetics.

Detailed biochemical characterization of the Cry1A.105 and Cry2Ab2 proteins produced in MON 89034 confirmed their identity and equivalence to the corresponding E. coli- produced proteins. The characterization was based on: a) the source organism from which the two Cry proteins are derived; 2) identity and function; and, 3) physicochemical and functional equivalence to the E. coli-produced protein standards. The donor organism for the Cry1A.105 and Cry2Ab2 proteins, Bacillus thuringiensis, has been used commercially in the U.S. for over four decades to produce microbial pesticides. Cry proteins have a history of safe use since 1958 as active ingredients either in Bt microbial pesticides or in biotechnology-derived food and feed crops. Cry1A.105 is a chimeric regime.and protein comprised of domains I and II from Cry1Ab and Cry1Ac,and do main III from Cry1F AG (Bt subsp. aizawai), and the C-terminal portion from Cry1Ac (Bt subsp. kurstaki). Domains I and II of Cry1A.105 are 100% identical in amino acid sequence to domains I and II of both Cry1Ab and Cry1Ac, domainBayer III is 99%property identifical protection to domainpublishing III of Cry1F, and the C-terminal portion is 100% identicalof to the C-terminal portion ofcontents Cry1Ac. The its overall amino acid sequence identity to the Cry1Ab,parties. Cry1Ac,data and Cry1Ftherefore proteins is 90.0%, 93.6%, and 76.7 % respectively. Bt microbial strainsand/or producingor Cry1Ac and affiliates. 1 Cry1Ab, and Cry1F proteinsproperty have been intellectual usedthird for decades as biopesticides.may The its Cry1A.105 protein produced in MONas 89034 is structurally and functionally similar to the of and Cry1A proteins producedis in a number of biotechnology-derivedregulatory cropsowner. (e.g., YieldGard ® a ® document Corn Borer corn, Bollgardany cotton such and Bollgard II cotton) thatits have demonstrated reproductiondocument history of safe use. owner this of under of rightsthe this The Cry2Ab2document proteinand/or produced in MON 89034 is derived fromrights the Bt subspecies kurstaki to of fall use of and its amino acid sequence differs from that of the wild-typethe protein by a single amino acid. TheThis Cry2Ab2 protein hasmay 88% distribution, aminoand acid sequence identity to the Cry2Aa protein rights owner which is presentsubject in commercial microbial pest control products such as Dipel® and Crymax®. The Cry2Ab2 proteins producedthe in violateMON 89034 and Bollgard II cotton have be copy an identical amino acid sequence. Bollgardof II cotton has been on the market since 2003 and and theremay have beendocument no adversepublication, reports regarding its safety. It exploitation this The characterizationany of the Cry1A.105 and Cry2Ab2 proteins produced in MON 89034 permission confirmed that these proteins arprohibitede equivalent to the respective E. coli-produced protein standards used in safety studies. Since the in planta expression of the Cry1A.105 and the be Cry2Ab2 proteinscommercial is low, it was necessary to produce these proteins in the high- expressingFurthermore, anyrecombinant host organism, E. coli, so that they could be used in safety studies.Consequently, The E.without coli -produced proteins were engineered to have the identical amino acid sequences as their counterparts expressed in MON 89034. The MON 89034- and E. coli- produced proteins were then evaluated to ensure that they were physicochemically and

1 Cry1Ab and Cry1Ac are components of the microbial product Dipel®, and a Cry1Ac/Cry1F chimeric protein is a component of the microbial product Lepinox® (Ecogen Inc.). ®Dipel is a registered trademark of Abbott Inc., Crymax and Lepinox are registered trademarks of Ecogen, Inc., and Bollgard and Bollgard II are registered trademarks of Monsanto Technology LLC.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 21 of 323 functionally equivalent based on the following analytical tests: a) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to estimate approximate molecular weight; b) western blot analysis to demonstrate identity and immunoreactivity; c) N- terminal sequence analysis or western blot analysis to examine the intactness of the N- terminus; d) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to establish protein identity by peptide mapping; e) glycosylation analysis to determine the presence or absence of covalently-linked carbohydrates; and, f) insect bioassay to assess functional equivalence. These analyses provided a detailed characterization of the Cry1A.105 and Cry2Ab2 proteins isolated from MON 89034 and confirmed their equivalence to the E. coli-produced Cry1A.105 and Cry2Ab2 proteins.

The assessment of potential allergenicity and toxicity showed there was a reasonable regime.and certainty of no harm to mammals from exposure to the Cry1A.105and and Cry2Ab2 proteins. AG These assessments were based on: a) an evaluation of potential allergenicity based on the source of the protein, structural similarities to known allergens, in vitro digestibility in simulated digestive fluids, and expectedBayer dietary exposure;property and,protection b)publishing an evaluation of potential toxicity based on history of ofuse, similarity to known toxins contentsor biologically its active proteins, and evaluation of acute toxicity to mammalsparties.data therefore and/oror affiliates. As mentioned previously, Bacillusproperty thuringiensis intellectualthird, the donor organism mayfor these two Cry its proteins, has been used commerciallyas in the U.S. for over four decades to produce the of and microbial pesticides, andis there are no confirregulatorymed cases of allergicowner. reactions to Cry a document proteins. Results of extensiveany such bioinfor matics assessments usingits FASTA sequence reproductiondocument alignment and eight-amino acid slidingowner window searches,this showedof that the Cry1A.105 and Cry2Ab2 proteins do not share anyunder amino acidof sequence similarities with known rightsthe this allergens, gliadins,document and/orglutenins, or protein toxins that haverights adverse effects to mammals. to of fall use of Assessment of the in vitro digestibility in simulated gastricthe fluid (SGF) showed that the Cry1A.105This and Cry2Ab2 proteinsmay aredistribution, rapidlyand dige sted, with greater than 95% to 99% of rights owner the proteins, respectively,subject being digested in less than 30 seconds. Proteins that are rapidly digestible have a lower risk of causithe ngviolate allergic reactions or resulting in toxicity be copy when consumed. Mice acute oral toxicityof studies demonstrate that the Cry1A.105 and and Cry2Ab2may proteins documentare notpublication, acutely toxic and do not cause any adverse effects even at It exploitation maximum attainablethis dose levels of 2072 and 2198 mg/kg body weight for the Cry1A.105 and Cry2Ab2 proteins,any respectively. The independent safety assessment for each of the permission Cry proteins in mice was consideredprohibited appropr iate and adequate based on the extensive history of safe use of mixtures of Cry proteins present in Bt microbial pesticides. the be commercial TheFurthermore, levels ofany the Cry1A.105 and Cry2Ab2 proteins estimated in tissues of MON 89034 showedConsequently, trends thatwithout were consistent for exposure calculations and intended uses. Tissues of MON 89034 were collected from field trials conducted at five sites in the U.S. during 2005. Tissues from the different growth stages of the corn plant were collected throughout the growing season and analyzed by enzyme-linked immunosorbent assay (ELISA). The mean Cry1A.105 levels across sites were 520 µg/g dwt in young leaf, 42 µg/g dwt in forage, and 5.9 µg/g dwt in grain. The mean Cry2Ab2 levels across sites were in 180 µg/g dwt in young leaf, 38 µg/g dwt in forage, and 1.3 µg/g dwt in grain. In general, the levels of the two Cry proteins declined over the growing season.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 22 of 323

A dietary safety assessment based on Cry1A.105 and Cry2Ab2 protein levels, expected dietary exposure, and the results of acute toxicology tests showed large MOEs for humans and animals. The MOEs for the overall U.S. population were greater than or equal to 199,000 and 981,000 for the Cry1A.105 and Cry2Ab2 proteins, respectively. For children aged 3-5 years old, an age group with the highest corn consumption (body weight basis), the MOEs were greater than or equal to 79,400 and 390,000 for the Cry1A.105 and Cry2Ab2 proteins, respectively. For poultry and livestock, the MOEs ranged between 1,930 – 13,500 and 2,160 – 47,600 for the Cry1A.105 and Cry2Ab2 proteins, respectively. These results are consistent with the extensive safety testing previously conducted for the Cry1A and Cry2A class of proteins.

regime.and The EPA further confirmed the safety of the Cry1A.105 and Cry2Ab2and proteins recently AG when they established temporary exemptions from the requirement of a tolerance for the Cry1A.105 and Cry2Ab2 proteins and the genetic material for their production in corn (40 CFR §174.453 and 40 CFR §174.454, Bayerrespectively). property protectionpublishing of contents its Compositional assessment of the grain and forage fromparties. MONdata 89034 demonstratedtherefore that it is nutritionally and biologically equivalent to its conventionaland/or counterpart,or LH198 x affiliates. LH172. Compositional data onproperty key nutrients, intellectual thirdanti-nutrients and othermay components were its collected for the forage and grain fromas MON 89034 and conventional control corn, the of and grown at five field sitesis in the U.S. duringregulatory 2004. Three differentowner. conventional, a document commercial corn referenceany hybrids such were also grown at each site,its for a total of 15 reproductiondocument references. Composition data fromowner the referencesthis was used ofto establish a range of variability described by a 99% toleranceunder intervalof for each component analyzed. rightsthe this Statistical comparisonsdocumentand/or of 61 components from MON rights89034 and the control were to of fall use of conducted for the combination of all five sites (i.e.,the the combined-site) and for each individualThis site. The overall datamay set distribution, was andexamined for evidence of biologically relevant rights owner changes. Evaluationsubject of the data, including the results of statistical analysis, leads to the conclusion that MON 89034 is compositionallythe violate and nutritionally equivalent to be copy conventional corn. of and may documentpublication, It exploitation No statistical differencesthis were observed in 58 of 61 combined-site site comparisons made between MON 89034any and the conventional control. The three differences observed were permission generally small (3.4 – 19.2%), consideringprohibited the range of natural variability, and the mean levels and ranges of MON 89034 were well within the 99% tolerance intervals for the be commercial corn.commercial For the individual site analyses, there were no statistical differences thatFurthermore, were consistentlyany observed across all sites. Furthermore, the means and ranges of all componentsConsequently, fromwithout MON 89034 showing a statistical difference were within the 99% tolerance intervals of conventional corn and/or within the International Life Sciences Institute Crop Composition Database.

In conclusion, the data and information presented in this summary demonstrate that the foods and feeds derived from MON 89034 are as safe and nutritious as the comparable foods and feeds derived from conventional corn. This conclusion is based on several lines of evidence including:

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 23 of 323

1. The detailed molecular characterization of the inserted DNA, which confirmed the presence of single functional copies of the cry1A.105 and cry2Ab2 cassettes, stably integrated at a single locus of the genome; 2. The biochemical characterization of the Cry1A.105 and Cry2Ab2 proteins produced in MON 89034, which confirmed their identity and equivalence to the same Cry1A.105 and Cry2Ab2 proteins produced by recombinant strains of Escherichia coli used in the various safety assessment studies; 3. A safety assessment of the Cry1A.105 and Cry2Ab2 proteins, which shows the lack of allergenic potential and acute toxicity when ingested; 4. A dietary safety assessment, which showed and no meaningful risks to humanand or and regime. animal health from dietary exposure to theAG Cry1A.105 and Cry2Ab2 proteins; and 5. Compositional and nutritional assessments, which demonstrate that the MON 89034 is compositionally equivalentBayer to and as propertysafe as conventional publishing corn. protectioncontents of its Therefore it is concluded that the consumption of MONparties. data89034 and the foodtherefore and feed and/oror derived from it will be as safe and nutritious as that of conventional corn. 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 FDA BNF No. 00105 / Monsanto 06-CR-161F Page 24 of 323

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 89034 is:

Monsanto Company 800 North Lindbergh Blvd. St. Louis, MO 63167 and and regime. Communications with regard to this submissionAG should be directed to Regulatory Affairs Manager, at the above address. She can also be contacted by telephone at . Bayer property protectionpublishing of contents its parties.data therefore and/oror SECTION 2. The subject of this Summaryaffiliates. and the Plant Species from which They property intellectualthird may were Derived its as the of and is regulatory owner. The subject of this summary is MON 89034,a a corn productdocument derived from a Monsanto any such its proprietary corn inbred. reproductiondocument of owner this under of rightsthe this SECTION 3.document Distinctiveand/or Designations Given to the Subjectrights of This Summary to of fall use of the The lepidopteran-protectedThis cornmay that distribution, is theand su bject of this summary has been designated rights owner MON 89034. Insubject accordance with OECD’s “Guidance for the Designation of a Unique Identifier for Transgenic Plants,” MON 89034 has been assigned the unique identifier be the violate MON-89Ø34-3. copy of and may documentpublication, It exploitation this SECTION 4. Identityany and Sources of the Genetic Material Introduced into permission MON 89034 prohibited the be MON 89034 wascommercial developed through Agrobacterium-mediated transformation of corn usingFurthermore, the binaryany plasmid vector, PV-ZMIR245 (Figure IV.1). Agrobacterium-mediated transformationConsequently, withoutis a well-documented process for the transfer and integration of exogenous DNA into a plant’s nuclear genome (Ishida et al., 1996). PV-ZMIR245 contains two separate T-DNAs (therein referred to as 2 T-DNA system). The first T- DNA, designated as T-DNA I, contains the cry1A.105 and the cry2Ab2 expression cassettes. The second T-DNA, designated as T-DNA II, contains the nptII expression cassette that encodes the neomycin phosphotransferase enzyme that confers tolerance to certain antibiotics such as neomycin and paromomycin. Each T-DNA was independently integrated into the corn genome. Plants containing both T-DNA’s I and II were identified

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 25 of 323 and then subjected to conventional plant breeding to identify segregating plants that only contained T-DNA I, which includes the cry1A.105 and cry2Ab2 genes. Only plants containing T-DNA I, but lacking T-DNA II, were developed further by breeding. A detailed description of the transformation and selection process is provided in Figure IV.2. The genetic elements present in PV-ZMIR245 are listed in Table IV.2.

The expression cassette for the coding sequence of the Cry1A.105 protein consists of the promoter (P-e35S) and leader sequence for the cauliflower mosaic virus (CaMV) 35S RNA (Odell et al., 1985) containing a duplicated enhancer region (Kay et al., 1987). It contains the 5' untranslated leader of the wheat chlorophyll a/b/ binding protein (L-Cab) (Lamppa et al., 1985), the intron from the rice actin gene (I-Ract1) (McElroy et al., 1991), the cry1A.105 coding sequence that was optimized for expression in monocots, regime.and and the 3’ nontranslated region of the coding sequence for wheatand he at shock protein 17.3 AG (T-Hsp17), which terminates transcription and provides the signal for mRNA polyadenylation (McElwain and Spiker, 1989). Bayer property protectionpublishing The cry2Ab2 gene expression cassette thatof produces the Cry2Ab2 proteincontents consists of the its 35S promoter from figwort mosaic virus (P-FMV; Rogers,parties. data2000) and the firsttherefore intron from the corn heat shock protein 70 gene (I-Hsp 70; Brown and Santino,and/oror 1995). It contains a affiliates. cry2Ab2 coding sequence withproperty a modified intellectual codonthird usage (CS-cry2Ab2may; Widner and its Whitely, 1989, Donovan, 1991) fusedas to a chloroplast transit peptide region of corn the of and ribulose 1,5-biphosphateis carboxy lase small subunitregulatory including the firstowner. intron (TS-SSU- a document CTP; Matsuoka et al., 1987).any Thesuch 3’ nontranslated region of the itsnopaline synthase (T- reproductiondocument nos) coding region from Agrobacteriumowner tumefaciens thisT-DNA terminatesof transcription and directs polyadenylation (Bevan et al., 1983).under of rightsthe this documentand/or rights to of fall use of The molecular analyses described in Part IV demonstratethe that MON 89034 contains a single copyThis of introduced T-DNAmay distribution,I (~9.3and kb) inserted at a single locus. This insert rights owner contains one intactsubject copy each of the cry1A.105 and cry2Ab2 gene expression cassettes. There are no detectable plasmid backbonethe sequences, violate except for sequences common with be copy the T-DNA I, and no additional elements,of linked or unlinked to intact cassettes, from and transformationmay vectordocument PV-ZMIR245.publication, It exploitation this These results supportany the conclusion that the insert in MON 89034 only encodes the two permission expected full-length proteins, Cry1A.105prohibited and Cry2Ab2.

the be commercial SECTIONFurthermore, any5. The Intended Technical Effect of MON 89034 Consequently,without MON 89034 produces two structurally different Bt proteins, Cry1A.105 and Cry2Ab2, which are both highly efficacious against a variety of lepidopteran insect pests. MON 89034 provides a level of protection from feeding damage caused by lepidopteran larvae that is superior to that offered by currently available conventional chemical insecticides or insect-protected, biotechnology-derived corn products. Further details regarding the benefits of MON 89034 are discussed in Section 6 below.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 26 of 323

Cry1A.105 is a chimeric protein comprised of domains I, II from Cry1Ab and Cry1Ac, domain III from Cry1F (Bt subsp. aizawai), and the C-terminal portion from Cry1Ac (Bt subsp. kurstaki). Domains I and II of Cry1A.105 are 100% identical in amino acid sequence to domains I and II of both Cry1Ab and Cry1Ac, domain III is 99% identifical to domain III of Cry1F, and the C-terminal portion is 100% identical to the C-terminal portion of Cry1Ac. Cry1A.105 has an overall amino acid sequence identity of 93.6%, 90.0%, and 76.7 % to the Cry1Ac, Cry1Ab, and Cry1F proteins, respectively. Cry1Ac, Cry1Ab, and Cry1F are well known Cry insecticidal proteins that have been used in a number of microbial Bt insect control products and biotechnology-derived crops. As with other Cry1A proteins, Cry1A.105 is active against major lepidopteran insect pests. and The spectrum of activity includes corn borers from the generaand Ostrinia regime. and Diatraea (such as European corn borer, Asian corn borer,AG sugarcane borer, southwestern corn borer), armyworms (Spodoptera spp. including fall armyworm), earworms (Helicoverpa spp. including corn earworm), and cutworms (e.g., Agrotis ipsilon, black cutworm). Bayer property protectionpublishing of contents The Cry2Ab2 protein produced in MON 89034 is derived from the Bt subspeciesits kurstaki parties.data therefore and its amino acid sequence differs from that of the wild-typeand/or proteinor by a single amino acid. The Cry2Ab2 protein has 88% affiliates.amino acid sequence identity to the Cry2Aa protein property intellectualthird may which is present in commercial itsmicrobial pest control products such as Dipel and as Crymax. The Cry2Ab2 proteinsthe of produced andin MON 89034 and Bollgard II cotton have an is regulatory owner. identical amino acid sequence. Bollgard II cottona has beendocument on the marketits since 2003 and there have been no concernsany regarding such its safety reproductionto date. The Cry2Ab2 protein is active this documentof against lepidopteran insects and protectsowner plants from damage due to larval feeding of under of Ostrinia, Spodoptera, and Dipterarightsthe species. this documentand/or rights to of fall use of Further details regarding the identity and function theof the Cry1A.105 and Cry2Ab2 This may distribution,and proteins are provided inrights Part VI. owner subject be the violate copy of and SECTIONmay 6. The documentApplicatiopublication, ns and Uses of MON 89034 It exploitation Corn (Zea maysthis L.) any is the largest crop grown in the U.S. in terms of acreage planted and net value. In 2005, 81.8 permissionmillion acres of corn were planted in the U.S. The U.S. production yielded 11 billion bushelsprohibited with an average national yield of 148 bushels per the acre (NCGA, 2006).commercial The majority be of corn acres (52% or 42.5 million acres) were planted using seed developed via biotechnology methods (USDA-NASS, 2006). In 2006, biotechnology-derivedFurthermore,any corn plantings increased to approximately 48 million acres (or Consequently,without 61% of total corn acres), with approximately 32 million acres planted with insect- protected Bt corn products (USDA-NASS, 2006). This rapidly expanding trend in the use of Bt corn also is seen globally, with 17.8 million hectares of insect-protected corn planted in 2005, up from 16.9 million hectares in 2004 (James, 2005).

In the U.S., insect pests cause significant damage to the corn crop resulting in poor grain quality and reduced yields (James, 2003). In 1997, Monsanto commercialized the

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 27 of 323 biotechnology-derived product, YieldGard® Corn Borer corn (hereafter referred to as MON 810) that contains the cry1Ab gene from Bacillus thuringiensis (Bt), which encodes the Cry1Ab protein that provides effective protection against damage caused by lepidopteran insect pests, especially the European corn borer (ECB, Ostrinia nubilalis) and the corn earworm (CEW, Helicoverpa zea). At that time in the U.S., the combination of crop losses and management costs resulting from lepidopteran pests were estimated to be over $1 billion per year (Mason et al., 1996). The introduction of MON 810 and other Bt corn products provided corn growers with a more effective solution for the control of lepidopteran larval pests such as ECB and CEW. In addition to the benefits of MON 810 as an effective pest management tool, the use of this product has also led to lower levels of harmful mycotoxins in Bt corn leading to improved food and feed safety. This results from the control of insects that can cause damage to the ear leading to fungalregime. infections.and The reduction in mycotoxin levels has been consistently demonstratedand in countries AG around the world where corn borers are the predominant insect pests (Clements et al., 2003; Dowd, 2000 and 2001; Hammond et al., 2002 and 2004; de la Campa et al., 2005; Bakan et al., 2002; Magg et al. 2002; MunkvoldBayer et al.,property 1999; Munkvold,protectionpublishing 2003; Papst et al., 2005; Pietri and Piva 2000; Wu, 2006).of Furthermore, the use of MONcontents 810 and other its Bt corn products has reduced the use of chemical insecticidesparties.data (Carpenter thereforeet al., 2004). and/or Within ten years of the first Bt corn product introduction, the safe andor effective use of Bt affiliates. corn has been adopted globallyproperty on over intellectual 17 millionthird hectares (James,may 2005) to control its several primary insect pests of corn inas 12 countries. In 2005, insect protected corn was the of and used on over 40% of the isU.S corn acres (USDA-NAAS,regulatory 2006; Monsanto,owner. 2006). a document any such its reproductiondocument Recently, Monsanto has developedowner MON 89034 thisas a second-generationof product to under of provide enhanced benefits rightsfor thethe control of lepidopteranthis pests of corn. MON 89034 will documentand/or rights effectively address a cornto grower’sof fall need to usecontrol of a wider spectrum of lepidopteran pests, strengthen insect resistance management, furtherthe reduce the potential for mycotoxinsThis in grain, enable moremay efficientdistribution,and plant breeding of this multi-genic trait into rights owner superior hybridssubject compared to MON 810, and potentially reduce the refuge acreage required for resistance management purposes.the violate By producing effective levels of two be copy insecticidal proteins, Cry1A.105 and Cry2Aof b2, MON 89034 increases the durability of and the productmay againstdocument the publication,primary lepidopteran pests of corn. In addition, the individual It exploitation proteins extendthis the spectrum of control against lepidopteran insects commonly present in corn fields. Specifically,any the Cry1A.105 protein provides increased activity against fall permission armyworm (FAW, Spodoptera spprohibited.) and black cutworm (BCW, Agrotis ipsilon) compared to Cry1Ab. The Cry2Ab2 protein provides improved control over Cry1Ab-containing the be products from damagecommercial caused by corn earworm. This wider spectrum of activity also willFurthermore, potentiallyany contribute to the further reduction of mycotoxins in grain that result from fungalConsequently, invasionwithout after in sect feeding damage.

Taken together, adoption of MON 89034 is likely to enhance the economic and other benefits to farmers and improve the quality of grain and the safety of derived food and feed products. In addition, MON 89034 was developed to allow the simultaneous and efficient introgression of two insect protection traits into improved corn germplasm,

® YieldGard is a registered trademark of Monsanto Technology LLC.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 28 of 323 which will reduce the time and costs for new improved variety introductions into the marketplace. MON 89034 was developed using a single transformation vector containing both the cry1A.105 and cry2Ab2 genes. This approach, known as vector stacking, increases the efficiency of breeding multiple traits into new corn hybrids, thereby providing growers an earlier access to improved germplasm containing these traits rather than through conventional inbred stacking.

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

• Extended Spectrum: A major benefit of MON 89034 is the protection of corn plants from feeding damage caused by lepidopteran insect pest larvae. MON 89034 provides regime.and outstanding control of Ostrinia species such as European cornand borer and Asian corn AG borer (ACB), and Diatraea species such as southwestern corn borer (SWCB) and sugarcane borer (SCB). Control of these insects provided by MON 89034 is comparable to MON 810. MON 89034Bayer also provipropertydes a high protection levelpublishing of control of fall armyworm throughout the season, whereasof MON 810 principally controlscontents damage its caused by FAW larvae during vegetative growth.parties. In addition,data MON 89034therefore provides significant protection from damage caused by corn earwormand/or. or Although earworm affiliates. control is not complete, it isproperty superior intellectualto thatthird provided by MON 810may and other Cry1- its containing corn products. Introductionas of MON 89034 will offer farmers a safe and the of and effective alternative isto the use of chemiregulatorycal insecticides for theowner. control of the a document lepidopteran pests. any such its reproductiondocument owner this of • Improved IRM: MON 89034 producesunder two structurallyof different Bt proteins, Cry2Ab2 rightsthe this and Cry1A.105,document and/orwhich are both highly efficacious againstrights a variety of lepidopteran to of fall use of pests, and act by different mechanisms. The mechanismthe of insecticidal activity or modeThis of action of Bt proteinsmay consists distribution,and of a number of steps e.g., proteolysis, binding, rights owner and pore formationsubject (English and Slatin, 1992; Gill et al., 1992; Schnepf et al., 1998; Zhuang and Gill, 2003). There arethe impor violatetant differences in each step of the be copy mechanism of activity that influenceof the interactions of these proteins with susceptible and organismsmay withoutdocument qualitativelypublication, influencing their host range. Several lines of evidence It exploitation establish thatthis Cry1A.105 and Cry2Ab2 have important differences in their mode of action, particularlyany in the way in which they bind to the lepidopteran midgut. These permission proteins have different structures,prohibited share only ~14% of amino acid sequence identity, and bind to distinct proteins in the midgut of target species at different rates with the be different affinities.commercial Therefore, the probability of cross-resistance between these Furthermore,proteins isany very low. Furthermore, in vitro and in planta studies of the Cry1A.105 and Cry2AbConsequently, proteinswithout demonstrate that both proteins are highly active against the primary lepidopteran pests of corn: ECB, SWCB, CEW, and FAW. In view of the dual effective dose and the distinct mode of action of the two proteins produced in MON 89034, the likelihood for the evolution of resistance is significantly reduced compared to products containing a single introduced protein. Therefore, MON 89034 is expected to be sustainable using a reduced structured refuge. Based on conservative mathematical models, a 5% structured refuge in the U.S. Corn Belt and a 20% structured refuge in cotton growing regions will ensure the durability of MON 89034.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 29 of 323

• Mycotoxin Reduction: Lepidopteran pests such as ECB, CEW and FAW cause substantial damage to stalks, ears, and leaves of developing corn plants resulting in reduced yields. MON 89034 has been shown to provide protection against a wider variety of lepidopteran pests than MON 810 and in addition to greater yield protection, this is expected to result in a reduced potential for mycotoxin contamination in diverse environments. Corn ears that are protected from feeding damage caused by ECB, CEW, and FAW would have fewer ports of entry for invasion by fungi that produce mycotoxins. As regulations are implemented globally that limit mycotoxin levels in food and feed, the economic impacts of mycotoxin contamination in grain will become increasingly important. In the U.S., the FDA has set guidelines for acceptable levels of the mycotoxin, fumonisin, in corn used for food and feed. In the recentregime. studyand by Wu (2006), the economic benefit of mycotoxin reduction inand Bt corn grain to meet this AG FDA standard resulted in an annual benefit in the U.S. of $23 million. With the expanded spectrum of MON 89034 compared to MON 810, it is expected that there will be a greater benefit to food and feedBayer consumed property globally. protectionpublishing of contents its • Efficient Trait Integration: The rapid developmentparties. of elitedata corn hybrids containingtherefore the cry1A.105 and cry2Ab2 genes is made possible by vector and/orstackor technology, i.e., the affiliates. incorporation of multiple genesproperty into intellectuala singlethird transformation event.may This approach its increases the efficiency of introductionas of both genes into new corn germplasm by the of and conventional breeding,is thereby providing growregulatoryers early access toowner. a variety of elite a document corn germplasm containingany suchboth insecticidal proteins. Cornits hybrids containing reproductiondocument multiple Cry proteins conferringowner insect protectionthis traits ofhave been developed under of previously using traditionalrightsthe breeding techniques,this i.e., two inbreds containing individual documenttraits wereand/or crosse d to produce the combined rightstrait product. However, such to of fall use of breeding programs are generally more costly becausethe of duplicated work to introgress two transformationThis events mayinto newdistribution, germplasms.and MON 89034 reduces the time and rights owner cost factors insubject typical breeding programs, which rely on the sequential introgression of events containing single traits. the violate be copy of and In summary,may the introductiondocumentpublication, of MON 89034 will provide superior corn hybrids with It exploitation higher yields, enhancedthis breeding efficiencies, better quality grain, and reduced potential for the developmentany of insect resistance to Cry proteins. permission prohibited the commercial be SECTION 7. Applications for which MON 89034 is not Suitable Furthermore,any MonsantoConsequently, Companywithout is not aware of food or feed uses of conventional corn that are not applicable to MON 89034.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 30 of 323 References

Bakan, B., D. Melcion, D. Richard-Molard, and B. Cahagnier. 2002. Fungal growth and Fusarium mycotoxin content in isogenic traditional maize and genetically modified maize grown in France and Spain. J. Agric. Food. Chem. 50:728-731.

Bevan, M., W.M. Barnes, and M. Chilton. 1983. Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res. 11:369-385.

Brown, S.M. and C.G. Santino. 1995. Enhanced expression in plants. United States Patent No. 5,424,412. and Carpenter, J.E., S. Sankula, C.E. silvers,and L.P. Gianessi. 2004.and Insecticidalregime. Bacillus thuringiensis plants versus chemical insecticAGides. ACS Symposium Series 866. Agricultural Biotechnology 3:37-51. Bayer property protectionpublishing Clements, M.J., K.W. Campbell, C.M. Maragos,of C. Pilcher, J.M. Headrick,contents J.K. Patacky, its and D.G. White. 2003. Influence of Cry1Abparties. proteindata and hybrid thereforegenotype on fumonisin contamination and fusarium ear rot of corn. Cropand/or Sci.or 43:1283-1293. affiliates. property intellectualthird may its De la Campa, R., D.C. Hooker, J.D. Milleras , A.W. Schaafsma, and B.G. Hammond. 2005. the of and Modeling effects ofis environment, insect damage,regulatory and Bt genotypesowner. on fumonisin a document accumulation in maize anyin Argentina such and The Philippines. Mycopathologiaits 159:539- reproductiondocument 552. owner this of of rights under this Donovan, W.P. 1991.and/or CryIIB thecrystal protein gene from Bacillus thuringiensis. United document to of fall of rights States Patent No. 5,073,632. use the This may distribution,and rights owner Dowd, P.F. 2000.subject Indirect reduction of ear molds and associated mycotoxins in Bacillus thuringiensisbe corn under controlled andthe open violate field conditions: utility and limitations. J. Econ. Entomol.copy 93:1669-1679. of document and may publication,exploitation Dowd,It P.F. 2001. Biotic and abiotic factors limiting efficacy of Bt corn in indirectly this reducing mycotoxinany levels in commercial fields. J. Econ. Entomol. 94:1067-1074. permission prohibited English, L., and S.L. Slatin. 1992. The mode of action of ∆-endotoxins from Bacillus the thuringiensiscommercial—a comparison be with other bacterial toxins. Insect Biochem. Mol. Biol. 22:1-7. Furthermore,any Consequently,without Gill, S.S., E.A. Cowles, and P.V. Pietrantonio. 1992. The mode of action of Bacillus thuringiensis endotoxins. Annu. Rev. Entomol. 37:615-636.

Hammond, B., K. Campbell, T. Degooyer, A. Robinson, J. Richard, J. Segueira, C. Rubinstein, J. Cea, M. Plancke, L. Pinson, C. Radu, H. Esin, F. Tatli, and R. Grogna. 2002. Reduction of fumonisin levels in grain from YieldGard Cornborer. Proceedings of the 2nd Fungal Genomics, 3rd Fumonisin Elimination, and 15th Aflatoxin Elimination Workshops, October 23-25, 2002. San Antonio, Texas.

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Hammond, B.G., K.W. Campbell, C.D. Pilcher, T.A. Degooyer, A.E. Robinson, B.L. McMillen, S.M. Spangler, S.G. Riordan, L.G. Rice, and J.L. Ricahrd. 2004. Lower fumonisin mycotoxin levels in the grain of Bt corn grown in the United States in 2000-2002. J. Agric. Food Chem. 53:1390-1397.

Ishida, Y., H. Saito, S. Ohta, Y. Hiei, T. Komari, and T. Kumashiro. 1996. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotech. 14:745-750.

James, C. 2003. Global review of commercialized transgenic crops: 2002. Feature: Bt maize. ISAA Briefs No. 24. ISSAA, Ithaca, New York. regime.and and AG James, C. 2005. Global review of commercialized biotech/GM crops: ISAA Briefs No. 34. ISSAA, Ithaca, New York. Bayer property protectionpublishing Kay, R., A. Chan, M. Daly, and J. McPhof erson. 1987. Duplication contentsof CaMV 35S its promoter sequences created a strong enhancer forparties. plantdata genes. Sciencetherefore 236:1299- 1302. and/oror affiliates. property intellectualthird may its Lamppa, G., G. Morelli, and N. Chua.as 1985. Structure and developmental regulation of the of and a wheat gene encodingis the major chlorophylregulatoryl a/b-binding polypeptide.owner. Mol. Cell. a document Biol. 5:1370-1378. any such its reproductiondocument owner this of Magg, T., A.E. Melchinger, D. Klein,under and M. ofBohn. 2002. Relationship between rightsthe this European documentcorn and/orborer resistance and concentration rightsof mycotoxins produced by to of fall use of Fusarium spp. In grains of transgenic Bt maize hybrids,the their isogenic counterparts and commercialThis varieties. mayPlant Breed.distribution,and 121:146-154. rights owner subject Mason, C.E., M.E. Rice, D.d. Calvin, J.W.the Van violate Duyn, W.B. Showers, W.D. Hutchinson, be copy J.F. Witkowski, R.A. Higgins, D.W.of Onsta d, and G.P. Dively. 1996. European corn and borer:may Ecology documentand Management.publication, North Central Regional Extension Publication 327. It exploitation Iowa State University,this Ames, Iowa. any permission Matsuoka, M., Y. Kano-Murakami,prohibited Y. Tanaka , Y. Ozeki, and N. Yamamoto. 1987. Nucleotide sequence of the cDNA encoding the small subunit of ribulose-1,5- the be bisphosphatecommercial carboxylase from maize. J. Biochem. 102(4):673-676. Furthermore,any McElroy,Consequently, D., A.D.without Blowers, B. Jenes, and R. Wu. 1991. Construction of expression vectors based on the rice actin 1 (Act1) 5’region for use in monocot transformation. Mol. Gen. Genet. 231:150-160.

McElwain, E. and S. Spiker. 1989. A wheat cDNA clone which is homologous to the 17 kd heat-shock protein gene family of soybean. Nucl. Acids Res. 17:1764.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 32 of 323 Monsanto. 2006. http://www.monsanto.com/monsanto/content/investor/financial/reports (01 Aug 2006).

Munkvold, G.P., R.L. Hellmich, and L.G. Rice. 1999. Comparison of fumonisin concentration in kernels of transgenic Bt maize hybrids and nontransgenic hybrids. Plant Disease 83:130-138.

Munkvold, G.P. 2003. cultural and genetic approaches to managing mycotoxins in maize. Annu. Rev. Phytopathol. 41:99-116.

NCGA. 2006. The World of Corn. National Corn Growers Association. http://www.ncga.com/03world/main/index.html (01 Aug 2006). regime.and and AG Odell, J.T., F. Nagy, and N. Chua. 1985. Identificatin of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810-812. Bayer property protectionpublishing Rogers, S.G. 2000. Promoter for transgenof ic plants. United States Patent No.contents 6,018,100. its parties.data therefore Papst, C., H.F. Utz, A.E. Melchinger, J. Elder, T. Magg, D. Kleiand/orn, orand M. Bohn. 2005. affiliates. Mycotoxins produced by propertyFusarium intellectualspp. Inthird isogenic Bt vs. non-mayBt maize hybrids its under European corn borer pressure.as Agronomy J. 97:219-224. the of and is regulatory owner. a document Pietri, A. and G. Piva. 2000.any Occurrencesuch and control of mycotoxinsits in maize grown in reproductionth document Italy. Pp 226-236. In Proceedingsowner of the this6 Internationalof Feed Production Conference, Food Safety, Currentunder Situati on,of and Perspectives in the Eujropean rightsthe this Community.document Piva,and/or G. and F. Masoero (eds.). Novemberrights 27-28, 2000, Piacenza, Italy. to of fall use of the Schnepf,This E., N. Crickmore, J. mayVan Rie,distribution, D.and Le reclus, J. Baum, J. Feitelson, D.R. Zeigler, rights owner and D.H. Dean.subject 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:775-806.the violate be copy of and USDA-NASS.may 2006.document Acreagepublication, Report. United States Department of Agriculture, It exploitation Washington,this D.C. http://usda.mannlib.cornell.edu/usda/nass/Acre/2000s/2006/Acre- 06-30-2006.pdf any(01 Aug 2006). permission prohibited Widner, W.R. and H.R. Whitely. 1989. Two highly related insecticidal crystal proteins the be of Bacillus thuringiensiscommercial subsp. kurstaki possess different host range specificities. J. Furthermore,Bacteriol.any 171(2):965-974. Consequently,without Wu, F. 2006. Mycotoxin reduction in Bt corn: potential economic, health, and regulatory impacts. Transgenic Res. 15:27-289.

Zhuang, M. and S.S. Gill. 2003. Mode of action of Bacillus thuringiensis toxins. Pp 213-236. In Chemistry of Crop Protection, Progress and Prospects in Science and Regulation. Voss, G. and G. Ramos (eds.). Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 33 of 323 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 89034, including all progenies derived from crosses between MON 89034 and other corn, from the Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture (USDA) in 2006. Under regulations administered by USDA-APHIS (7 CFR 340), MON 89034 is currently considered a “regulated article.” Monsanto will continue and to conduct all field tests for MON 89034 in strict complianceand withregime. USDA field regulations until a Determination of NonregulatedAG Status is obtained for MON 89034.

Bayer property protectionpublishing SECTION 2. Status of Submission to ofU.S. EPA contents its parties.data therefore The U.S. Environmental Protection Agency (EPA) has authorityand/or overor the use of pesticidal affiliates. substances under the Federal propertyInsecticide, intellectual Fungicidethird and Rodenticidemay Act (FIFRA), as amended (7 U.S.C. § 136 et seq.).its An application for the registration of Bt Cry1A.105 as and Cry2Ab2 proteins and thethe geneticof materialand (vector PV-ZMIR245) necessary for their is regulatory owner. production in corn was submitted to the EPA ina Septemberdocument 2006. any such its reproductiondocument owner this of Pursuant to §408(d) of the Federal Foodunder Dr ug andof Cosmetic Act [21 U.S.C. 346 a(d)] rightsthe this Monsanto petitioneddocumentand/or EPA for temporary exemptionsof from rightsthe requirement of a tolerance for Bt Cry1A.105 andto Cry2Ab2 of fallproteins inuse 2005. In July 2006, EPA established the temporaryThis exemptions from themay requirement distribution,and of a tolerance for: a) the Cry1A.105 protein rights owner and the genetic materialsubject necessary for its production in the food and feed commodities of field corn, sweet corn and popcorn (40 CFR §174.453), and b) for the Cry2Ab2 protein be the violate and the genetic materialcopy necessary for itsof production in the food and feed commodities of field corn, sweet corn and popcorn (40 CFRand §174.454). may documentpublication, It exploitation this any permission SECTION 3. Status of Submissiprohibitedons to Foreign Governments the be Regulatory submissionscommercial for import and/or production approvals will be made to countries thatFurthermore, import anysignificant U.S. corn grain or derived food and feed products and have regulatoryConsequently, approvalwithout processes in place. These will include submissions to a number of foreign government regulatory agencies including, but not limited to, Japan’s Ministry of Agriculture, Forestry and Fisheries (MAFF), the Ministry of Health, Labor and Welfare (MHLW), the Canadian Food Inspection Agency (CFIA) and Health Canada, and the European Commission of the European Union. As appropriate, notifications of import will be made to importing countries that do not have a formal approval process.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 34 of 323 PART IV: DEVELOPMENT OF MON 89034

SECTION 1. Corn as a Crop

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. In the U.S., corn is a highly productive crop, yielding an average of 148 bushels per acre in the U.S. during 2005 (NCGA, 2006). Its high yield makes it one of the most economical sources of metabolizable energy for feeds, and of starch and sugar for food and industrial products. In 2005, approximately 55% of the corn grain produced in the U.S. wasand used as animal feed (NCGA, 2006). Therefore, indirect consumption isand much greaterregime. than direct consumption for humans. AG

Corn, the host plant, has been a staple of the human diet for centuries. Corn grain and its Bayer property protectionpublishing processed fractions are consumed in a multitudeof of food and animal feed products.contents Corn its forage is extensively consumed as animal feed by ruminants.parties.data therefore and/oror affiliates. A thorough description of theproperty anti-nutrients intellectual prthirdesent in corn has beenmay presented in an its OECD consensus document (OECD, as2002). These anti-nutrients include phytic acid, 2,4-dihydroxy-7-methoxy-2theH-1,4-benzoxazin-3(4 of and H)-one (DIMBOA), raffinose, and is regulatory owner. a document trypsin and chymotrypsin inhibitors.any such Phytic acid is considered an importantits anti-nutrient reproductiondocument for animals, especially nonruminants,owner since it reducesthis the bioavailabof ility of phosphorus in corn tissues to levels below 15%. Feedunder formulators of addthis the enzyme phytase to swine and poultry diets to improveand/or therights utilizationthe of phosphorus. The OECD does not recommend document to fall of rights analysis of DIMBOA due to thofe variable levelsuse found across corn hybrids. Raffinose is a the low molecularThis weight carbohydratemay distribution, presentand in corn grain that is considered an anti- rights owner nutrient due tosubject the gas production and resulting flatulence caused by consumption. Trypsin and chymotrypsin inhibitors occur at low levels in corn and are not considered be the violate nutritionally significantcopy for human healthof (White and Pollak, 1995) and may documentpublication, Corn Itis not a common allergenicexploitation food and there have been few reports of allergenic reactions to thethis consumption any of corn products (OECD, 2002). In the U.S. there have been only two reported casespermission of documented allergy to the ingestion of corn (Pauls and Cross, 1998; Tanaka et al., 2001)prohibited over the past seven years. Jones et al. (1995) found that the many individualscommercial with apparent be grain allergy are really sensitized to pollens and that a food challenge in ~80% of these individuals did not provoke clinical symptoms. Further, Furthermore, those with bonaany fide grain allergy are typically allergic to wheat proteins (~75%) and that Consequently,without allergies to corn (corn) are rare (less than six in over one hundred patients referred to a pediatric allergy specialty center for grain allergy).

Two recent reports from Italy (Pasterollo, 2000; Pasini et al., 2002) indicate that at least a few food allergic individuals have reactions consistent with allergies resulting from the consumption of corn products, including polenta, with mild food challenge positive results in six individuals from Naples (Pasini et al., 2002). However, all of the patients in the Italian studies have multiple sensitivities, usually to grass pollen, other grains, spices

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 35 of 323 and/or from members of the prunidae. The multiple allergies make clinical histories less reliable in identifying the causative agent of a particular episode, and also make diagnosis by skin prick test and in vitro IgE binding more complicated due to potentially weak and irrelevant cross-reactivity. At this time there is not enough evidence to understand the prevalence of allergies to corn products in Italy or the Mediterranean region. There have been no other reported instances of food allergic reactions to corn products from other parts of the world.

This evaluation suggests that the toxic or allergenic risk to humans from the consumption of corn and derived products is likely to be very low.

regime.and 1.1. Scientific name and taxonomic classification of corn and AG Corn (Zea Mays L.) is a member of the tribe Maydae, which is included in the subfamily Panicoideae of the grass family Gramineae. The genera included in the tribe Maydae Bayer property protectionpublishing include Zea and Tripsacum in the Westernof Hemisphere, and Coix, Polytocacontents, Chionachne , Schlerachne, and Trilobachne in Asia. Although some researchers haveits implicated the parties.data therefore Asian genera in the origin of corn, the evidence for themand/or is notor as extensive and affiliates. convincing as for the genera locatedproperty in theintellectual Westernthird Hemisphere. Tablemay IV.1 summarizes the taxonomic classification of cornits and its close relatives. 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 FDA BNF No. 00105 / Monsanto 06-CR-161F Page 36 of 323 Table IV.1. Taxonomic Classification of Corn and Its Close Relatives

Family - Gramineae Subfamily - Panicoideae Tribe - Maydae Western Hemisphere: I. Genus - Zea A. Subgenus - Luxuriantes 1. Zea luxurians (2n = 20) 2. Zea perennis (2n = 40) 3. Zea diploperennis (2n = 20) B. Subgenus - Zea 1. Zea mays (2n = 20) regime.and Subspecies and AG 1. Z. mays parviglumis (2n = 20) 2. Z. mays huehuetenangensis (2n = 20) 3. Z. mays mexicana (Schrad.) (2n = 20) Bayer property protectionpublishing of contents II. Genus – Tripsacum its parties.data therefore A. Section – Tripsacum B. Section and/or– Fasciculataor Species affiliates. Species property intellectualthird may 1. T. andersomiiits (2n = 64) 1. T. jalapense (2n = 72) as 2. T. australethe of (2n = 36) and 2. T. lanceolatum (2n = 72) Varietiesis regulatory 3. T. fasciculatumowner. (2n = 36) a document a) T.any australe such var. australe 4. T. maizarits (2n = 36, 72) reproductiondocument b) T. australeowner var. hirstum this5. T. pilosumof (2n = 72) 3. T. bravum (2n = 36,under 72) of Varieties rightsthe this 4.and/or T. cundinamarce (2n = 36) a). T. pilosum var. guatemalense document to of fall of rights 5. T. dactyloides (2n = 72) use b). T. pilosum var. pilosum the This Varietiesmay distribution,and a)rights T. dactyloides var. hispidumowner subject b) T. dactyloides var. dactyloides be the violate copy c) T. dactyloides ofvar. meridonale d) T. dactyloides var. mexicanum document and may 6. T.publication, floridanum (2n = 36) It exploitation this 7. T. intermedium (2n = 72 8.any T. manisuroides (2n = 72) permission 9. T. latifoliumprohibited (2n = 36) 10. T.the percuvianum (2n = 72, 90, 108) commercial11. T. zopilotense be (2n = 36, 72) Asia: Furthermore, any I. Genera— without Consequently, Chionachne (2n = 20) Schlerachne (2n = 20) Coix (2n = 10, 20) Trilobachne (2n = 20) Polytoca (2n = 20) Tribe—Andropogoneae I. Genus - Manisuris

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 37 of 323 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 developedand using simple mass selection by the indigenous native peopleand prior to regime.the arrival of Columbus. Their methods of selection were simpleAG compared to present-day standards, but they were obviously effective in developing races, varieties, and strains to satisfy their food, fuel, feed, and cultural needs. Hybridization occurred between varieties as Bayer property protectionpublishing cultures moved within the Western Hemisphereof , releasing genetic variabilitycontents to develop other unique varieties. its parties.data therefore and/oror affiliates. The fundamental concepts for propertydevelopment intellectual of hybridthird corn were definedmay by 1920. Basic studies on the genetic compositionits of a corn variety were conducted to determine the as effects of self pollinating (orthe inbreeding,of whicand h is the opposite of outcrossing) within a is regulatory owner. a document corn variety (Shull, 1909).any suchBecause corn naturally cross-fertilizes,its the genetic reproductiondocument composition of each plant is not known.owner Continuousthis self-pollination of of individuals for seven to ten generations resulted in pureunder lines (or ofinbred this lines) within which every plant had similar traits. and/orThe correctrights the interpreta tion of what occurred during inbreeding was document to fall of rights based on Mendelian genetics:of the heterozygoususe loci were eliminated by inbreeding to the homozygousThis loci of either onemay of the distribution, twoand alleles at each locus. The fixation of alleles in rights owner pure lines causedsubject a general reduction in vigor and productivity.

be the violate It was found uponcopy crossing two pureof lines that vigor was restored. If no selection occurred during inbreeding, the average perfandormance (e.g., grain yield) of all possible may documentpublication, crossesIt was similar to performanceexploitation of the original variety in which inbreeding was initiated. Somethis crosses, any however, were better than the original open-pollinated variety and could be reproduced frompermission the cross of the pure-line parents of the cross. Hence, the concept of hybrid corn was determined:prohibited self to develop pure lines, cross the pure lines to the produce hybrids,commercial evaluate hybrids be to determine the best hybrid, and use pure-line parents to reproduce the superior hybrid and distribute it for use by growers (Shull, 1909). Furthermore,any Consequently,without 1.3. History of corn development Corn likely originated in the highlands of Mexico 7,000 to 10,000 years ago where it was first domesticated as a crop. Recent genotyping studies suggest that corn originated from a single domestication in southern Mexico 9000 years ago (Matsuoka et al., 2002). The putative parents of corn have not been recovered, but it is likely that teosinte played an important role in the genetic background of corn. The transformation from a wild, weedy species to one dependent on humans for its survival probably evolved over a long period

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 38 of 323 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.

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 corn is a regime.and significant component of the human diet. and AG

The original corn-growing areas did not include the Northcentral region (U.S. Corn Belt) of the United States. The highly productiveBayer U.S. Cornproperty Belt dent protection cornpublishing was derived after the colonization of North America. Theof European settlers accepted thecontents local native its American varieties and incorporated them with otherparties. cropsdata to provide food, thereforefeed, and fuel for their survival. The current U.S. Corn Belt dent corn evolvedand/oror from the gradual affiliates. mingling of those settlements propertythat spread intellectual norththird and west from the Southeasternmay North its America, and those settlements that asspread south and west from Northeastern North the of and America. is regulatory owner. a document any such its reproductiondocument The corn types grown in the northeastowner are called northernthis flints;of their origin is not clear, but races from the highlands of Guatemalaunder have similarof ear morphology (Goodman and rightsthe this Brown, 1988).document Northernand/or flints are largely eight-rowed withrights cylindrical ears, are early to of fall use of maturing, and are short-statured plants with tillers. Thethe southern dent corn grown in the southeastThis U.S. seemed to havemay originated distribution,and from the southeast coast of Mexico. Southern rights owner dent corn is characterizedsubject as having tall, late maturing, non-tillered, poorly rooted plants with soft-textured white kernels on many-rothe wed,violate tapering ears. It seems the Tuxpeno be copy race contributed to the developmentof of southern dents. The intentional and/or and unintentionalmay crossingdocument betweenpublication, the early northern flints and late southern dents eventually It exploitation led to the highlythis productive U.S. Corn Belt dent corn that is used extensively throughout the world today. any permission prohibited The origin of corn has been studied extensively and four main hypotheses have been the be suggested (OECD,commercial 2003): Furthermore,any 1.Consequently, Descentwithout from teosinte - corn originated by direct selection from teosinte; 2. The tripartite hypothesis: 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); 3. The common origin hypothesis - corn, teosinte, and Tripsacum descended independently from a common, unknown ancestor; and, 4. The catastrophic sexual transmutation hypothesis – that modern corn originated from teosinte by an epigenetic sexual mutation causing ear development.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 39 of 323

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

Evidence has been reported to support the different hypotheses, but it seems that the preponderance of evidence supports the hypothesis that corn descended from teosinte (Galinat, 1988). The teosinte genome is similar to corn, teosinte easily crosses with corn, and teosinte has several plant morphological traits similar to corn. Teosinte has a more weedy appearance and more tillers than modern corn varieties. The one major regime.and distinguishing difference between corn and teosinte is the femaleand inflorescence, or ear. AG Modern corn varieties have one to three lateral branches that terminate in an ear with 8 to 24 kernel rows of 50 seeds, and the ear is enclosed in modified leaves or husks. Teosinte also has lateral branches, but they terminatBayere in two-rowedproperty spikesprotection ofpublishing perhaps 12 fruit cases, with each fruit case having one seedof enclosed by an indurated glumecontents (Goodman, its 1988). parties.data therefore and/oror affiliates. property intellectualthird may SECTION 2. Characterization ofits th e Vector Used in Transformation the as of and owner. Molecular analyses are anis integral part of the characterizationregulatory of crop products with new a documentits traits introduced by methodsany of suchbiotechnol ogy. Vectors and methods are selected for reproductiondocument of transformation to achieve high probabilityowner of obtainingthis the trait of interest and integration under of of the introduced DNA intorights a singlethe locus in the plantthis genome. This helps ensure that documentand/or rights only the intended DNA toencoding of thfalle desired usetrait(s) of is integrated into the plant genome and facilitates the molecular characterization of the product.the This may distribution,and rights owner This section describessubject the plasmid vector PV-ZMIR245 and the method of transformation that was usedbe to produce MON 89034. Thethe next violate section describes the molecular analyses copy of of the insert in MON 89034. and may documentpublication, It exploitation 2.1. Method of transformation this any permission MON 89034 was developed throughprohibited Agrobacterium -mediated transformation of corn using the binary plasmid vector, PV-ZMIR245 (Figure IV.2). Agrobacterium-mediated the be transformation commercialis a well-documented process for the transfer and integration of exogenousFurthermore, anyDNA into a plant’s nuclear genome (Ishida et al., 1996). PV-ZMIR245 containsConsequently, two separatewithout T-DNAs (herein referred to as 2 T-DNA system). The first T- DNA, designated as T-DNA I, contains the cry1A.105 and the cry2Ab2 expression cassettes. The second T-DNA, designated as T-DNA II, contains the nptII expression cassette that encodes the neomycin phosphotransferase enzyme that confers tolerance to certain antibiotics such as neomycin and paromomycin.

The use of the 2 T-DNAs system is the basis for an effective approach to generate marker-free plants. It allows for insertion of the T-DNA with the traits of interest (e.g., T-

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 40 of 323 DNA I) and the T-DNA encoding the selectable marker (e.g., nptII, T-DNA II) into two independent loci within the genome of the plant. Following selection of the transformants that contain both T-DNAs, the inserted T-DNA encoding the selectable marker (e.g., T-DNA II) can be segregated from progeny through subsequent conventional breeding and genetic selection, while the inserted T-DNA containing the trait(s) of interest is maintained (e.g., T-DNA I). This 2T-DNA binary vector approach has been successfully used in tobacco (Komari et al., 1996); soybean (Xing et al., 2000), barley (Matthews et al., 2001), corn (Miller et al., 2002); and rice (Komari et al., 1996; Breitler et al., 2004). MON 89034 was developed using such 2T-DNA vector transformation and selection techniques.

Freshly isolated immature corn embryos were used in the transformation (Ishida et al., regime.and 1996; Rout and Armstrong, 1997) that resulted in productiand on of MON 89034. AG Agrobacterium tumefaciens strain ABI, containing plasmid PV-ZMIR245 was induced to be virulent by the use of acetosyringone. Strain ABI also contains a helper plasmid that does not contain any T-DNA but allows forBayer the transfer property of T-DNAs protection I andpublishing II into the plant cells. Following inoculation with Agrobacteriumof , the immature corn contentsembryos were its transferred to a co-culture medium for one to threeparties. daysdata to ensure transformationtherefore of individual cells. This process of Agrobacterium-mediatedand/or transformationor of corn affiliates. involves the attachment of theproperty bacterium intellectualto thethird corn cells, which leadmays to transfer of the its region of DNA between the Left and asRight Borders of the binary plasmid (i.e., the T- the of and DNA) into the corn genomicis DNA. Each T-DNAregulatory was integrated into theowner. plant genome at a document separate loci. any such its reproductiondocument owner this of Following the incubation period on theunder co-cu ltureof medium, the immature embryos were rightsthe this transferred todocument selectiand/oron medium containing carbenicillin torights eliminate Agrobacterium, and to of fall use of paromomycin to eliminate cells that were not transformed,the so that only cells containing T-DNA ThisII and/or T-DNA I + mayT-DNA distribution, II andsurvived. The resulting transformed cells were rights owner then subculturedsubject several times on a selection medium and regenerated into the R0 plants containing both T-DNA’s I and II, accordingthe toviolate the protocol described by Armstrong and be copy Phillips (1988). of and may documentpublication, It exploitation During subsequentthis breeding at the F1 generation the unlinked insertions of T-DNA I or T- DNA II were segregated.any The plants containing only the insert that contains the permission cry1A.105 and cry2Ab2 gene cassettesprohibited were selected using molecular analysis, while the plants containing the nptII cassette (T-DNA II) were eliminated from subsequent the be breeding. The absencecommercial of the nptII gene and the NPTII protein was further confirmed by bothFurthermore, Southernany blot and ELISA analyses. Figure IV.2 displays the process map of the majorConsequently, steps involvedwithout in the transformation, selection, and development of MON 89034.

2.2. Plasmid PV-ZMIR245 The section describes the different genetic elements present in plasmid PV-ZMIR245.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 41 of 323 2.2.1. T-DNA I This section describes the elements contained on T-DNA I that were integrated into the corn genome to produce MON 89034.

2.1.1.1. The cry1A.105 gene and Cry1A.105 protein The cry1A.105 coding sequence encodes the 133 kDa Cry1A.105 insecticidal protein that provides protection against feeding damage by lepidopteran insect pests. Cry1A.105 is a chimeric protein comprised of domains I, II from Cry1Ab and Cry1Ac, domain III from Cry1F (Bt subsp. aizawai), and the C-terminal portion from Cry1Ac (Bt subsp. kurstaki). Domains I and II of Cry1A.105 are 100% identical in amino acid sequence to domains I and II of both Cry1Ab and Cry1Ac, domain III is 99% identifical to domain III ofand Cry1F, and the C-terminal portion is 100% identical to the C-terminaland portion of regime.Cry1Ac. The Cry1A.105 is a modified Cry1A protein with overallAG amino acid sequence identity to Cry1Ab, Cry1Ac and Cry1F proteins of 90.0%, 93.6% and 76.7%, respectively (Crickmore et al., 1998). The deduced amino acid sequence of the Cry1A.105 protein Bayer property protectionpublishing produced in MON 89034 is presented inof Figure IV.3. contents its parties.data therefore 2.1.1.2. The cry1A.105 regulatory sequences and/oror affiliates. The expression cassette for theproperty coding sequenintellectualcethird of the Cry1A.105 proteinmay consists of the its promoter (P-e35S) and leader for the caulifloas wer mosaic virus (CaMV) 35S RNA (Odell the of and et al., 1985) containing ais duplicated enhancer reregulatorygion (Kay et al., 1987).owner. It contains the 5' a document untranslated leader of the wheatany chsuchlorophyll a/b/ binding protein (L-itsCab ) (Lamppa et al., reproductiondocument 1985), the intron from the rice actinowner gene ( I-Ract1) (McElroythis et al.,of 1991), the cry1A.105 under of coding sequence that was optimizedrightsthe for expression in monocots,this and the 3’ nontranslated region of thedocument codingand/or sequence for wheat heat shock proteinrights 17.3 (T-Hsp17), which to of fall use of terminates transcription and provides the signal for mRNAthe polyadenylation (McElwain and Spiker,This 1989). may distribution,and rights owner subject 2.1.1.3. Thebe cry2Ab2 gene and Cry2Ab2the protein violate copy of The Cry2Ab2 protein present in MON 89034and is a member of the Cry2Ab class of may documentpublication, proteinsIt that share >95% aminoexploitation sequence homology (Crickmore et al., 1998). It has an amino acid sequence identity of >99% with the wild-type Cry2Ab2 protein derived from this any Bt subsp. kurstaki, differing by a single amino acid. The deduced amino acid sequence of permission the Cry2Ab2 protein produced inprohibited MON 89034 is presented in Figure IV.4. the commercial be 2.1.1.4. The cry2Ab2 regulatory sequences TheFurthermore, cry2Ab2any gene expression cassette that produces the Cry2Ab2 protein consists of the 35SConsequently, promoter fromwithout figwort mosaic virus (P-FMV; Rogers, 2000), the first intron from the corn heat shock protein 70 gene (I-Hsp 70; Brown and Santino, 1995). It contains a cry2Ab2 coding sequence with a modified codon usage (CS-cry2Ab2; Widner and Whitely, 1989, Donovan, 1991) fused to a chloroplast transit peptide region of corn ribulose 1,5-biphosphate carboxylase small subunit including the first intron (TS-SSU- CTP; Matsuoka et al., 1987). The 3’ nontranslated region of the nopaline synthase (T- nos) coding region from Agrobacterium tumefaciens T-DNA terminates transcription and directs polyadenylation (Bevan et al., 1983).

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 42 of 323

2.1.1.5. T-DNA borders Plasmid vector PV-ZMIR245 contains sequences that are necessary for transfer of T- DNA into the plant cell. These sequences are termed the Right and Left Border regions. The Right and Left Border regions each contains a border sequence that is a 24-26 bp sequence that defines the extent of the DNA that should be transferred into the plant genome. They flank both T-DNAs I and II, allowing for independent transfer and integration of each T-DNA into the plant genome during transformation. The Right Borders present in PV-ZMIR245 are made of a 24 bp nucleotide sequence that was originally derived from plasmid pTiT37, which was isolated from A. tumefaciens (Depicker et al., 1982). The Left Borders present in PV-ZMIR245 are made of a 25 bp nucleotide sequence from the A. tumefaciens plasmid pTi5955, a derivative of andplasmid and regime. pTiA6 (Barker et al., 1983). AG

2.2.2. T-DNA II Bayer property protectionpublishing This section describes the elements containedof on T-DNA II that were notcontents integrated into the corn genome to produce MON 89034. its parties.data therefore and/oror affiliates. 2.2.2.1. nptII gene and NPTIIproperty protein intellectualthird may its The nptII genes encodes the neomycinas phosphotransferase II enzyme (NPTII) that the of and inactivates certain aminoglycosideis antibioticsregulatory such as kanamycin,owner. neomycin and a document paromomycin. The use ofany selectable such marker genes, such as nptII,its is essential to select reproductiondocument transformed cells under selective growthowner conditions.this In the presenceof of paromomycin; under of cells transformed with nptIIrights survive,the while those that dothis not contain npt II are removed due to the actiondocument ofand/or the paromomyci n. The T-DNA II, andrights therefore the nptII gene, is to of fall use of segregated out at the F1 generation. the This may distribution,and rights owner 2.2.2.2. nptII regulatorysubject sequences The nptII genebe cassette that produces the NPTIIthe violate protein consists of the promoter (P-e35S) copy of from the the cauliflower mosaic virus (CandaMV) 35S RNA (Odell et al., 1985). The may documentpublication, sequenceIt coding for the NPTIIexploitation protein (Beck et al., 1982) is followed by the 3’ nontranslated region of the nopaline synthase (T-nos) coding region from Agrobacterium this any tumefaciens T-DNA that ends transcription and directs polyadenylation (Bevan et al., permission 1983). prohibited the commercial be 2.2.2.3. T-DNA borders TheFurthermore, right andany left T-DNA borders are described under Section 2.1.1.5. Consequently,without 2.2.3. Genetic elements outside the T-DNA borders The backbone region outside of the inserted DNA, contains two origins of replication necessary for replication and maintenance of the plasmid in bacteria. In addition, it contains a bacterial selectable marker gene, aad, which encodes an aminoglycoside- modifying enzyme that confers resistance to the action of the antibiotics spectinomycin

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 43 of 323 and streptomycin. Detailed descriptions of all elements in the plasmid backbone region are presented in Table IV.2.

and and regime. AG

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 44 of 323

aadA BstE II 458

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

CS-rop BstE II 2464 OR-ori V I-Ract1 Ssp I 15077

B-Left Border T-DNA II P-35S Nco I 2940 and and regime. AG CS-nptII PV-ZMIR245 17600Bayer bp BstE II 4613 property protectionCS-cry1A.105publishing T-nos of contents BstE II 4894its parties.data therefore and/oror B-Right Border affiliates. property intellectualthird may its T-Hsp17 as B-Left Borderthe of and P-FMV T-DNA I is regulatory owner. a document any such its T-nos reproductionI-Hsp70 document owner this of Nco I 10511 Ssp I 7921 under TS-SSU-CTPof this and/orrightsBstE II 10267the document CS-cry2Ab2 Nco I 8589 rights to of fall use of the This may distribution,and rights owner subject the be violate copy of and may documentpublication, It exploitation Figure IV.1. Plasmidthis any Map of Vector PV-ZMIR245

permission A circular map of the plasmid vectorprohibited PV-ZMIR245 used to develop corn MON 89034 is shown. PV-ZMIR245 containsthe two T-DNA regions designated as T-DNAs I and II. The commercial be genetic elements and restriction sites used in Southern blot analyses (with positions relativeFurthermore, to theany size of the plasmid vector) are shown on the exterior of the map. Consequently,without

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 45 of 323

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

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

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

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

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

Figure IV.2. Process Map for Transformation, Selection, Regeneration and Evaluation of MON 89034

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 46 of 323 Table IV.2. Summary of Genetic Elements in Vector PV-ZMIR245

Genetic Location in Element Plasmid Function (Reference) Vector Backbone Intervening Sequence 1-257 Sequences used in DNA cloning Bacterial promoter, coding sequence, and terminator sequence for an aminoglycoside-modifying enzyme, 3’(9)-O-nucleotidyltransferase from the transposon Tn7 (Fling et al. 1985) (GenBank accession X03043).and aad A and regime. aadA 258-1146 confers resistanceAG to streptomycin and spectinomycin Intervening Sequence 1147-1261 Sequences used in DNA cloning Bayer property protectionpublishing ofT-DNA I contents DNA region from Agrobacteriumits tumefaciens parties.data therefore B1-Right containing the right border sequenceand/oror used for transfer 1262-1618 of theaffiliates. T-DNA (Depicker et al. 1982) Border property intellectualthird may its Intervening as the of and Sequence 1619-1728is Sequences usedregulatory in DNA cloning owner. a document any suchThe promoter and 9 bp leader for theits cauliflower mosaic reproductiondocument virusowner (CaMV) 35S RNAthis (Odellof et al., 1985) containing 2 P -e35S 1729-2349 the duplicatedunder enhancerof region (Kay et al. 1987) rightsthe this documentand/or rights Intervening to of fall use of Sequence 2350-2375 Sequences used in DNAthe cloning This may distribution,and rights The 5' untranslatedowner leader of the wheat chlorophyll L3-Cab subject2376-2436 a/b-binding protein (Lamppa et al. 1985) be the violate Intervening copy of Sequence document2437-2452 Sequencesand used in DNA cloning may publication,exploitation I4-Ract1It 2453-2932 Intron from the rice actin gene (McElroy et al. 1991) this any Intervening permission Sequence 2933-2941 prohibited Sequences used in DNA cloning the commercial be Coding sequences for the Bacillus thuringiensis CS5-cry1A.1056 2942-6475 Cry1A.105 protein (Monsanto unpublished data) Furthermore,any Consequently,without

1 B – border region 2 P - promoter 3 L - leader 4 I - intron 5 CS – coding sequence 6 The cry1A.105 coding sequence includes one stop codon, which accounts for three base pairs.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 47 of 323 Table IV.2 (cont.). Summary of Genetic Elements in Vector PV-ZMIR245

Genetic Location in Element Plasmid Function (Reference) Intervening Sequence 6476-6506 Sequences used in DNA cloning The 3' nontranslated region of the coding sequence for wheat heat shock protein 17.3, which ends transcription T1-Hsp17 6507-6716 and directs polyadenylation (McElwain and Spiker, 1989) Intervening and Sequence 6717-6783 Sequences used in DNA cloningand regime. AG P-FMV 6784-7347 The Figwort Mosaic Virus 35S promoter (Rogers, 2000) Bayer Intervening property protectionpublishing of contents Sequence 7348-7369 Sequences used in DNA cloning its parties.data therefore The first intron from the maizeand/or heator shock protein 70 I-Hsp70 7370-8173 geneaffiliates. (Brown and Santino, 1995) property intellectualthird may its Intervening as the of and Sequence 8174-8189is Sequences usedregulatory in DNA cloning owner. a document any suchDNA region containing the targetingits sequence for the reproductiondocument transitowner peptide regionthis of maize ribuloseof 1,5-bisphosphate carboxylase smallof subunit and the first intron (Matsuoka rights under this TS2-SSU-CTP and/or8190-8590 theet al. 1987) document to of fall of rights Coding sequenceuse for a Cry2Ab2 protein from Bacillus the This maythuringiensis distribution,and (Widner and Whitely, 1989; Donovan, rights 1991). Thisowner coding sequence uses a modified codon 3 subject CS-cry2Ab2 8591-10498 usage the violate be copy Intervening 10499- of and Sequencemay document10524publication, Sequences used in DNA cloning It exploitation this 3' transcript termination sequence of the nopaline any synthase (nos) gene from Agrobacterium tumefaciens 10525-permissionwhich terminates transcription and directs prohibited T-nos 10777 polyadenylation (Bevan et al., 1983) the be Intervening commercial10778- Furthermore,Sequence any 10844 Sequence used in DNA cloning Consequently,without DNA region from Agrobacterium tumefaciens 10845- containing the 25 bp left border sequence used for B-Left Border 11286 transfer of the T-DNA (Barker et al. 1983)

1 T – transcript termination sequence 2 TS – targeting sequence 3 The cry2Ab2 coding sequence includes two stop codons, which accounts for six base pairs.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 48 of 323 Table IV.2 (cont.). Summary of Genetic Elements in Vector PV-ZMIR245

Location in Genetic Element Plasmid Function (Reference) Vector Backbone Intervening 11287- Sequence 12489 Sequences used in DNA cloning T-DNA II DNA region from Agrobacterium tumefaciens regime.and 12490- containing the 24 bp right andborder sequence used for B-Right Border 12846 transfer of the AGT-DNA (Depicker et al. 1982) Intervening 12847- Sequence 12971 SequencesBayer used in DNAproperty cloning publishing protectioncontents 3' transcriptof termination sequence of the nopaline its synthase (nos) geneparties. fromdata Agrobacterium therefore tumefaciens and/oror 12972- which terminates transcription and directs affiliates. T-nos 13224 propertypolyadenylation intellectualthird (Bevan et al., 1983) may its as Intervening 13225-the of and is regulatory owner. Sequence 13255 Sequences useda in DNAdocument cloning any such its Coding sequencereproduction for neomycindocument of phosphotransferase II 13256- proteinowner that confersthis resistance to neomycin and under of CS-nptII 14050rights the kanamycin (Beck et al.,this 1982) documentand/or of rights Intervening 14051-to of fall use Sequence 14083 Sequence used in DNAthe cloning This may distribution,and rights owner subject14084- Promoter and 31 bp leader for the cauliflower mosaic P-35S be 14407 virus (CaMV)the violate 35S RNA (Odell et al. 1985) copy of Intervening 14408- and Sequencemay document14457publication, Sequences used in DNA cloning It exploitation DNA region from Agrobacterium tumefaciens this any 14458- containing the left border sequence used for transfer of permission B-Left Border 14899 prohibitedthe T-DNA (Barker et al. 1983) the commercial be Vector Backbone Furthermore,Interveningany 14900- SequenceConsequently, without 14985 Sequences used in DNA cloning 14986- Origin of replication for Agrobacterium derived from the OR1-ori V2 15382 broad host range plasmid RK2 (Stalker et al. 1981)

1 OR – origin of replication.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 49 of 323 Table IV.2 (cont.). Summary of Genetic Elements in Plasmid Vector PV-ZMIR245

Genetic Location in Element Plasmid Function (Reference) Intervening 15383- Sequence 16119 Sequence used in DNA cloning Coding sequence for repressor of primer protein for 16120- maintenance of plasmid copy number in E. coli (Giza CS-rop 16311 and Huang 1989) Intervening 16312- regime.and Sequence 16738 Sequence used in DNA cloningand AG 16739- Origin of replication from pBR322 for maintenance of OR-ori-PBR322 17327 plasmid in E. coli (Sutcliffe 1978) Bayer property protectionpublishing Intervening 17328- of contents Sequence 17600 Sequence used in DNA cloning 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 FDA BNF No. 00105 / Monsanto 06-CR-161F Page 50 of 323

0001 MDNNPNINEC IPYNCLSNPE VEVLGGERIE TGYTPIDISL SLTQFLLSEF 0051 VPGAGFVLGL VDIIWGIFGP SQWDAFLVQI EQLINQRIEE FARNQAISRL 0101 EGLSNLYQIY AESFREWEAD PTNPALREEM RIQFNDMNSA LTTAIPLFAV 0151 QNYQVPLLSV YVQAANLHLS VLRDVSVFGQ RWGFDAATIN SRYNDLTRLI 0201 GNYTDHAVRW YNTGLERVWG PDSRDWIRYN QFRRELTLTV LDIVSLFPNY 0251 DSRTYPIRTV SQLTREIYTN PVLENFDGSF RGSAQGIEGS IRSPHLMDIL 0301 NSITIYTDAH RGEYYWSGHQ IMASPVGFSG PEFTFPLYGT MGNAAPQQRI 0351 VAQLGQGVYR TLSSTLYRRP FNIGINNQQL SVLDGTEFAY GTSSNLPSAV 0401 YRKSGTVDSL DEIPPQNNNV PPRQGFSHRL SHVSMFRSGF SNSSVSIIRA 0451 PMFSWIHRSA EFNNIIASDS ITQIPLVKAH TLQSGTTVVR GPGFTGGDIL 0501 RRTSGGPFAY TIVNINGQLP QRYRARIRYA STTNLRIYVT VAGERIFAGQ 0551 FNKTMDTGDP LTFQSFSYAT INTAFTFPMS QSSFTVGADT FSSGNEVYIDand 0601 RFELIPVTAT LEAEYNLERA QKAVNALFTS TNQLGLKTNVand TDYHIDQVSNregime. 0651 LVTYLSDEFC LDEKRELSEK VKHAKRLSDEAG RNLLQDSNFK DINRQPERGW 0701 GGSTGITIQG GDDVFKENYV TLSGTFDECY PTYLYQKIDE SKLKAFTRYQ 0751 LRGYIEDSQD LEIYSIRYNA KHETVNVPGT GSLWPLSAQS PIGKCGEPNR Bayer property protectionpublishing 0801 CAPHLEWNPD LDCSCRDGEK CAHHSHHFSLof DIDVGCTDLN EDLGVWVIFKcontents 0851 IKTQDGHARL GNLEFLEEKP LVGEALARVK RAEKKWRDKR EKLEWETNIVits 0901 YKEAKESVDA LFVNSQYDQL QADTNIAMIH AADKRVHSIRparties.data EAYLPELSVItherefore and/oror 0951 PGVNAAIFEE LEGRIFTAFS LYDARNVIKN GDFNNGLSCW NVKGHVDVEE affiliates. 1001 QNNQRSVLVV PEWEAEVSQEproperty VRVCPGRGYI intellectualthird LRVTAYKEGY GEGCVTIHEImay its 1051 ENNTDELKFS NCVEEEIYPN NTVTCNDYTVas NQEEYGGAYT SRNRGYNEAP the of and 1101 SVPADYASVY EEKSYTDGRRis ENPCEFNRGYregulatory RDYTPLPVGY VTKELEYFPEowner. 1151 TDKVWIEIGE TEGTFIVDSV ELLLMEEa document any such its reproductiondocument owner this of Figure IV.3. Deduced Amino Acid Sequenunder ce ofof the Cry1A.105 Protein Produced in rightsthe this documentMONand/or 89034 of rights to of fall use the This may distribution,and rights owner subject 001 MAPTVMMASSbe ATAVAPFQGL KSTASLPVARthe RSSRSLGNVSviolate NGGRIRCMQV WPAYGNKKFE 061 IRTLSYLPPL copySTGGRCMQA M DNSVLNSGRTof TICDAYNVAA HDPFSFQHKS LDTVQKEWTE 121 WKKNNHSLYL DPIVGTVASF LLKKVGSLVGand KRILSELRNL IFPSGSTNLM QDILRETEKF may documentpublication, 181 LNQRLNTDTLIt ARVNAELTGLexploitation QANVEEFNRQ VDNFLNPNRN AVPLSITSSV NTMQQLFLNR 241 LPQFQMQGYQthis LLLLPLFAQA ANLHLSFIRD VILNADEWGI SAATLRTYRD YLKNYTRDYS 301 NYCINTYQSA FKGLNTRLHDany MLEFRTYMFL NVFEYVSIWS LFKYQSLLVS SGANLYASGS 361 GPQQTQSFTS QDWPFLYSLFpermission QVNSNYVLNG FSGARLSNTF PNIVGLPGST TTHALLAARV 421 NYSGGISSGD IGASPFNQNF prohibitedNCSTFLPPLL TPFVRSWLDS GSDREGVATV TNWQTESFET the 481 TLGLRSGAFTcommercial ARGNSNYFPD be YFIRNISGVP LVVRNEDLRR PLHYNEIRNI ASPSGTPGGA 521 RAYMVSVHNR KNNIHAVHEN GSMIHLAPND YTGFTISPIH ATQVNNQTRT FISEKFGNQG 681Furthermore, DSLRFEQNNTany TARYTLRGNG NSYNLYLRVS SIGNSTIRVT INGRVYTATN VNTTTNNDGV 701 NDNGARFSDIConsequently,without NIGNVVASSN SDVPLDINVT LNSGTQFDLM NIMLVPTNIS PLY

Figure IV.4. Deduced Amino Acid Sequence of the Cry2Ab2 Protein Produced in MON 89034

The chloroplast transit peptide (CTP) region is underlined.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 51 of 323

SECTION 3. Characteriztion of the Introduced Genetic Material

Molecular analysis was performed to characterize the inserted DNA in MON 89034. This analysis was performed to confirm that only the DNA associated with the desired trait is present in the final product.

Specifically Southern blot analyses were performed to assess:

a) Number of inserts in the genome, b) Number of copies of insert, c) Intactness of the genetic elements within the insert, and and regime. d) Absence of backbone sequences, AG e) Absence of the T-DNA II encoding the selectable marker, f) Stability of the inserted DNA across multiple generations, and g) Organization of the insert Bayer property protectionpublishing of contents its parties.data therefore Genomic DNA from corn MON 89034 was digested usingand/or restrictionor enzymes and subjected to Southern blot analyses. affiliates. Maps of plasmid vector PV-ZMIR245 annotated property intellectualthird may with the probes used in the Southernits analysis are presented in Figures IV.6 and IV.7. Figure IV.8 provides a schematicthe of theas insert present in MON 89034. The Southern blot of and owner. analyses are presented inis Figures IV.9 – IV.20regulatory and the breeding generations used for a documentits analysis are depicted in Figureany IV.21.such For estimating the sizes of bands present in the reproductiondocument of long-run lanes of Southern blots, theowner molecula r size markersthis on the left of the figure were under of used. For estimating the sizesrights ofthe bands present in the short-runthis lanes, the molecular size documentand/or rights markers on the right of tothe figureof fallwere used.use Materials of and methods for the molecular analysis are provided in Appendix A. the This may distribution,and rights owner subject Molecular analyses confirmed that MON 89034 contains one insert derived from T-DNA be the violate I at a single locuscopy of the nuclear genome.of The insert contains one functional copy of the cry1A.105 and of the cry2Ab2 expression cassettes.and All genetic elements from T-DNA I may documentpublication, are intactIt and present in theexploitation expected order, except for the 5’ end of the insert as discussed below.this anyThese analyses also confirmed the absence of plasmid backbone sequence, nptII and T-DNApermission II derived elements, other those in common with the T-DNA I (i.e., 35S promoter, nos 3´ endprohibited sequence, and the Left Border sequence). The Southern the blot fingerprint ofcommercial MON 89034 be was maintained through seven generations of conventional breeding, thereby confirming the stability of the insert over multiple generations. These generationsFurthermore, anydid not contain any detectable T-DNA II elements other than those in common Consequently,without with T-DNA I, nor did they contain any detectable backbone sequence from plasmid PV- ZMIR245. PCR and DNA sequence analyses confirmed the organization of the elements within the insert, the 5' and 3' insert-to-plant junctions, and the complete DNA sequence of the insert.

DNA sequence analyses of the MON 89034 insert showed that the e35S promoter that regulates expression of the cry1A.105 gene has been modified and that the Right Border sequence present in PV-ZMIR245 is replaced by a Left Border sequence (Part IV,

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 52 of 323 Section 3.5). This molecular rearrangement can be explained by a recombination, either prior to or during the process of T-DNA transfer to the plant cell, between the shared DNA sequences of the 35S promoters in T-DNA’s I and II (see Figure IV.5). Due to fidelity of this recombination event, the reconstituted 35S promoter, e35S89, was found to be identical to the e35S promoter, except that there is a deletion in the 5’ portion from the duplicated enhancer elements (see Figure IV.5). The enhancer elements are not critical for expression of the Cry1A.105 protein in MON 89034 and the levels of this protein are sufficient to provide the expected efficacy against target lepidopteran pests.

and and regime. AG

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 53 of 323

and and regime. AG

Bayer property publishing protectioncontents of 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 Figure IV.5. Description of the Recombination Process that Explains the Modifiedthe 5’End of the Insert This may distribution,and rights owner A) Illustration of the T-DNA II of plasmid PV-ZMIR245.subject be the violate B) Illustration of the T-DNA I of plasmid PV-ZMIR245.copy of C) Illustration of the modified T-DNA I in MON 89034. and may documentpublication, Abbreviations and symbols: DERIt = duplicated enhancerexploitation region; L = leader sequence; I = intron sequence; P = promoter; T = termination sequence. Detailed descriptionthis of any all the genetic elements are described in Tables IV.2 and IV.3. The diagram illustrates a recombination event, which likelypermission occurred prior to or during the process of T-DNA transfer to the plant cells, between the DNA sequences near the 35S promoters in T-DNAs I and II. prohibited Due to this recombination event, the reconstituted e35S promoter in MON 89034 89 the (referred to as modified e35S or e35S ) no longercommercial has the duplicated be enhancer elements compared to the original e35S promoter in PV-ZMIR245. Despite the deletion of the enhance elements, the Cry1A.105 protein expression levels in MON 89034 are still sufficiently high under the regulation of the modified e35SFurthermore, promoterany to deliver the required efficacy against target insect pests. Consequently,without

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 54 of 323 aadA BstE II 458

OR-ori-PBR322 B-Right Border P-e35S Nde I 16501 L-Cab CS-rop BstE II 2464 OR-ori V I-Ract1 Ssp I 15077 B-Left Border 16 17 P-35S Nco I 2940 T-DNA II 15 1 2 13 CS-nptII 3 12 PV-ZMIR245 11 17600 bp BstE II 4613 CS-cry1A.105 T-nos 4 and 14 regime. andBstE II 4894 10 5 AG6 9 8 7 B-Right Border Bayer propertyT-Hsp17 protectionpublishing B-Left Border of P-FMV contentsT-DNA I its therefore T-nos parties.data I-Hsp70 and/oror Nco I 10511 affiliates. Ssp I 7921 property intellectualthirdTS-SSU-CTP may BstE II 10267its CS-cry2Ab2as Nco I 8589 the of and is regulatory owner. a document any such its reproductiondocument Probe DNA Probe ownerStart Position (bp)this End Positionof (bp) Total Length (~kb) 1 P-e35S/L-Cab under 1714of this 2447 0.7 and/orrights the 2 documentI-Ract1 fall 2427 of rights2941 0.5 3 CS-cry1A.105to probeof 1 use 2942 4923 2.0 4 CS-cry1A.105 probe 2 4726 the 6505 1.8 This may distribution,and 5 T-Hsp17rights owner6490 6797 0.3 6 subjectP-FMV 6755 7366 0.6 7 be I-Hsp70 the 7347violate 8179 0.8 8 TS-SSU-CTPcopy/CS- cry2Ab2 probe 1 of 8173 9516 1.3 9 TS-SSU-CTP/CS-cry2Ab2 probe 2 and 9296 10509 1.2 may documentpublication, 10 It T-nos exploitation 10525 10778 0.3 11 thisT-DNA II probe 1 12458 13391 0.9 12 T-DNA II anyprobe 2/CS-nptII probe 13256 14050 0.8 13 T-DNA II probepermission 3 13973 14916 0.9 14 Backbone 1 prohibited 11287 12489 1.2 the 15 commercialBackbone 2 be 14900 16511 1.6 16 Backbone 3 16289 136 1.4 Furthermore,17 any Backbone 4 48 1261 1.2 Consequently,without Figure IV.6. Genetic Elements and Restriction Sites of Vector PV-ZMIR245 Used in Southern Blot Analyses (Probes 1-17) A circular map of the plasmid vector PV-ZMIR245 used to develop corn MON 89034 is shown. Genetic elements and restriction sites used in Southern analyses (with positions relative to the size of the plasmid vector) are shown on the exterior of the map. The probes used in the Southern analyses are shown on the interior of the map. PV-ZMIR245 contains two T-DNA regions designated as T-DNA’s I and II.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 55 of 323

aadA BstE II 458 B-Right Border

P-e35S OR-ori-PBR322 Nde I 16501 L-Cab CS-rop BstE II 2464 OR-ori V I-Ract1 Ssp I 15077 B-Left Border T-DNA II Nco I 2940 P-35S regime.and 18 and AG CS-nptII PV-ZMIR245 19 17600 bp BstE II 4613 CS-cry1A.105 T-nos Bayer property 20 protectionpublishing of BstE II 4894 contents 23 21 its B-Right Border 22 parties.data therefore and/oror T-Hsp17 affiliates. B-Left Borderproperty intellectualthird P-FMV may its T-DNA I theT-nos as I-Hsp70 of and regulatory owner. is Nco I 10511 Ssp I 7921 a document BstEanyII 10267such its CS-cry2Ab2 reproductiondocument owner this of under of rightsthe TS-SSU-CTP this documentand/or of rights to of fall NcouseI 8589 the This may distribution,and rights owner subject the violate be copy of and may documentpublication, ProbeIt DNA Probe exploitation Start Position (bp) End Position (bp) Total Length (~kb) 18 thisT-DNA any I probe 1 1210 2753 1.5 19 T-DNA I probe 2 2649 4676 2.0 permission 20 T-DNA I probe 3 prohibited 4518 6505 2.0 21 T-DNA I probe 4 6371 8179 1.8 the 22 commercialT-DNA I probe be 5 8004 9863 1.9 23 T-DNA I probe 6 9780 11354 1.6 Furthermore,any FigureConsequently, IV.7. Geneticwithout Elements and Restriction Sites of Vector PV-ZMIR245 Used in Southern blot Analyses (Probes 18 - 23)

A circular map of the plasmid vector PV-ZMIR245 used to develop corn MON 89034 is shown. Genetic elements and restriction sites used in Southern analyses (with positions relative to the size of the plasmid vector) are shown on the exterior of the map. The overlapping T-DNA I probes used in the Southern analyses are shown on the interior of the map.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 56 of 323 3' Genomic Flank DNA 5' Genomic Flank DNA BstE II 5196 Ssp I 8223 Nco I 3242 BstE II 4915 Nco I 8891 BstE II 10569 Nde I 452 Nde I 1531 BstE II 2766 Nco I 10813 Nco I 11920 Ssp I 3 BstE II 12274

and 1 and regime. 12282 AG

cry2Ab2 cry2Ab2 cry1A.105cry1A.105 SSU-CTPSSU-CTP nosnos Hsp17 Hsp17 Cab Cab FMVFMV

e35Se35S Bayer

propertyHsp70Hsp70 Ract1 Ract1 protectionpublishing TS- TS- CS-CS- T-T- I-I- CS-CS- T-T- P-P- B-Left Border Border B-Left B-Left I-I- Border Border B-Left B-Left P-P- L-L- of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and ~8.2is kb regulatory owner.>4.3 kb Ssp I a document any such its >2.8 kb ~2.1 kb ~0.3 kb reproduction ~1.7 kb ~5.4 kbdocument of BstE II owner this under of rightsthe this documentand/or >11.4 kb rights Nde I to of fall use of

>3.2 kb ~5.6 kb the ~1.9 kb ~1.1 kb Nco I This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation Figure IV.8. Schematic Representationthis of theany Ins ert and Genomic Flanking Sequences in MON 89034 The linear DNA derived from T-DNA I of vector PV-ZMIR245,permission which was incorporated into MON 89034 is shown. Arrows indicate the end of the insert and the beginning of corn genomic prohibitedflanking sequence. Identified on the map are genetic elements within the the insert, as well as restriction sites with positionscommercial relative be to the size of the linear map for enzymes used in the Southern blot analyses. A portion of Left Border sequence and a modified e35S promoter sequence is present at the 5' insert-to-flank junction in MON 89034. Furthermore,any Consequently,without

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 57 of 323 Table IV.3. Summary of Genetic Elements in MON 89034

Location Genetic in Element Sequence Function (Reference) Sequence flanking the 5′ end of the insert 1-2060 Corn genomic DNA B1-Left 239 bp DNA region from the B-left Border region Borderr1 2061-2299 remaining after integration Intervening Sequence 2300-2349 Sequence used in DNA cloning regime.and Modified e35s promoter and 9and bp leader resulting from a AG P-e35S89 2350-2651 recombination between the P-e35s and P-35s promoters Intervening Bayer Sequence 2652-2677 Sequence used in DNAproperty cloning protectionpublishing of contents 5' untranslated leader of the wheat chlorophyllits a/b-binding parties.data therefore L2-Cab 2678-2738 protein (Lamppa et al., 1985) and/oror affiliates. Intervening property intellectualthird may its Sequence 2739-2754 Sequenceas used in DNA cloning the of and I3-Ract1 2755-3234is Intron from the regulatoryrice actin gene (McElroyowner. et al. 1991) a documentits Intervening any such reproduction this documentof Sequence 3235-3243 Sequenceowner used in DNA cloning of rights under this and/or theCoding sequence for the Bacillus thuringiensis Cry1A.105 document to of fall of rights CS4-cry1A.1055 3244-6777 protein (Monsantouse unpublished data) the InterveningThis may distribution,and rights owner Sequence subject6778-6808 Sequence used in DNA cloning 3' nontranslatedthe violate region of the coding sequence for wheat be copy heat shockof protein 17.3, which ends transcription and 6 and T -Hsp17may document6809-7018publication, directs polyadenylation (McElwain and Spiker, 1989) It exploitation Intervening this Sequence any7019-7085 Sequence used in DNA cloning permission prohibited the commercial be Furthermore,any Consequently,without

1 B – border region 2 L - leader 3 I - intron 4 CS – coding sequence 5 Coding sequence of the cry1A.105 including stop codon 6 T – transcript termination sequence

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 58 of 323 Table 3 (cont.). Summary of Genetic Elements in MON 89034

Genetic Location in Element Sequence Function (Reference)

P1-FMV 7086-7649 Figwort Mosaic Virus 35S promoter (Rogers, 2000) Intervening Sequence 7650-7671 Sequence used in DNA cloning The first intron from the maize heat shock protein 70 I-Hsp70 7672-8475 gene (Brown and Santino, 1995) Intervening and and regime. Sequence 8476-8491 Sequence used AGin DNA cloning DNA region containing the targeting sequence for the transit peptide region of maize ribulose 1,5- bisphosphateBayer carboxylase property small protection subunitpublishing and the first TS2-SSU-CTP 8492-8892 intron.of (Matsuoka et al., 1987) contents its Coding sequence forparties. a Cry2Ab2data protein fromtherefore Bacillus thuringiensis (Widner and Whitely,and/oror 1989; Donovan, affiliates. property1991) Thisintellectual codingthird sequence uses amay modified codon its CS-cry2Ab2 8893-10800 usageas the of and is regulatory owner. Intervening a document Sequence 10801-10826any such Sequence used in DNA cloning its reproductiondocument 3'owner termination sequencethis of the ofnopaline synthase (nos) under of rightsthe coding sequence fromthis Agrobacterium tumefaciens documentand/or rights to of whichfall terminatesuse of transcription and directs T-nos 10827-11079 polyadenylation (Bevanthe et al., 1983) This may distribution,and Intervening rights owner Sequence subject 11080-11146 Sequence used in DNA cloning be the violate B-Left copy 230 ofbp DNA region from the B-Left Border region Borderr2 11147-11377 remainingand after integration may documentpublication, SequenceIt exploitation flanking the 3this′ any end of the permission insert 11378-12282prohibited Corn genomic DNA the commercial be Furthermore,any Consequently,without

1P – promoter 2 TS – targeting sequence

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 59 of 323 3.1. Insert and copy number Southern blot analyses were performed to assess copy number and insert number of the DNA inserted in MON 89034. Results of these analyses are presented in Figure IV.9. The insert number (the number of integration sites of T-DNA I in the corn genome) was evaluated by digesting the test and control DNA with Nde I, a restriction enzyme that does not cleave within T-DNA I. This enzyme generates a restriction fragment containing T-DNA I and adjacent, plant genomic DNA. Thus, the number of restriction fragments detected indicates the number of inserts present in MON 89034. It is noted that the long run (overnight runs) enabled greater separation of the higher molecular weight restriction fragments while the short run (runs for a few hours) allowed the smaller molecular weight restriction fragments to be retained on the gel. and and regime. The number of copies of the T-DNA present was AGdetermined by digesting test and control genomic DNA samples with Ssp I, which cleaves once within the insert. If MON 89034 contains one copy of the insert, probing with overlapping T-DNA I [probes 18 – 23 in Figure IV.7] should result in two bands, eachBayer representing property a portion publishingof the insert along protectioncontents with adjacent, plant genomic DNA. of its parties.data therefore and/oror Genomic DNA isolated from conventional corn digested with Nde I (lanes 2 and 6) or affiliates. Ssp I (lanes 4 and 8) producedproperty several hybridizintellectualthirdation signals. This wasmay expected because its as several of the genetic elementsthe ofcomprising and T-DNA I were originally derived from corn (see Table IV.2). Theseis hybridization signalsregulatory result from the probesowner. hybridizing to a document endogenous targets residingany in thesuch conventional corn genome and areits not specific to the reproductiondocument inserted DNA. These signals were ownerproduced in all lanes,this includingof those containing the under of conventional corn controlrights DNAthe material, and thereforethis they are considered to be documentand/or rights endogenous background.to Plasmidof fall PV-ZMIR use245 DNAof mixed with conventional corn control DNA and digested with Ssp I (lanes 9 and 10)the produced the expected bands at This may distribution,and ~10.4 and ~7.2 kb in additionrights to the endogenousowner background hybridization produced by the conventionalsubject corn control DNA (lane 8). be the violate copy of MON 89034 DNA digested with Nde I (lanesand 1 and 5) produced a single unique band of ~13 kbmay in additiondocument topublication, the endogenous background hybridization observed in the It exploitation conventional corn control DNA (lanes 2 and 6). This result confirms that MON 89034 this any contains one insert located on ~13 kb Nde I restriction fragment. permission prohibited The MON 89034 DNAthe digested be with Ssp I (lanes 3 and 7) produced two bands in addition to the endogenouscommercial background hybridization observed in the conventional corn controlFurthermore, DNAany (lanes 4 and 8). The ~8.2 kb band is the expected size for the border fragmentConsequently, containingwithout the 5' end of the inserted DNA (corresponding to T-DNA I) along with the adjacent genomic DNA flanking the 5' end of the insert (Figure IV.8). The ~7.4 kb band, which was expected to be >4.3 kb, represents the 3' border fragment containing the 3' end of the inserted DNA along with the adjacent genomic DNA flanking the 3' end of the insert.

The results presented in Figure IV.9 indicate that MON 89034 contains one copy of T-DNA I that resides at a single locus of integration on ~13 kb Nde I restriction fragment.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 60 of 323 LONG RUN SHORT RUN

1 2 3 4 5 6 7 8 9 10

40 20 15 10 40 8.1 7.1 20 6.1 15 5.1 4.1 regime.and 10 and AG 3.1 8.1 2.0 7.1 Bayer property 1.6 publishing 6.1 protection of contents its 5.1 parties.data 1.0 therefore and/oror affiliates. 4.1 property intellectualthird may its 0.5 the as of and regulatory owner. is 3.1 a documentits any such reproductiondocument of owner this under of this and/orrights the document to of fall of rights 2.0 use the This may distribution,and rights owner subject

be the violate Figure IV.9. Southerncopy Blot Analysis of MON 89034: Insert and Copy Number The blot was examined with overlapping and32P-labeled probes that spanned the T-DNA I may documentpublication, sequenceIt (probes 18 - 23, Figureexploitation IV.7). Each lane contains ~10 µg of digested genomic this DNA isolated from anygrain. Lane designations are as follows: Lane 1: MON 89034 (Ndepermission I) 2: Conventional corn (Ndeprohibited I) the 3: MONcommercial 89034 (Ssp beI) 4: Conventional corn (Ssp I) Furthermore,any Consequently, 5: MONwithout 89034 (Nde I) 6: Conventional corn (Nde I) 7: MON 89034 (Ssp I) 8: Conventional corn (Ssp I) 9: Conventional corn spiked with PV-ZMIR245 (Ssp I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Ssp I) [1.0 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 61 of 323

3.2. Intactness of the cry1A.105 and cry2Ab2 expression cassettes The presence and intactness of all the elements of the inserted cry1A.105 and cry2Ab2 expression cassettes was assessed by digestion of MON 89034 genomic DNA with the restriction enzyme Ssp I, Nco I, or BstE II. This is necessary to ensure that only expected proteins are produced in MON 89034.

Digestion with Ssp I releases two border fragments with the expected size of ~8.2 and >4.3 kb (Figure IV.8). The ~8.2 kb fragment contains genomic DNA flanking the 5' end of the insert, Left Border sequence, modified e35S promoter sequence, Cab leader, Ract1 intron, cry1A.105 coding sequence, Hsp17 3' end sequence, FMV promoter, and a portion of the Hsp70 intron. The border fragment >4.3 kb contains the remaining portionand of the and regime. Hsp70 intron, SSU-CTP targeting sequence, cry2Ab2AG coding sequence, nos 3' end sequence, Left Border sequence, and genomic DNA flanking the 3' end of the inserted DNA (Figure IV.8). Bayer property publishing protectioncontents Digestion of MON 89034 genomic DNAof with Nco I releases two internal restriction its fragments and two border fragments (Figure IV.8). Theparties. 5' databorder fragment thereforeis expected to and/oror be >3.2 kb and contains genomic DNA flanking the 5' end of the insert, the Left Border1 affiliates. sequence, modified e35S promoterproperty sequence, intellectual thethird Cab leader, and the mayRact1 intron. The its ~5.6 kb internal fragment contains asthe cry1A.105 coding sequence, Hsp17 3' end the of and sequence, FMV promoter,is Hsp70 intron, and theregulatory SSU-CTP targeting sequence.owner. The ~1.9 a document kb internal fragment containsany the such cry2Ab2 coding sequence. The its3' border fragment is reproductiondocument expected to be ~1.1 kb and containsowner the nos 3' endthis sequence, of a second Left Border under of sequence, and genomic DNArights flankingthe the 3' end of the insertedthis DNA. documentand/or rights to of fall use of Digestion of MON 89034 genomic DNA with BstE IIthe generates two border fragments and threeThis internal fragments (Figuremay distribution,IV.8).and The 5' border fragment is expected to be >2.8 rights owner kb and containssubject genomic DNA flanking the 5' end of the insert, Left Border1 sequence, the violate modified e35Sbe promotercopy sequence, and the Cab leader sequence. The 3' border fragment is expected to be ~1.7 kb and contains aof portion of the cry2Ab2 coding sequence, the nos and 3' end, Leftmay Border documentsequence,publication, and genomic DNA flanking the 3' end of the inserted DNA. It exploitation this Plasmid PV-ZMIR245any DNA was combined with conventional corn control DNA and permission digested with Nco I or BstE II prohibited(BstE II was used for the T-nos and T-DNA II Southern blots) and loaded on thethe gelbe to serve as a positive hybridization control. Individual Southern blots werecommercial examined with the following probes: e35S promoter including the CabFurthermore, leader, anythe Ract1 intron, the cry1A.105 coding sequence, the Hsp17 3' end sequence, the Consequently,FMV promoter,without the Hsp70 intron, the SSU-CTP targeting sequence/cry2Ab2 coding sequence, and the nos 3' end sequence (probes 1-10, Figure IV.6).

1 See section 3.1.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 62 of 323 3.2.1. e35S promoter/Cab leader The results of this analysis are presented in Figure IV.10. Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) probed with the e35S promoter and Cab leader probe showed no hybridization bands, as expected for the negative control. Conventional corn control DNA spiked with plasmid PV-ZMIR245 DNA digested with Nco I (lanes 9 and 10) produced the expected size band at ~10 kb.

MON 89034 DNA digested with Ssp I (lanes 1 and 5) produced the expected single unique band of ~8.2 kb. MON 89034 DNA digested with Nco I (lanes 3 and 7) produced the single unique band of ~5.4 kb. This is consistent with the expected band being >3.2 kb. No unexpected bands were detected, indicating that MON 89034 contains no additional e35S promoter and Cab leader elements other than those associated with the regime.and cry1A.105 cassette. and AG 3.2.2. Ract1 intron Bayer The results of this analysis are presented in Figure propertyIV.11. Conventionalprotectionpublishing corn control of contents DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) probedits with the Ract1 parties.data therefore intron probe showed no hybridization bands, as expected for theand/or negativeor control. affiliates. property intellectualthird may Conventional corn control DNA spikedits with plasmid PV-ZMIR245 DNA digested with as Nco I (lanes 9 and 10) producedthe of the expectedand size band at ~10 kb. The migration of the is regulatory owner. ~10 kb fragments is slightly higher than indiacated by thedocument molecular marker band sizes. any such its The altered migrations may be due to the differencereproduction in saltdocument concentrations of between the corn DNA sample and the molecularowner marker (Sambrookthis and Russell, 2001). under of rightsthe this documentand/or rights MON 89034 DNA digestedto ofwith fallSsp I (lanesuse 1 andof 5) produced the expected single unique band of ~8.2 kb. MON 89034 DNA digested withthe Nco I (lanes 3 and 7) produced This may distribution,and the single unique ~ 5.4 rightskb band. This is consisteownernt with the expected band being >3.2 kb. No unexpected subjectbands were detected, indicating that MON 89034 contains no additional the Ract1 intron beelements other than those associatedviolate with the cry1A.105 cassette. copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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40 20 15 40 10 8.1 20 7.1 regime.and 15 and6.1 AG 5.1 4.1 10 3.1 8.1 Bayer property protectionpublishing 7.1 of contents 2.0 its 6.1 parties.data therefore and/or1.6 or 5.1 affiliates. property intellectualthird may its 1.0 4.1 the as of and regulatory owner. is a documentits any such reproduction 0.5 3.1 documentof owner this under of rightsthe this documentand/or rights to of fall use of

2.0 the This may distribution,and rights owner subject Figure IV.10.be Southern Blot Analysis ofthe MON violate 89034: e35S Promoter/Cab Leader The blot was examinedcopy with a 32P-labeledof probe that spanned the e35S promoter and Cab leader region (probedocument 1, Figure IV.6). Eachand lane contains ~10 µg of digested genomic may publication,exploitation DNA Itisolated from grain. Lane designations are as follows: this Lane 1: MON 89034any (Ssp I) 2: Conventional cornpermission (Ssp I) prohibited 3: MON 89034 (Nco I) the 4: Conventionalcommercial corn be ( Nco I) 5: MON 89034 (Ssp I) Furthermore,any Consequently, 6: Conventionalwithout corn (Ssp I) 7: MON 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 64 of 323

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1 2 3 4 5 6 7 8 9 10

40 20 15

40 10 8.1 7.1 20 6.1 15 5.1 4.1 and 10 and regime. AG 3.1 8.1 7.1 2.0 6.1 Bayer property 1.6protectionpublishing of contents 5.1 its parties.data 1.0 therefore and/oror 4.1 affiliates. property intellectualthird 0.5 may its the as 3.1 of and regulatory owner. is a documentits any such reproductiondocument of owner this under of 2.0 rightsthe this documentand/or rights to of fall use of 1.6 the This may distribution,and rights owner subject be the violate Figure IV.11. Southerncopy Blot Analysisof of MON 89034: Ract1 Intron The blot was examineddocument with a 32P-labeledand probe that spanned the Ract1 intron (probe 2, may publication,exploitation FigureIt IV.6). Each lane contains ~10 µg of digested genomic DNA isolated from grain. this Lane designations areany as follows: Lane 1: MON 89034 (Ssppermission I) prohibited 2: Conventional corn (Ssp I) the 3: MONcommercial 89034 (Nco be I) 4: Conventional corn (Nco I) Furthermore,any Consequently, 5: MONwithout 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 65 of 323 3.2.3. cry1A.105 coding sequence The results of this analysis are presented in Figure IV.12. Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) hybridized simultaneously with overlapping probes spanning the cry1A.105 coding sequence showed no hybridization bands as expected for the negative control. Conventional DNA spiked with plasmid PV-ZMIR245 DNA digested with Nco I (lanes 9 and 10) produced the expected size band at ~5.6 kb. The migration of the ~ 5.6 kb fragments is slightly higher than indicated by the molecular marker band sizes. The altered migrations may be due to the difference in salt concentrations between the corn DNA sample and the molecular marker (Sambrook and Russell, 2001).

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40 20 15 10 40 8.1 7.1 20 6.1 15 5.1 4.1 10 and and3.1 regime. 8.1 AG 7.1 2.0 6.1 1.6 Bayer property protectionpublishing of contents 5.1 1.0 its parties.data therefore and/oror 4.1 affiliates. 0.5 property intellectualthird may its as 3.1 the of and is regulatory owner. a documentits any such reproduction this documentof owner under of rightsthe this document2.0and/or rights to of fall use of the This 1.6 may distribution,and rights owner Figure IV.12. Southernsubject Blot Analysis of MON 89034: cry1A.105 Coding Sequence be the32 violate The blot was examinedcopy with overlappingof P-labeled probes that spanned the cry1A.105 coding sequence (probes 3 and 4, Figure IV.6).and Each lane contains ~10 µg of digested may documentpublication, genomicIt DNA isolated from exploitationgrain. Lane designations are as follows: Lane 1: MONthis 89034 any (Ssp I) 2: Conventional corn (Ssp I) permission 3: MON 89034 (Nco I) prohibited the 4: Conventionalcommercial corn be ( Nco I) 5: MON 89034 (Ssp I) Furthermore, 6: Conventionalany corn (Ssp I) Consequently, 7: MONwithout 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

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

6.1 Bayer property2.0 protectionpublishing of 1.6 contents

5.1 its therefore parties.data 1.0 and/oror 4.1 affiliates. property intellectualthird may its the as 0.5 of and owner. 3.1 is regulatory a documentits any such reproduction this documentof owner under of rightsthe this document2.0 and/or rights to of fall use of the This may distribution,and rights owner Figure IV.13. Southernsubject Blot Analysis of MON 89034: Hsp17 3' End Sequence be 32 the violate The blot was examinedcopy with a P-labeledof probe that spanned the Hsp17 3' end sequence (probe 5, Figure IV.6). Each lane containsand ~10 µg of digested genomic DNA isolated may documentpublication, from grain.It Lane designationsexploitation are as follows: Lane 1: MONthis 89034 any (Ssp I) 2: Conventional corn (Ssp I) permission 3: MON 89034 (Nco I) prohibited the 4: Conventionalcommercial corn be ( Nco I) 5: MON 89034 (Ssp I) Furthermore, 6: Conventionalany corn (Ssp I) Consequently, 7: MONwithout 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 68 of 323 3.2.5. FMV promoter The results of this analysis are presented in Figure IV.14. Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) examined with the FMV promoter probe showed no hybridization bands, as expected for the negative control.

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

MON 89034 DNA digested with Ssp I (lanes 1 and 5) produced the expected single regime.and unique band of ~8.2 kb. MON 89034 DNA digested with Nco andI (lanes 3 and 7) produced the expected single unique band of ~5.6 kb. AG No unexpected bands were detected, indicating that MON 89034 contains no additional FMV elements other than those associated with the cry2Ab2 cassette. Bayer property protectionpublishing of contents its 3.2.6. Hsp70 intron parties.data therefore and/oror The results of this analysis are presenteaffiliates.d in Figure IV.15. Conventional corn control property intellectualthird may DNA digested with Ssp I (lanes 2its and 6) or Nco I (lanes 4 and 8) examined with the as Hsp70 intron probe producedthe ofseveral hybridizand ation signals. This is not unexpected is regulatory owner. because the Hsp70 intron was originally deriveda from corn.document These hybridization signals any such its result from the probes hybridizing to endogenousreproduction sequencesdocument residing of in the corn genome and are not specific to the inserted ownerDNA. These signalsthis were produced in both test and under of control lanes, and thereforerights the bandsthe are considered to thisbe endogenous background. documentand/or rights to of fall use of Conventional corn control DNA spiked with plasmid thePV-ZMIR245 DNA digested with This may distribution,and Nco I (lanes 9 and 10)rights produced the expectedowner size band at ~5.6 kb in addition to the endogenous bands.subject The migration of the ~5.6 kb fragments is slightly higher than the indicated by bethe molecular marker band sizes. violateThe altered migrations may be due to the copy of difference in salt concentrations between theand corn DNA sample and the molecular marker may documentpublication, (SambrookIt and Russell, 2001).exploitation this any MON 89034 DNA digested with Ssp I (lanes 1 and 5) produced two expected bands of permission ~8.2 and ~7.4 kb in addition to prohibitedthe endogenous bands. The ~8.2 kb band is the expected size for the border fragmentthe containing the 5' end of the inserted DNA (T-DNA I) along commercial be with the adjacent genomic DNA flanking the 5' end of the insert (see Figure IV.8). The ~7.4Furthermore, kb bandany represents the 3' border fragment containing the 3' end of the inserted DNA alongConsequently, with thewithout adjacent genomic DNA flanking the 3' end of the insert which was expected to be >4.3 kb. MON 89034 DNA digested with Nco I (lanes 3 and 7) produced the expected band of ~5.6 kb in addition to the endogenous bands that resulted from non- specific hybridization as shown on lanes 4 and 8. No unexpected bands were detected, indicating that MON 89034 contains no additional Hsp70 intron elements other than those associated with the cry2Ab2 cassette.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 69 of 323 3.2.7. SSU-CTP targeting sequence/cry2Ab2 coding sequence The results of this analysis are presented in Figure IV.16. Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) and examined with overlapping probes spanning the SSU-CTP/cry2Ab2 coding sequence probe (probes 8 and 9, Figure IV.6) produced several hybridization signals. This is expected because the SSU-CTP targeting sequence was originally derived from corn. These hybridization signals result from the probes hybridizing to endogenous targets residing in the corn genome and are not specific to the inserted DNA. These signals were produced with both test and control material, therefore they are considered to be endogenous background. Endogenous bands were not detected in the long runs of the Nco I digests because they ran off the gel, as expected.

regime.and Conventional corn control DNA spiked with plasmid PV-ZMIR245and DNA digested with Nco I (lanes 9 and 10) produced the expected sizeAG bands at ~1.9 and ~5.6 kb in addition to the endogenous bands. Bayer property protectionpublishing MON 89034 DNA digested with Ssp Iof (lanes 1 and 5) produced one expectedcontents band of its ~7.4 kb in addition to the endogenous bands. The parties.~7.4 kbdata band is consistenttherefore with the expected band of >4.3 kb (see Figure IV.8). MON 89034 DNAand/or ordigested with Nco I affiliates. (lanes 3 and 7) produced twpropertyo bands inintellectual additionthird to the endogenousmay bands that are its consistent with the expected sizes of ~5.6as and ~1.9 kb. The position of the these bands the of and was slightly higher thanis indicated by the molecularregulatory marker band sizesowner. in the long run a document (lane 3) but was concurrentany with such the bands produced by PV-ZMIR245its in the short run reproductiondocument (lanes 7, 9, and 10). The alteredowner migrations maythis be due toof the difference in salt concentrations between the corn DNA undersample andof the molecular marker (Sambrook and rightsthe this Russell, 2001).document No unexpectedand/or bands were detected, indicatingrights th at MON 89034 contains to of fall use of no additional SSU-CTP/cry2Ab2 elements other than those associated with the cry2Ab2 the cassette.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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40 20 15 40 10 8.1 20 7.1 6.1 15 5.1 regime.and and4.1 10 AG 8.1 3.1 7.1 Bayer property protectionpublishing 6.1 of 2.0 contents 1.6 its 5.1 parties.data therefore and/oror affiliates. 1.0 4.1 property intellectualthird may its the as of and regulatory 0.5 owner. 3.1 is a documentits any such reproduction this documentof owner under of rightsthe this documentand/or2.0 rights to of fall use of Figure IV.14. Southern Blot Analysis of MON 89034:the FMV Promoter This may32 distribution,and The blot was examinedrights with a P-labeled probeowner that spanned the FMV promoter (probe subject 6, Figure IV.6). Each lane contains ~10 µg of digested genomic DNA isolated from be the violate grain. Lane designaticopyons are as follows:of Lane 1: MON 89034 (Ssp I) and may documentpublication, It 2: Conventional cornexploitation (Ssp I) 3: MONthis 89034 any (Nco I) 4: Conventional corn (Nco I) permission 5: MON 89034 (Ssp I) prohibited the 6: Conventionalcommercial corn be (Ssp I) 7: MON 89034 (Nco I) Furthermore, 8: Conventionalany corn (Nco I) Consequently, 9: Conventionalwithout corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

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40 20 15 40 10 20 8.1 15 7.1 6.1 5.1 regime.and and4.1 10 AG 8.1 3.1 7.1 Bayer property 2.0protectionpublishing 6.1 of contents 1.6 its parties.data therefore 5.1 and/oror affiliates. 1.0 may 4.1 property intellectualthird its as the of and 0.5 is regulatory owner. a documentits 3.1 any such reproduction this documentof owner under of rightsthe this documentand/or of rights 2.0 to of fall use the This may distribution,and Figure IV.15. Southernrights Blot Analysis of MONowner 89034: Hsp70 Intron The blot was examinedsubject with a 32P-labeled probe that spanned the Hsp70 intron (probe 7, be the violate Figure IV.6). Eachcopy lane contains ~10 µofg of digested genomic DNA isolated from grain. Lane designations are as follows: and may documentpublication, Lane It 1: MON 89034 (Ssp I)exploitation 2: Conventionalthis any corn (Ssp I) 3: MON 89034 (Nco I) permission 4: Conventional corn (Ncoprohibited I) the 5: MONcommercial 89034 (Ssp beI) 6: Conventional corn (Ssp I) Furthermore, 7: MONany 89034 (Nco I) Consequently, 8: Conventionalwithout corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

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40 20 15 10 40 8.1 7.1 20 6.1 15 5.1 4.1 10 and and3.1 regime. 8.1 AG 7.1 2.0 6.1 1.6 Bayer property publishing 5.1 protection of 1.0 contents its parties.data therefore 4.1 and/oror 0.5 affiliates. property intellectualthird may its 3.1 as the of and is regulatory owner. a documentits any such reproduction this documentof 2.0 owner under of this and/orrights the document 1.6 rights to of fall use of the This may distribution,and Figure IV.16. Southernrights Blot Analysis of MONowner 89034: SSU-CTP Targeting Sequence/subject cry2Ab2 Coding Sequence be the32 violate The blot was examinedcopy with overlappingof P-labeled probes that spanned the SSU-CTP targeting sequence and cry2Ab2 coding sequenceand (probes 8 and 9, Figure IV.6). Each may documentpublication, lane containsIt ~10 µg of digestedexploitation genomic DNA isolated from grain. Lane designations are as follows: this any Lane 1: MON 89034 (Ssp I) permission 2: Conventional corn (Sspprohibited I) the 3: MONcommercial 89034 (Nco be I) 4: Conventional corn (Nco I) Furthermore, 5: MONany 89034 (Ssp I) Consequently, 6: Conventionalwithout corn (Ssp I) 7: MON 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 73 of 323 3.2.8. nos 3' end sequence The results of this analysis are presented in Figure IV.17. Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or BstE II (lanes 4 and 8) examined with the nos 3' end sequence probe showed no detectable hybridization bands, as expected for the negative control. Conventional corn control DNA spiked with plasmid PV-ZMIR245 DNA digested with BstE II (lanes 9 and 10) produced the expected size band at ~7.8 kb.

MON 89034 DNA digested with Ssp I (lanes 1 and 5) produced a single, unique band of ~7.4 kb that is consistent with the expected band >4.3 kb (see Figure IV.8). MON 89034 DNA digested with BstE II (lanes 3 and 7) produced the expected single unique band of ~1.7 kb. No unexpected bands were detected, indicating that MON 89034 contains no additional nos 3'end elements other than those associated with the cry2Ab2 cassette. regime.and and AG

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40 20 15 40 10 20 8.1 7.1 15 6.1 5.1 regime.and 10 and4.1 AG 8.1 3.1 7.1 6.1 Bayer property 2.0protectionpublishing of 1.6 contents 5.1 its parties.data therefore and/oror 4.1 1.0 affiliates. property intellectualthird may its 3.1 the as 0.5 of and regulatory owner. is a documentits any such reproductiondocument of owner this 2.0 under of this and/orrights the document1.6 rights to of fall use of the This may distribution,and rights owner subject Figure IV.17.be Southern Blot Analysis ofthe MON violate 89034: nos 3' end Sequence copy of The blot was examineddocument with a 32P-labeledand probe that spanned the nos 3' end sequence may publication,exploitation (probeIt 10, Figure IV.6). Each lane contains ~10 µg of digested genomic DNA isolated this from grain. Lane designationsany are as follows: Lane 1: MON 89034 (Ssppermission I) prohibited 2: Conventional corn (Ssp I) the 3: MONcommercial 89034 (BstE be II) 4: Conventional corn (BstE II) Furthermore,any Consequently, 5: MONwithout 89034 (Ssp I) 6: Conventional corn (Ssp I) 7: MON 89034 (BstE II) 8: Conventional corn (BstE II) 9: Conventional corn spiked with PV-ZMIR245 (BstE II) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (BstE II) [1.0 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 75 of 323 3.2.9. Analysis to confirm the absence of plasmid PV-ZMIR245 backbone Southern blot analysis was used to confirm that the plasmid backbone sequences necessary for transformation are no longer present in MON 89034. This is important to ensure that only the desired proteins are produced in MON 89034.

The results of this analysis are presented in Figure IV.18. Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) examined with overlapping probes spanning the vector backbone of PV-ZMIR245 (probes 14-17, Figure IV.6) showed no hybridization bands as expected for the negative control.

Conventional corn control DNA spiked with plasmid PV-ZMIR245 DNA digested with Nco I (lanes 9 and 10) produced the expected size band of ~10 kb. regime.and and MON 89034 DNA digested with Ssp I (lanes 1 andAG 5) or Nco I (lanes 3 and 7) produced no detectable hybridization bands, indicating that MON 89034 contains no PV-ZMIR245 backbone elements. Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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9.4

23 6.6

4.4

regime.and 9.4 and

AG 2.3

6.6 Bayer property protectionpublishing of contents its parties.data therefore 4.4 and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a documentits any such reproduction this documentof owner under of 2.3 rightsthe this documentand/or rights to of fall use of the This may distribution,and Figure IV.18. Southernrights Blot Analysis of MONowner 89034: PV-ZMIR245 Backbone Sequencesubject be 32 the violate The blot was examinedcopy with P-labeledof probes that spanned the PV-ZMIR245 backbone sequence (probes 14-17, Figure IV.6). Eachand lane contains ~10 µg of digested genomic may documentpublication, DNA Itisolated from grain. Laneexploitation designations are as follows: Lane 1: MONthis 89034 any (Ssp I) 2: Conventional corn (Ssp I) permission 3: MON 89034 (Nco I) prohibited the 4: Conventionalcommercial corn be ( Nco I) 5: MON 89034 (Ssp I) Furthermore, 6: Conventionalany corn (Ssp I) Consequently, 7: MONwithout 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 77 of 323 3.3. Analysis to confirm the absence of T-DNA II 3.3.1. Analysis to confirm the absence of the nptII coding sequence Southern blot analysis was used to confirm that the nptII sequence necessary for transformation is no longer present in MON 89034. This is important to ensure that only the desired proteins are produced in MON 89034.

The results of this analysis are presented in Figure IV.19. Conventional corn control DNA digested with Ssp I (lanes 2 and 6) or Nco I (lanes 4 and 8) examined with the nptII coding sequence probe (probe 12, Figure IV.6) showed no detectable hybridization bands, as expected for the negative control.

and Conventional corn control DNA spiked with plasmid PV-ZMIR245and DNAregime. digested with Nco I (lanes 9 and 10) produced the expected sizeAG band of ~10 kb.

MON 89034 DNA digested with Ssp I (lanes 1 and 5) or Nco I (lanes 3 and 7) produced Bayer property protectionpublishing no detectable hybridization bands, indicatingof that MON 89034 contains nocontents nptII-derived elements. its parties.data therefore and/oror 3.3.2. Analysis to confirm the absenceaffiliates. of the overall T-DNA II region property intellectualthird may its This Southern blot analysis confirmsas the absence of the nptII coding sequence and the of and demonstrates the absenceis of any additional regulatoryT-DNA II sequencesowner. except for those a document elements that are in commonany with such the T-DNA I expression cassetteits (i.e., 35S promoter, reproductiondocument of nos 3´ end sequence, Left Border sequence)owner . This analysisthis confirms that the F1 plants that under of contained T-DNA II elementsrights werethe segregated out and thisthat the plants selected for further documentand/or rights development only containedto T-DNAof fall I. use of the The resultsThis of this analysis aremay presente distribution,andd in Figure IV.20. Conventional corn control rights owner DNA digested withsubject Ssp I (lanes 2 and 6) and BstE II (lanes 4 and 8) examined with overlapping probes spanning T-DNA theII (pviolaterobes 11-13, Figure IV.6) showed no be copy hybridization bands. of and may documentpublication, It exploitation Conventional cornthis control DNA spiked with plasmid PV-ZMIR245 DNA digested with BstE II (lanes 9 andany 10) produced the two expected size bands at ~7.8 and ~2 kb. The permission overlapping probes spanning T-DNAprohibited II contain the 35S promoter, nos 3' end, and Left Border sequences, which are contained on T-DNA I. Therefore, the T-DNA II probe is the be expected to hybridizecommercial to bands derived from T-DNA I. Furthermore,any MONConsequently, 89034 DNAwithout digested with Ssp I (lanes 1 and 5) produced two bands of ~8.2 and ~7.4 kb The presence and size of these bands is consistent with the 8.2 kb band detected during the analysis of the e35S promoter/Cab leader region (Figures IV.10, lanes 1 and 5), and the 7.4 kb band detected during the analysis of the nos 3' end sequences (Figure IV.17, lanes 1 and 5).

MON 89034 DNA digested with BstE II (lanes 3 and 7) produced two bands of ~4.2 and ~1.7 kb. The ~4.2 kb band is consistent with the >2.8 kb expected band for sequence

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 78 of 323 from T-DNA I digested with BstE II (Figure IV.8), and the ~1.7 kb band is consistent with the T-DNA I specific band observed in Figure IV.17, lanes 3 and 7. No unexpected bands were detected, indicating that MON 89034 contains no additional elements other than those in common with T-DNA I.

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8.1 3.1

7.1 Bayer property publishing 2.0protection 6.1 of contents 1.6 its parties.data therefore 5.1 and/oror affiliates. 1.0 4.1 property intellectualthird may its as the of and 0.5 is regulatory owner. 3.1 a documentits any such reproduction this documentof owner under of rightsthe this document2.0and/or rights to of fall use of 1.6 the This may distribution,and rights owner Figure IV.19. Southernsubject Blot Analysis of MON 89034: npt II Coding Sequence be 32 the violate The blot was examinedcopy with a P-labeledof probe that spanned the npt II coding sequence (probe 12, Figure IV.6). Each lane containsand ~10 µg of digested genomic DNA isolated may documentpublication, from grain.It Lane designationsexploitation are as follows: Lane 1: MONthis 89034 any (Ssp I) 2: Conventional corn (Ssp I) permission 3: MON 89034 (Nco I) prohibited the 4: Conventionalcommercial corn be ( Nco I) 5: MON 89034 (Ssp I) Furthermore, 6: Conventionalany corn (Ssp I) Consequently, 7: MONwithout 89034 (Nco I) 8: Conventional corn (Nco I) 9: Conventional corn spiked with PV-ZMIR245 (Nco I) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (Nco I) [1.0 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

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1 2 3 4 5 6 7 8 9 10

40 20 40 15

20 10 15 8.1 7.1 6.1 and and regime. 10 5.1 AG 4.1 8.1 7.1 3.1 Bayer 6.1 property protectionpublishing of contents 2.0 5.1 its parties.data therefore and/or1.6or 4.1 affiliates. property intellectualthird may its 1.0 the as of and regulatory owner. 3.1is a documentits any such 0.5 reproductiondocument of owner this under of rightsthe this documentand/or2.0 rights to of fall use of

1.6 the This may distribution,and rights owner subject Figure IV.20.be Southern Blot Analysis ofthe MON violate 89034: T-DNA II The blot was examinedcopy with overlappingof 32P-labeled probes that spanned the T-DNA II and sequencemay (probes 11-13,documentpublication, Figure IV.6). Each lane contains ~10 µg of digested genomic It exploitation DNA isolated fromthis grain. Lane designations are as follows: Lane 1: MON 89034any (Ssp I) 2: Conventional cornpermission (Ssp I) prohibited 3: MON 89034 the(BstE II) 4: Conventionalcommercial corn be ( BstE II) 5: MON 89034 (Ssp I) Furthermore,any Consequently, 6: Conventionalwithout corn (Ssp I) 7: MON 89034 (BstE II) 8: Conventional corn (BstE II) 9: Conventional corn spiked with PV-ZMIR245 (BstE II) [0.5 copy] 10: Conventional corn spiked with PV-ZMIR245 (BstE II) [1.0 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

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3.4. Insert stability across generations of MON 89034 3.4.1. Summary To demonstrate the stability of the MON 89034 insert during breeding, additional Southern blot analyses were performed using DNA samples obtained from multiple generations of the MON 89034 breeding history. For reference, the breeding history of MON 89034 is presented in Figure IV.21. The specific generations tested are indicated in the legends of Figures IV.22-24. For these analyses, DNA samples were digested with the restriction enzyme Ssp I, which cleaves once within the inserted DNA and in both the 5' and 3' genomic flanking sequences of MON 89034. This produces two DNA fragments of ~8.2 and >4.3 kb (Figure IV.8). The ~8.2 kb fragment contains genomic and DNA flanking the 5' end of the insert, Left Border sequence,and modified e35Sregime. promoter sequence, Cab leader, Ract1 intron, cry1A.105 codingAG sequence, Hsp17 3' end sequence, FMV promoter, and a portion of the Hsp70 intron. The >4.3 kb fragment contains the remaining portion of the Hsp70 intron, SSU-CTP targeting sequence, cry2Ab2 coding Bayer property protectionpublishing sequence, nos 3' end sequence, Left Borderof sequence, and genomic DNAcontents flanking the 3' end of the inserted DNA (Figure IV.8). its parties.data therefore and/oror Plasmid PV-ZMIR245 DNA was spikedaffiliates. into the conventional corn control DNA, property intellectualthird may digested with Ssp I, and loaded onits the gel to serve as a positive hybridization control. as Individual Southern blots thewere of examined and with three-probe sets. The stability of the is regulatory owner. MON 89034 insert across generations was confirmeda usingdocument overlapping T-DNA I probes any such its spanning the entire inserted DNA sequence (probesreproduction 18 – 23 documentin Figure IV.7). The absence owner this of of the nptII selectable marker and uniqueunder T-DNA ofII genetic elements not contained in T- DNA I was confirmed usingrights overlappingthe probes spanningthis T-DNA II (probes 11, 12, and documentand/or of rights 13, Figure IV.6). The toabsence of offall plasmi d usePV-ZMIR245 backbone sequence across generations was confirmed using overlapping probes thespanning the vector backbone of This may distribution,and PV-ZMIR245 (probes rights14 - 17, Figure IV.6)owner. A second conventional corn control subject (referred to as conventional corn A) was used in these Southern blots to ensure that the be the violate genetic backgroundscopy of all the generationsof were accurately represented. and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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R0 x LH172

LH172 BC0 F1

⊗

LH172 BC0 F2

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

LH172 BC0 F4

⊗ Bayer property protectionpublishing of contents LH172 BC0 F5 its parties.data therefore ⊗ and/oror 1 affiliates. TI: BC1:F1 x RP property intellectualthird may LH172its BC0 F as 6 the of and is ⊗ regulatory owner. a document any such its x LH198 reproductiondocument owner this of LH172 BC0 F under7 of this and/orrights the document [LH172rights BC0 F6 x LH198]F1H to of fall use of

x LH198 the This may distribution,and ⊗ rights owner subject the be violate LH172 BC0 F6 x F2H1 copy of [LH172 BC0 F7 x LH198]F1H document and may publication,exploitation It this any R0 = transformed plant; F(#)permission = filial generation; ⊗ = self-pollination; BC(#) = backcross prohibited generation.; RP = recurrent parent; H = hybrid; TI - Trait Integration the commercial be

Furthermore,any FigureConsequently, IV.21. withoutBreeding History of MON 89034 The LH172 BC0 F6 x F2H1 generation was used for all molecular analyses. Generations used for stability analysis are shown in bold in the breeding tree.

1 Recurrent parent (RP) that was used in the analyses depicted in Figures IV.22 and IV.23 is referred to as Conventional corn A.

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3.4.2. Stability of the T-DNA I insert across generations of MON 89034 The results of this analysis are presented in Figure IV.22. Conventional corn control DNA digested with Ssp I (lane 7 and 9) and examined with overlapping T-DNA I probes spanning the entire inserted DNA sequence (probes 18 - 23, Figure IV.7) showed several faint hybridization bands. This was expected because T-DNA I contain several genetic elements originally derived from corn. These endogenous bands were only detected in conventional corn or upon longer exposure and were seen previously when blots were probed with the overlapping T-DNA I probe (see Figure IV.9), the Hsp 70 intron probe (Figure IV.15), and the SSU-CTP/cry2Ab2 probe (Figure IV.16).

regime.and Conventional corn control DNA spiked with plasmid PV-ZMIR245and DNA digested with Ssp I (lanes 10 and 11) produced the two expectedAG size bands at ~10.4 and ~7.2 kb in addition to the expected endogenous bands. The migration of the ~10.4 kb fragment is slightly higher than indicated by the moleculaBayerr marker property band sizes protection in thepublishing lanes 10 and 11. The altered migration may be due to theof diffe rence in salt concentration betweencontents the corn its DNA samples and the molecular marker (Sambrook andparties. Russell,data 2001). therefore and/oror affiliates. DNA extracted from seven MONproperty 89034 generationsintellectualthird digested with Sspmay I (lanes 1, 2, 3, 4, its 5, 6, and 8) each produced two bandsas of ~8.2 and ~7.4 kb in addition to the expected the of and endogenous hybridization.is The ~8.2 kb bandregulatory is the expected sizeowner. for the 5' border a document fragment and the ~7.4 kb bandany issuch consistent with the expected bandits size of >4.3 kb for reproductiondocument the 3' border fragment. These bandsowner are consistent withthis the bandsof detected in Figure IV.9 (lanes 3 and 7). under of rightsthe this documentand/or rights to of fall use of Two faint bands of ~15.6 and ~12 kb were observed in lanes 3 and 4 (Figure IV.22). the These bandsThis likely are the resultmay of partialdistribution,and dige stion because they are not seen in prior or rights owner subsequent generationssubject that were produced by self pollination. In support of this conclusion thebe presence of the ~15.6 kb bandthe violateis consistent with the lack of digestion at the internal Ssp I copysite. This band wouldof be expected as a combination of the 7.4 and 8.2 kb bands observed documentfor Ssp I digestion. Theand ~12 kb band is consistent with the partial may publication,exploitation digestionIt of the Ssp I site in the 5' flanking genomic sequence which would result in an this ~11.8 kb band. any permission prohibited No additional unexpected bands were detected, indicating that the single copy of the T-DNA I in MONcommercial 89034 is stable be in the selected generations.

Furthermore,any Consequently,without

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Bayer 2.0 property protectionpublishing of contents 1.6 its parties.data therefore and/oror affiliates. may 1.0 property intellectualthird its as the of and is regulatory owner. a documentits 0.5 any such reproduction this documentof owner under of rightsthe this documentand/or rights to of fall use of the This may distribution,and Figure IV.22. Insert Stabilityrights of MON 89034:owner T-DNA I The blot was examinedsubject with overlapping 32P-labeled probes that spanned the T-DNA I be the violate sequence (probes copy18 - 23, Figure IV.7).of Each lane contains ~10 µg of digested genomic DNA isolated from grain. Lane designationsand are as follows: may documentpublication, Lane It 1: MON 89034 (LH172exploitation BC0F6 x F2H1, Ssp I) this 2: MON 89034any (LH172 BC0 F3, Ssp I) 3: MON 89034 (LH172 BC0 F , Ssp I) permission 4 4: MON 89034 (LH172prohibited BC0 F5, Ssp I) the 5: MONcommercial 89034 (LH172 be BC0 F6, Ssp I) 6: MON 89034 ([LH172 BC0 F7 x LH198]F1H, Ssp I) Furthermore, 7: Conventionalany corn (LH172, Ssp I) Consequently,without 8: MON 89034 (TI: BC1: F1 x RP, Ssp I) 9: Conventional corn A1 (Ssp I) 10: Conventional corn spiked with PV-ZMIR245 (Ssp I) [1.0 copy] 11: Conventional corn spiked with PV-ZMIR245 (Ssp I) [0.5 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

1 Monsanto proprietary conventional corn hybrid

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 85 of 323 3.4.3. Confirmation of the absence of T-DNA II in multiple generations of MON 89034 The results of this analysis are presented in Figure IV.23. Conventional corn control DNA digested with Ssp I (lane 7 and 9) and examined with three overlapping probes spanning T-DNA II (probes 11-13, Figure IV.6) showed no detectable hybridization bands. Conventional corn control DNA spiked with plasmid PV-ZMIR245 DNA digested with Ssp I (lanes 10 and 11) produced the two expected size bands at ~10 and ~7.2 kb. The migration of the ~10 kb fragment is slightly higher than indicated by the molecular marker band sizes in the lanes 10 and 11. The altered migration may be due to the difference in salt concentration between the corn DNA samples and the molecular marker (Sambrook and Russell, 2001). The overlapping probes spanning T-DNA II contains the 35S promoter, nos 3' end, and the Left Border which are contained on regime.and T-DNA I. Therefore, the T-DNA II probe is expected to hybridizeand to fragments derived from T-DNA I. AG

DNA extracted from seven generations of BayerMON 89034property digested protection with publishingSsp I (lanes 1, 2, 3, 4, 5, 6, and 8) each produced two bandsof of ~8.2 and ~7.4 kb which are contentsconsistent with its those observed with the overlapping T DNA I probes.parties. Thedata ~ 8.2 kb band istherefore the expected size for the 5' border fragment and the ~7.4 kb band is consistentand/or withor the expected band affiliates. size of > 4.3 kb for the 3' borderproperty fragment. intellectual thirdOn longer exposures, twomay faint bands of its ~15.6 and ~12 kb were observed in aslanes 3 and 4 (Figure IV.23). These bands are the of and consistent with the bandsis observed in lanes 3 regulatoryand 4 of Figure IV.22owner. and are likely the a document result of partial digestion becauseany such they are not seen in prior generationsits produced by self- reproductiondocument pollination. The presence of the 15.6owner kb band is consistentthis withof the possibility that the internal Ssp I site was not digested andunder a combinationof of the 7.4 and 8.2 kb bands rightsthe this produced the document15.6 kband/or band. The 12.0 kb band is consistentrights with the partial digestion of to of fall use of the Ssp I site in the 5' flanking genomic sequence which would result in an approximately the 11.8 kb band.This may distribution,and rights owner subject No additionalbe bands were detected, indicatithe ngviolate that the seven generation tested do not contain any additionalcopy T-DNA II elementsof other than those associated with T-DNA I and that therefore those documentT-DNA II elements segregatedand as expected. may publication,exploitation It this 3.4.4. Confirmationany of the absence of plasmid PV-ZMIR245 backbone sequence in multiple generationspermission of MON 89034 prohibited The results of this analysisthe are presented in Figure IV.24. Conventional corn control commercial be DNA obtained from LH172, digested with Ssp I (lanes 7 and 9), and examined with four overlappingFurthermore,any probes spanning the PV-ZMIR245 backbone sequence (probes 14-17, Figure IV.6)Consequently, showed nowithout detectable hybridization bands. Although difficult to observe in Figure IV.24, overexposures of Southern blots showed that conventional corn A control DNA, digested with Ssp I (lanes 7 and 9), produced two faint hybridization bands at 6.0 and 3.5 kb. These are likely the result of endogenous hybridization to corn genetic elements specific to this background. Conventional control DNA spiked with plasmid PV- ZMIR245 DNA digested with Ssp I (lanes 10 and 11) produced the two expected size bands at ~10.4 and ~7.2 kb. DNA samples extracted from the seven generation digested

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 86 of 323 with Ssp I (lanes 1, 2, 3, 4, 5, 6, and 8) produced no hybridization bands. This confirmed that the backbone sequences from PV-ZMIR245 are not present in MON 89034.

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2.0 Bayer property protectionpublishing 1.6 of contents its parties.data therefore and/oror 1.0 affiliates. property intellectualthird may its as 0.5 the of and is regulatory owner. a document any such its reproductiondocument of owner this under of rightsthe this documentand/or rights to of fall use of the This may distribution,and Figure IV.23. Analysisrights of Multiple Generationsowner of MON 89034: T-DNA II The blot was examinedsubject with overlapping 32P-labeled probes that spanned the T-DNA II sequence (probesbe 11-13, Figure IV.6). Eachthe laneviolate contains ~10 µg of digested genomic copy of DNA isolated from grain. Lane designationsand are as follows: may documentpublication, Lane It 1: MON 89034 (LH172exploitation BC0F6 x F2H1, Ssp I) 2: MON 89034 (LH172 BC0 F , Ssp I) this any 3 3: MON 89034 (LH172 BC0 F4, Ssp I) permission 4: MON 89034 (LH172prohibited BC0 F5, Ssp I) 5: MON 89034 the(LH172 BC0 F , Ssp I) commercial be 6 6: MON 89034 ([LH172 BC0 F7 x LH198]F1H, Ssp I) Furthermore, 7: Conventionalany corn (LH172, Ssp I) Consequently, 8: MONwithout 89034 (TI: BC1: F1 x RP, Ssp I) 9: Conventional corn A1 (Ssp I) 10: Conventional corn spiked with PV-ZMIR245 (Ssp I) [1.0 copy] 11: Conventional corn spiked with PV-ZMIR245 (Ssp I) [0.5 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on ethidium bromide stained gel.

1 Monsanto proprietary conventional corn hybrid

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1 2 3 4 5 6 7 8 9 10 11

9.4

6.6

4.4

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2.3 2.0 Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as 0.5 the of and regulatory owner. is a documentits any such reproductiondocument of owner this under of rightsthe this documentand/or rights to of fall use of Figure IV.24. Backbone Analysis of Multiple Generationsthe of MON 89034: PV- This ZMIR245 Backbonemay distribution, Sequenceand rights owner The blot was examinedsubject with overlapping 32P-labeled probes that spanned the PV- the violate ZMIR245 backbonebe copy sequence (probes 14-17, Figure IV.6). Each lane contains ~10 µg of digested genomic DNA isolated from graiof n. Lane designations are as follows: document and Lane 1:may MON 89034 (LH172publication, BC0F 6 x F2H1, Ssp I) It exploitation 2: MON 89034 (LH172 BC0 F3, Ssp I) this any 3: MON 89034 (LH172 BC0 F4, Ssp I) permission 4: MON 89034 (LH172prohibited BC0 F5, Ssp I) 5: MON 89034 the(LH172 BC0 F6, Ssp I) commercial be 6: MON 89034 ([LH172 BC0 F7 x LH198]F1H, Ssp I) Furthermore, 7: Conventionalany corn (LH172, Ssp I) Consequently, 8: MONwithout 89034 (TI: BC1: F1 x RP, Ssp I) 9: Conventional corn A1 (Ssp I) 10: Conventional corn spiked with PV-ZMIR245 (Ssp I) [1.0 copy] 11: Conventional corn spiked with PV-ZMIR245 (Ssp I) [0.5 copy] Symbol denotes size of DNA, in kilobase pairs, obtained from molecular markers on the ethidium bromide stained gel.

1 Monsanto proprietary conventional corn hybrid

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3.5. Organization and sequence of the insert DNA in MON 89034 The reported organization of the elements within the insert in MON 89034 was confirmed by DNA sequencing analyses. PCR primers were designed to amplify seven overlapping regions of DNA that span the entire length of the insert (9.3 kb). The amplified DNA fragments were subjected to DNA sequencing analyses to further confirm the organization of the elements within the insert. The results confirmed that the sequence of the DNA insert in MON 89034 matched the designed, corresponding sequences in PV-ZMIR245 with one exception. The e35S promoter that regulates expression of the cry1A.105 gene has been modified and that the Right Border sequence present in PV-ZMIR245 was replaced by a Left Border sequence in MON 89034. This molecular rearrangement can be explained by a recombination event, which occurredand either prior to or during the process of T-DNA transfer to theand pl ant cell,regime. between the DNA sequences near the 35S promoters in T-DNAAG I and T-DNA II (Figure IV.5). Due to this recombination event, the reconstituted e35S promoter in MON 89034 (referred to as modified e35S or e35S89) no longer has the duplicated enhancer elements compared to the Bayer property protectionpublishing original e35S promoter in PV-ZMIR245.of Despite the deletion of the enhancercontents elements, the modified e35S promoter in MON 89034 regulates Cry1A.105 proteinits expression to parties.data therefore provide sufficiently high levels for efficacious control of target and/orinsector pests. affiliates. property intellectualthird may 3.6. Inheritance of the lepidopteranits protection trait in MON 89034 as the of and is regulatory owner. Significance of the segregation pattern (positives/negatives)a document was assessedits by Chi square analysis over four generationsany ofsuch MON 89034 toreproduction determine the heritability and stability this documentof of the trait (cry1A.105 and cry2Ab2owner genes, and Cry1A.105 and Cry2Ab2 proteins) in under of (Table IV.4). The confirmationrightsthe of the presence of the thisgene and stability of the trait was documentand/or rights based on one of severalto assays of : 1)fall ELISA touse detect of the Cry2Ab2 protein; 2) ELISA to detect the Cry1A.105 protein; 3) PCR assay to detectthe presence of the cry genes; and 4) This may distribution,and GeneCheck® immunoassaysrights to detect Cry2Ab2owner prot ein (Cry2A QuickStix Lateral Flow bulk strips, Envirologixsubject Inc). be the violate copy of As described in Part IV, Section 2.1, MONand 89034 was developed using a 2 T-DNA may documentpublication, vectorIt transformation systemexploitation that employe d two separate T-DNA regions. One T-DNA (T-DNA I) containedthis any the cry2Ab2 and cry1A.105 genes while the other T-DNA (T-DNA II) contained the nptII expression cassette. F1 plants were generated in a LH172 permission 1 germplasm by making crossesprohibited of the R 0 plant with the LH172 inbred. From the the population of producedcommercial F1 beplants, selections were made for the absence of nptII and plants were screened for copy number of the cry1A.105 and cry2Ab2 inserted cassettes usingFurthermore, Southernany blot analysis. Plants selected in the F1 generation were either used to Consequently,without make BC1F1 seed, or were self-pollinated to generate F2 seed (Figure IV.21). The overall goal in the F1 population of plants was to select single copy, marker–free plants. A final plant was selected from the F1 generation, designated as event MON 89034, and progeny derived from this plant showed the expected patterns for genetic segregation (Table IV.4).

® GeneCheckTM is an Envirologix Inc Trademark. 1 LH172 is a commercial corn line produced by Holdens Company, a Monsanto affiliate.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 90 of 323

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

Expected Generation Ratioa Comment LH172 BC0F1 n.a. screened for copy number and absence of nptII (segregation data not shown) LH172 BC0F2 3:1 positive:negative (product of self pollination) LH172 BC0F3 1:0 positive:negative (homozygous plant selection) LH172 BC0F4 1:0 positive:negative (homozygous plant selection) b LH172 BC1F1 1:1 positive:negative (product of backcrossing) and c and regime. LH172 BC1F2 3:1 positive:negative (product of self pollination) c AG LH172 BC1F2 3:1 positive:negative (product of self pollination) an.a. = not applicable. bTo confirm segregation, LH172 BC0F plants were backcrossed to the recurrent parent (LH172) to 1 Bayer property protectionpublishing produce this generation (not shown on the breedingof tree, Figure IV.21). contents c To confirm segregation, The LH172 BC1F1 plants were selfed to produce two differentits plant populations parties.data therefore for this generation (not shown on the breeding tree, Figure IV.21). and/oror affiliates. property intellectualthird may its as The Chi-square test was computedthe of as: and is regulatory owner. a document any χ2such = Σ [ (| o – e | – 0.5)2 / e] its reproductiondocument of owner this under of where o = observed frequencyrights ofthe the genotype, e = expectedthis frequency of the genotype, documentand/or rights and 0.5 = Yates correctionto factorof fallfor analysuseis with of one degree of freedom (Little and Hills, 1978). the This may distribution,and rights owner Results of the Chi-squaresubject test are summarized in Table IV.4. All χ2 values indicated no significant differencesbe between observedthe and violateexpected genetic ratios across all tested copy of generations of MON 89034. These resultsand are consistent with molecular characterization may documentpublication, data indicatingIt a single insertionexploitation site for the cry1A.105 and cry2Ab2 expression cassettes. this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 91 of 323

Table IV.4. Segregation Analyses of MON 89034a

Number Observed Observed Expected Expected Chi- Generation Probability of plants Positives1 Negatives Positives Negatives Square LH172 BC0F2 11 7 4 8.25 2.75 0.2727 >0.05 LH172 BC0F3 24 24 0 24 0 Fixed + n.a. LH172 BC0F4 30 30 0 30 0 Fixed + n.a. LH172 BC1F1 28 13 15 14 14 0.0357 >0.05 LH172 BC1F2 24 20 4 18 6 0.5000 >0.05 and LH172 BC1F2 24 17 7 18 and 6 0.0556regime. >0.05 AG aThe confirmation of the trait was based on one of several assays: 1) ELISA to detect the Cry2Ab2 protein; 2) ELISA to detect the Cry1A.105 protein; 3) PCR assay to detect presence of the cry genes; and, 4) GeneCheck immunoassays to detect the Cry2Ab2Bayer protein. property publishing protectioncontents of its parties.data therefore and/oror 3.7. Conclusion affiliates. property intellectualthird may its Molecular analyses confirmed that MONas 89034 contains one insert derived from T-DNA the of and I at a single locus of theis nuclear genome. The regulatoryinsert contains one functionalowner. copy of the a document cry1A.105 and of the cry2Ab2any expressionsuch cassettes. All genetic elementsits from T-DNA I reproductiondocument are intact and present in the expectedowner order. These analysesthis alsoof confirmed the absence of plasmid backbone sequence, nptII and T-DNAof II derived elements, other those in rights under this common with the T-DNAand/or I (i.e.,the 35S promoter, nos 3´ end sequence, and the Left Border document to of fall of rights sequence). The Southern blot fingerprint of MONuse 89034 was maintained through seven the generationsThis of conventional breeding,may distribution, therebyand confirming the stability of the insert over rights owner multiple generations.subject These generations did not contain any detectable T-DNA II elements other than thosebe in common with T-DNA theI, no rviolate did they contain any detectable backbone sequence from plasmidcopy PV-ZMIR245. ofPCR and DNA sequence analyses confirmed the and organizationmay of the documentelementspublication, within the insert, the 5' and 3' insert-to-plant junctions, and the completeIt DNA sequence exploitationof the insert. this any permission prohibited the commercial be Furthermore,any Consequently,without

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SECTION 4. Other data and information about the development of MON 89034

All relevant information regarding development of MON 89034 is described in Parts II – VII of this summary.

References

Armstrong, C.L. and R.L. Phillips. 1988. Genetic and cytogenetic variation inand plants and regime. regenerated from organogenic and friable, AGembryogenic tissue cultures of maize. Crop Sci. 28:363-369.

Barker, R.F., K.B. Idler, D.V.Thompson, andBayer J.D. Kemp.property 1983. Nucleotidepublishing sequence of protectioncontents the T-DNA region from the Agrobacteriumof Tumefaciens octopine Ti plasmid its pTil6966. Plant Mol. Biol. 2:35-350. parties.data therefore and/oror

affiliates. Beck, E., G. Ludwig, E.A. Auerswald,property B.intellectual Rethirdiss, and H. Schaller. may1982. Nucleotide its sequence and exact localization asof the neomycin phosphotransferase gene from the of and transposon Tn5. Geneis 19:327-336. regulatory owner. a document any such its reproductiondocument Bevan, M., W.M. Barnes, and M. ownerChilton. 1983. this Structure ofand transcription of the under of nopaline syunthase generights regionthe of T-DNA. Nucl. Acidsthis Res. 11:369-385. documentand/or rights to of fall use of Breitler, J-C, D. Meynard, J. Van Boxtel, M. Royer,the F. Bonnot, L. Cambillau, and E. This may distribution,and Guiderdoni. 2004. rights A novel two T-DNA ownerbinary vector allows efficient generation of marker-free subjecttransgenic plants in three elite cultivars of rice (Oryza sativa L.). Transgenicbe Res. 13:271-287. the violate copy of and Brown, mayS.M. and C.G.document publication,Santino. 1995. Enhanced expression in plants. United States It exploitation Patent No. 5,424,412.this any permission Crickmore, N., D.R. Ziegler, J. prohibitedFeitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D.H. Dean. 1998.the beRevision of the nomenclature for the Bacillus thuringiensis pesticidal crystalcommercial proteins. Microbiol. Biol. Rev. 62:807-813. Furthermore,any Depicker,Consequently, A., S.without Stachel, P. Dhaese, P. Zambryski, and H.M. Goodman. 1982. Nopaline synthase: transcript mapping and DNA sequence. J. Mol. Appl. Genet. 1:561-573.

Depicker, A., L. Herman, A. Jacobs, J. Schell, and M. Van Montague. 1985. Frequencies of simultaneous transformkation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction. Mol. Gen. Genet. 201:477-484.

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Donovan, W.P. 1991. CryIIB crystal protein gene from Bacillus thuringiensis. United States Patent No. 5,073,632.

Fling, M., J. Kopf, and C. Richards. 1985. Nucleotide sequence of the transposon Tn7 gene encoding an aminoglycoside-modifying enzyme, 3’(9)-O-nucleotidyltrnasferase. Nucleic Acids Res. 13:7095-7106.

Galinat, W.C. 1988. The origin of corn. Pp 1-31. In Corn and Corn Improvement. Sprague, G.F. and J.W. Dudley (eds.). American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Madison, Wisconsin.

Giza, P.E. and R.C. Huang. 1989. A self-inducing runaway-replication plasmid regime.and expression system utilizing the Rop protein. Gene 78:73-84.and AG

Goodman, M.M. 1988. The history and evolution of maize. CRC Critical Rev. Plant Sci. 7:197-220. Bayer property protectionpublishing of contents its Goodman, M.M. and W.L. Brown. 1988. Races ofparties. corn. data Pp 33-79. In Corntherefore and Corn Improvement. Sprague, G.F. and J.W. Dudley (eds.).and/or Americanor Society of affiliates. Agronomy, Crop Science Societyproperty of America,intellectualthird and Soil Science Societymay of America, its Madison, Wisconsin. as the of and is regulatory owner. a document Ishida, Y., H. Saito, S. Ohta,any Y.such Hiei, T. Komari, and T. Kumashiro.its 1996. High reproductiondocument efficiency transformation of maizeowner (Zea maysthis L.) mediatedof by Agrobacterium tumefaciens. Nature Biotech. 14:745-750.under of rightsthe this documentand/or rights to of fall use of Jones, S.M., C.F. Magnolfi, S.K. Cooke, and H.A. Sampson.the 1995. Immunologic cross- reactivityThis among cereal grainsmay anddistribution, grassesand in children with food hypersensitivity. J. rights owner Allergy Clin.subject Immunol. 96:341-351. the violate be copy Kay, R., A. Chan, M. Daly, and J. McPhof erson. 1987. Duplication of CaMV 35S and promotermay sequencesdocumentpublication, created a strong enhancer for plant genes. Science 236:1299- It exploitation 1302. this any permission Komari, T., Y. Hiei, Y. Saito, N.prohibited Murai, and T. Kumashiro. 1996. Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium the be tumefaciens commercialand segregation of transformants free from selection markers. Plant J. Furthermore,10(1):165-174.any Consequently,without Lamppa, G., G. Morelli, and N. Chua. 1985. Structure and developmental regulation of a wheat gene encoding the major chlorophyll a/b-binding polypeptide. Mol. Cell. Biol. 5:1370-1378.

Little, T.M. and F.J. Hills. 1978. Agricultural Experimentation: Design and Analysis. John Wiley and Sons, New York.

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Mangelsdorf, P.C. 1974. Corn—Its Origin, Evolution, and Improvement. Harvard University Press, Cambridge, Massachusetts.

Matsuoka, M., Y. Kano-Murakami, Y. Tanaka, Y. Ozeki, and N. Yamamoto. 1987. Nucleotide sequence of the cDNA encoding the small subunit of ribulose-1,5- bisphosphate carboxylase from maize. J. Biochem. 102(4):673-676.

Matsuoka, Y., Y. Vigouroux, M.M. Goodman, J. Sanchez, G.E. Buckler, and J. Doebley. 2002. A single domestication for maize shown by multilocus microsatellite genotyping. Proc. Natl. Acad. Sci. 99:6080-6084.

Matthews, P.R., M-B Wang, P.M. Waterhouse, S. Thornton, S.J. Fieg, F. Gubler, and regime.and J.V. Jacobsen. 2001. Marker gene elimination from transgenicand barley, using co- AG transformation with adjacent “twin T-DNAs” on a standard Agrobacterium transformation vector. Mol. Breed. 7:195-202. Bayer property protectionpublishing McElroy, D., A.D. Blowers, B. Jenes, ofand R. Wu. 1991. Constructioncontents of expression its vectors based on the rice actin 1(Act) 5’ regionparties. for usedata in monocot transformation.therefore Mol. Gen. Genet. 231:150-160. and/oror affiliates. property intellectualthird may its McElwain, E. and S. Spiker. 1989. A aswheat cDNA clone which is homologous to the 17 the of and kd heat-shock proteinis gene family of soybean.regulatory Nucleic Acids. Res.owner. 17:1764. a document any such its reproductiondocument Miller, M., L. Tagliani, N. Wang, ownerB. Be rka, D. Bidney,this and Z-Yof Zhao. 2002. High efficiency transgene segregationunder in co-transformedof maize plants using an rightsthe this Agrobacteriumdocument tumefaciensand/or 2 T-DNA binary system. Transgenicrights Res. 11:381-396. to of fall use of the NCGA. This 2006. The Worldmay ofdistribution, Corn.and National Corn Growers Association. rights owner http://www.ncga.com/03world/main/index.htmlsubject (01 Aug 2006). the violate be copy Odell, J.T., F. Nagy, and N. Chua. 1985.of Identificatin of DNA sequences required for and activitymay of the cauliflowerdocumentpublication, mosaic virus 35S promoter. Nature 313:810-812. It exploitation this OECD. 2002. Consensusany document on compositional considerations for new varieties of permission maize (Zea mays): Key foodprohibited and feed nutrients, anti-nutrients and secondary plant metabolites. Organization for Economic Co-operation and Development. Series of the be the Safety commercialof Novel Foods and Feeds, No. 6. ENV/JM/MONO(2002)25. Furthermore,http://www.olis.oecd.org/olis/2002doc.nsf/43bb6130e5e86e5fc12569fa005d004c/ef5aany cb7d10c9cd66c1256c1b004323e8/$FILE/JT00130429.PDFConsequently,without (26 Sep 2006).

OECD. 2003. Consensus document on the biology of Zea mays subsp. mays (maize). Organization for Economic Co-operation and Development. Series on Harmonisation of Regulatory Oversight in Biotechnology, No. 27. ENV/JM/MONO(2003)11. http://www.olis.oecd.org/olis/2003doc.nsf/LinkTo/env-jm-mono(2003)11 (5 Oct 2006).

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Pasini, G., B. Simonato, A. Curioni, S. Vincenzi, A. Cristaudo, B. Santucci, A. Dal Belin Peruffo, and M. Giannattasio. 2002. IgE-mediated allergy to corn: a 50 kDa protein belonging to the reduced soluble proteins is a major allergen. Allergy 57:98-106.

Pastorello, E.A., L. Farioli, V. Pravettoni, M. Ispano, E. Scibola, C. Trambaioli, M.G. Giuffrida, R. Ansaloni, J. Godovac-Zimmermann, A. Conti, D. Fortunato, and C. Ortolani. 2000. The maize major allergen, which is responsible for food-induced allergic reactions, is a lipid transfer protein. J. Allergy Clin. Immunol. 106(4):744- 751.

regime.and Pauls, J.D. and D. Cross. 1998. Food-dependent exercise-inducedand anaphylaxis to corn. AG J. Allergy Clin. Immunol. 101:853-854.

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Widner, W.R. and H.R. Whitely. 1989. Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities. J. Bacteriol. 171(2):965-974.

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Xing, A., Z. Zhangyuan, S. Sato, P. Staswick, and T. Clemente. 2000. The use of the two T-DNA binary system to obtain marker free transgenic soybeans. In Vitro Cell Dev. Biol. 36:456-463.

and and regime. AG

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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 89034. Molecular characterization data presented in Part IV, Section 3 confirms the absence of the add and nptII genes encoding antibiotic resistance markers that were used in cloning and transformation processes.

and and regime. AG

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PART VI: CHARACTERIZATION OF THE PROTEINS INTRODUCED INTO MON 89034

SECTION 1. Identity and Characterization of the Cry1A.105 and Cry2Ab2 Proteins Produced in MON 89034

Safety assessment of biotechnology-derived crops includes characterization of introduced proteins, confirmation of their functions and physicochemical properties, and confirmation of the safety of each protein produced from the inserted DNA. Expression and of introduced proteins in planta is usually too low to allowand purification regime. of sufficient quantities for use in safety assessment studies. Therefore,AG it is necessary to produce the proteins in high-expressing recombinant host systems (such as bacteria) in order to obtain larger quantities of the protein(s) of interest. Proteins produced by the bacterial systems Bayer property protectionpublishing are engineered to have exactly the sameof amino acid sequences as theircontents counterparts expressed in the biotechnology-derived plants. Thus, physicochemicalits and functional parties.data therefore equivalence of plant- and bacteria-produced proteins need to beand/or examinedor as part of the protein characterization and safety assessment.affiliates. property intellectualthird may its as The lepidopteran-protectedthe corn of MON 89034and produces the Cry1A.105 and Cry2Ab2 is regulatory owner. proteins. The bacteria-derived Cry1A.105 aand Cry2Ab2document proteins were produced in any such its Escherichia coli and subsequently purified. Smreproductionall quantitiesdocument ofof the Cry1A.105 and Cry2Ab2 proteins were purified fromowner the grain of MONthis 89034. The identities of the under of MON 89034-derived proteinsrights wethe re confirmed and thethis physi cochemical properties and documentand/or rights functional activities wereto comparedof fall to those useof the ofbacteria-produced protein standards. The characteristics that were analyzed to establishthe the equivalence between the This may distribution,and recombinant E. coli- andrights the MON 89034-producedowner Cry1A.105 and Cry2Ab2 proteins included: subject be the violate copy of 1. SDS-PAGE analysis to estimate molecularand weight; may documentpublication, 2. It Western blot analysis exploitationto demonstrate identity and immunoreactivity; 3. Confirmationthis any of intactness of N-terminus of the protein with an antibody specific to the N-terminal peptide; permission 4. N-terminal sequencing byprohibited Edman degradation chemistry; 5. MALDI-TOF MSthe to establish protein identity by peptide mapping; commercial be 6. Glycosylation analysis to examine the presence or absence of covalently linked Furthermore,carbohydrates;any and 7. Consequently, Insect activitywithout bioassay to assess functional equivalence.

Results obtained for the characterization of the Cry1A.105 and Cry2Ab2 proteins are discussed in the sections below.

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1.1. Identity and function of the Cry1A.105 protein Cry1A.105 is a full-length, insecticidal protein consisting of 1,177 amino acids with a molecular weight (MW) of 133 kDa. Cry1A.105 is a chimeric protein that consists of domains I and II from Cry1Ab or Cry1Ac1, domain III from Cry1F, and the C-terminal portion from Cry1Ac (Figure VI.1). Cry1Ac, Cry1Ab and Cry1F are all well known and well characterized insecticidal proteins derived from the soil bacterium Bacillus thuringiensis (Bt), that have been used both in microbial insect control products and biotechnology-derived crops. Cry1A.105 was designed using domain exchange strategy to achieve high levels of activity against the target lepidopteran insect pests. Data in Table VI.1 shows the extremely high homology of the various domains of the Cry1A.105 protein to the respective domains in the Cry1Ab, Cry1Ac and Cry1F proteins. Domainsand I and II of Cry1A.105 are 100% identical in amino acid sequenceand to Domainsregime. I and II of both Cry1Ab and Cry1Ac. Domain III of Cry1A.105AG is 99% identical in amino acid sequence to Domain III of Cry1F. The C-terminal portion has 100% homology to the C- terminal portion of the Cry1Ac protein The overall amino acid sequence identity of Bayer property protectionpublishing Cry1A.105 to Cry1Ac, Cry1Ab, and Cry1Fof is 93.6%, 90.0%, and 76.7 %,contents respectively (Table VI.1). According to an accepted phylogram (Crickmore et al., 1998)its for Bt crystal parties.data therefore proteins, Cry1A.105 can be clustered with Cry1Ac and Cry1Aband/or dueor the high degree of affiliates. homology (Crickmore, 2004; seeproperty Part VI, intellectualSectionthird 5.4.2 and Figure VI.1).may its as Domain exchange is a well-knownthe of naturallyand occurring mechanism that increases Cry is regulatory owner. a document protein diversities (De Maagdany suchet al., 2001; Masson 2002; De itsMaagd et al., 2003). reproductiondocument Domain exchange strategies with modernowner molecularthis biological oftools have been used to switch the functional domains of Cry1under prot einsof tothis develop commercial microbial biopesticides with improvedand/orrights specificitythe to lepidopteran insect pests. Microbial pesticides document to fall of rights that contain a Cry1Ac/Cry1F ofchim eric proteinuse have been used for control of lepidopteran the pests sinceThis 1997 (Baum, 1998;may Baum distribution, et andal., 1999), and a biotechnology-derived cotton rights owner product expressingsubject a chimeric protein consisting of domains or sequences from Cry1F, Cry1C, and Cry1Ab has been commercialized (Gao et al., 2006). be the violate copy of The general mechanism of insecticidal activityand of Cry proteins is well understood (Gill et may documentpublication, al., 1992;It Schnepf et al., 1998;exploitation Zhuang and Gill, 2003). Cry proteins are comprised of several functionalthis domainsany that have highly conserved regions among the classes. For example, the amino acid sequencepermission of Cry1A proteins is highly conserved in domains I, II and III. These functional domainsprohibited have been shown to determine the activity and the specificity of thecommercial Cry proteins. be Domain I is involved in membrane insertion and pore formation. Domain II is involved in specific receptor recognition and binding. Domain IIIFurthermore, maintainsany the structural integrity of the protein molecule (Li et al., 1991) and also Consequently,without contributes to specificity (De Maagd et al., 2001). The C-terminal domain is implicated in crystal formation, which does not directly contribute to the insecticidal activity (De Maagd et al., 2001). The C-terminal domain is cleaved upon entry into the insect midgut or by certain proteases in vitro. Only insects with specific receptors are affected and no toxicity is observed in species that lack these receptors (Crickmore et al., 1998; De Maagd et al., 2001). As with other Cry1A proteins, Cry1A.105 is active against major

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

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lepidopteran insect pests. The spectrum of activity includes corn borers from the genera Ostrinia and Diatraea (such as European corn borer, Asian corn borer, sugarcane borer, southwestern corn borer), armyworms (Spodoptera spp. including fall armyworm), earworms (Helicoverpa spp. including corn earworm), and cutworms (e.g., Agrotis ipsilon, black cutworm).

Domain I Domain II Domain III C-Terminal

Cry1Ab

Cry1Ac

regime.and and AG Cry1F

Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. Cry1Ab or Cry1Ac propertyCry1F intellectual thirdCry1Ac may its as 100% 100%the of 99%and 100% Cry1A.105 is regulatory owner. a document any such its reproductiondocument owner this of under of Figure VI.1. Schematic Representationrightsthe of the Originthis of Cry1A.105 Protein Domains documentand/or rights Different color and patternsto of are fallused to differentiateuse of the origin of domains. For simplicity, the lengths of domains in this illustration arethe not in proportion to the lengths This may distribution,and of amino acid sequencerights of the respective domains.owner subject be the violate Table VI.1. Aminocopy Acid Sequence Identityof Between the Cry1A.105 and Cry1Ac,document Cry1Ab, and Cry1Fand Proteins may publication,exploitation It this any permissionAmino acid identity to Cry1A.105 (%) Domain prohibited Cry1Ac Cry1Ab Cry1F the be I commercial 100 100 57 Furthermore,anyII 100 100 37 Consequently,III without 57 46 99 C-terminal 100 92 93 Overall 93.6 90 76.7

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1.2. Characterization of the Cry1A.105 protein The Cry1A.105 protein was purified from the grain of MON 89034 as well as from a fermentation of a recombinant E. coli strain. A panel of analytical tests was used to identify, characterize and compare the MON 89034-produced and the E. coli-produced Cry1A.105 proteins including: (1) SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) to examine molecular weight equivalence, (2) western blot analysis to demonstrate immunoreactivity, (3) confirmation of N-terminus intactness with an antibody specific to the N-terminal peptide, (4) MALDI-TOF MS analysis to establish protein identity by tryptic peptide mapping, (5) glycosylation analysis to examine the presence or absence of covalently linked carbohydrates, and (6) activity in an insect bioassay to assess functional equivalence. and and regime. 1.2.1. Cry1A.105 protein molecular weight equivalenceAG The equivalence in molecular weight of the purified MON 89034- and E. coli-produced Cry1A.105 proteins was demonstrated usinBayerg SDS-PAGE. property The full-lengthpublishing MON 89034- protectioncontents produced Cry1A.105 protein migrated ofto a similar position compared to that of the E. its coli-produced protein standard which was analyzed concurrentlyparties.data (Figure VI.2.).therefore and/oror

affiliates. Based on the comparable electrophoreticproperty mobility,intellectualthird the MON 89034- andmay E. coli-produced its Cry1A.105 proteins were determined toas have equivalent molecular weights. the of and is regulatory owner. a document 1.2.2. Cry1A.105 protein anyimmunoreactivity such its reproductiondocument owner this of Western blot analysis using a polyclonal anti-Cry1A.105 antibody was conducted to under of this determine the relative rightsimmunoreactivitythe of the purified MON 89034-produced documentand/or of rights Cry1A.105 protein and tothe E.of coli -producedfall Cry1A.105use reference standard. The results demonstrated that the anti-Cry1A.105 antibody recognizedthe the full-length MON 89034- This may distribution,and produced Cry1A.105 proteinrights that migrated similarlyowner to the full-length E. coli-produced subject reference Cry1A.105 protein (Figure VI.3). As expected, the immunoreactive signal be the violate increased with increasingcopy levels of loadingof for both MON 89034- and E. coli-produced proteins. The immunoreactive bands withand lowe r molecular weight were present in both may documentpublication, proteinIt samples. These bandsexploitation most likely represent proteolytic fragments of the Cry1A.105 protein.this any It is common to observe such proteolytic fragments of Cry1 proteins due to the cleavage by proteasespermission in vivo or in vitro. A faint immunoreactive band with molecular weight above 250 kDaprohibited was obs erved in the samples of the MON 89034- the produced and E.commercial coli-produced be Cry1A.105 protein. This band most likely represents the aggregate of the Cry1A.105 protein. Furthermore,any TheConsequently, western blotwithout analysis confirmed the identity of the MON 89034-produced Cry1A.105 protein and demonstrated that the MON 89034- and E. coli-produced Cry1A.105 protein have equivalent immunoreactivity with the anti-Cry1A.105 antibody.

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kDa

Full-length Cry1A.105

Proteolytic fragment of Cry1A.105

regime.and and AG

Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its Figure VI.2. SDS-PAGE of the MONas 89034- and E. coli-produced Cry1A.105 the of and Proteinsis regulatory owner. a documentits Aliquots of the MON 89034-produany suchced Cry1A.105reproduction protein and the E. coli-produced this documentof Cry1A.105 reference standard wereowner separated by a Tris-glycine 4→20% polyacrylamide under of gradient gel and stained withrights an the Invitrogen SilverXpressthis silver staining kit. Approximate documentand/or of rights molecular weights (kDa)to areof shown fall on the useleft side of the gel and correspond to the markers loaded in lanes 1 and 9. the This may distribution,and rights owner subject Lanebe Samplethe violate Amount (ng) copy of 1 Broad Range molecularand weight markers (Bio-Rad) ⎯ may documentpublication, It 2 E. coli-producedexploitation Cry1A.105 reference standard 96 3 this any MON 89034-produced Cry1A.105 protein 48 4 MON 89034-producedpermission Cry1A.105 protein 48 5 MON 89034-producedprohibited Cry1A.105 protein 72 the 6 commercial MON 89034-produced be Cry1A.105 protein 72 7 MON 89034-produced Cry1A.105 protein 96 Furthermore,any 8 MON 89034-produced Cry1A.105 protein 96 Consequently,without 9 Broad Range molecular weight markers (Bio-Rad) ⎯

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

kDa

250 250 150 100 Full-length Cry1A.105 Proteolytic fragment 75 of Cry1A.105 50

37 regime.and and 25 AG 20 15 10 Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and Figure VI.3. Western Blotis Analysis of MON regulatory89034- and E. coli-producedowner. a document Cry1A.105any Proteins such its reproductiondocument of Aliquots of the purified, MON 89034-producedowner and thisE. coli -produced Cry1A.105 protein under of were separated by SDS-PAGE,rightsthe and electrotransferredthis to a PVDF membrane. The documentand/or rights membrane was probed towith ofrabbit fall polycl onaluse anti-Cry1A.105 of antibody and developed using an ECL system (Amersham Pharmacia). Approximatethe molecular weights (kDa) are This may distribution,and shown on the left side ofrights the blot, which correspondowner to the markers loaded in lanes 1 and 12. subject be the violate copy of Lane Sample and Amount Amount may documentpublication, It exploitation Loaded (ng) Loaded (µl) 1 Precisionthis Plusany Dual Color molecular weight markers ⎯ ⎯ 2 E. coli-produced Cry1A.105permission standard 1 ⎯ 3 E. coli-produced Cry1A.105prohibited standard 2 ⎯ the 4 E. coli-commercialproduced Cry1A.105 be standard 4 ⎯ 5 E. coli-produced Cry1A.105 standard 6 ⎯ Furthermore,any 6Consequently, MON without89034-produced Cry1A.105 protein ⎯ 7.5 7 MON 89034-produced Cry1A.105 protein ⎯ 7.5 8 MON 89034-produced Cry1A.105 protein ⎯ 10 9 MON 89034-produced Cry1A.105 protein ⎯ 10 10 MON 89034-produced Cry1A.105 protein ⎯ 20 11 MON 89034-produced Cry1A.105 protein ⎯ 20 12 Precision Plus Dual Color molecular weight markers ⎯ ⎯

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1.2.3. N-terminus intactness of the Cry1A.105 protein The N-terminus of the plant-produced proteins could be blocked by chemical modifications, which would not allow N-terminal analysis by the typical Edman degradation method (Brown and Roberts, 1976; Arfin and Bradshaw, 1988). Therefore, the intactness of the N-terminus of the MON 89034-produced Cry1A.105 was examined using western blot analysis with an N-terminal peptide-specific antibody. The anti-N- terminal peptide antibody was raised against a synthetic peptide consisting of the first 14 amino acids (MDNNPNINECIPYN) at the N-terminus of the Cry1A.105 protein (see Figure IV.3).

The E. coli-produced Cry1A.105 containing the intact N-terminal sequence, and the and Cry1A.105 trypsin-resistant core lacking the N-terminus of theand full-length regime. protein were used as positive and negative reference standards,AG respectively. As expected, no immunoreactive bands were observed in the lanes loaded with Cry1A.105 trypsin- resistant core (Figure VI.4, lanes 6 and 7). The band corresponding to the full-length Bayer property protectionpublishing Cry1A.105 protein (approximately 130 ofkDa) was observed in the lanes loadedcontents with both the MON 89034- and E. coli-produced Cry1A.105 proteins. As expected,its the intensity of parties.data therefore the bands increased in a manner dependent of the loading quantiand/ororties. Additionally, a fragment of lower molecular weightaffiliates. (approximately 85 kDa) was observed in both property intellectualthird may protein preparations (Figure VI.4,its lanes 2-5). This fragment likely represents a as proteolytic product of C-terminthe ofal degradationand of Cry1A.105 protein. These results are is regulatory owner. consistent with published literature that the intermediatea document proteolytic fragments of Cry1 any such its proteins have intact N-termini, while the trypsin-resistantreproduction coredocument isof formed by the cleavage of the N-terminal peptide (approximatelownery 25-30 aminothis acids; Gao et al., 2006). under of rightsthe this documentand/or rights In conclusion, the intacttoness of of thefall N-terminususe ofof the MON 89034- and the E.coli- produced Cry1A.105 proteins was confirmed. the This may distribution,and rights owner 1.2.4. Cry1A.105subject tryptic peptide mapping by MALDI-TOF MS be the violate The MON 89034-produced,copy full-lengthof Cry1A.105 protein was further identified by tryptic peptide mappingdocument analysis using MALDand I-TOF MS. The protein sample was heat- may publication,exploitation denatured,It chemically reduced, alkylated and digested with trypsin, and the masses of the this tryptic peptides wereany measured. permission prohibited There were 52 protein peptide masses identified that matched the expected tryptic the peptides generatedcommercial in silico bebased on the predicted cleavage sites in the sequence. The identified masses were used to assemble a coverage map that displays those matched Furthermore,any peptideConsequently, sequenceswithout for the protein (Figure VI.5). Overall, the confirmed sequence accounts for 43.8% (516 out of 1,177 amino acids) of the full-length sequence of Cry1A.105 protein. In general, a protein identification made by peptide mapping is considered to be reliable if the measured coverage of the sequence is 15% or higher with a minimum of five matched peptides (Jensen et al., 1997). In the current case, the detected peptide coverage was 43.8% with 52 matched peptides, therefore, the protein identity of the MON 89034-produced Cry1A.105 was confirmed.

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kDa

Full-length Cry1A.105

Proteolytic fragment of Cry1A.105

and and regime. AG

Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its Figure VI.4. N-terminus Intactness byas Immunoblot Analysis Using Anti-N-terminal the of and Peptide isAntibody regulatory owner. a document any such its reproductiondocument Aliquots of the MON 89034–producedowner Cry1A.105 protein,this E. coli-of produced Cry1A.105 reference standard, and Cry1A.105 trypsin-resistantof core standard were separated by rights under this SDS-PAGE and electrotransferredand/or the to a PVDF membrane. The membrane was probed document to of fall of rights with the anti-N-terminal peptide antibody, anduse immunoreactive bands were visualized the using anThis ECL system (5 minmay exposure). distribution,and Approximate molecular weights (kDa) are rights owner shown on the leftsubject side of the blot and correspond to the markers loaded in lanes 1 and 8. be the violate copy of Lane Sample and Amount (ng) may documentpublication, It exploitation 1 this Precisionany Plus Dual Color molecular weight markers ⎯ 2 MON 89034-produced Cry1A.105 protein 20 permission 3 MON 89034-producedprohibited Cry1A.105 protein 40 4 E. colithe-produced Cry1A.105 reference standard 20 commercial be 5 E. coli-produced Cry1A.105 reference standard 40 Furthermore, 6 any Cry1A.105 trypsin-resistant core standard 20 Consequently, 7 without Cry1A.105 trypsin-resistant core standard 40 8 Precision Plus Dual Color molecular weight markers ⎯

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under of rightsthe this Figure VI.5. Sequenceand/or Coverage Map of the MON 89034-produced Cry1A.105 document to of fall of rights Protein by MALDI-TOF MSuse Analysis the This may distribution,and rights owner Shaded regions subjectcorrespond to 52 fragments of tryptic peptide masses that were identified from the full-lengthbe protein band. Overall,the 43.8% violate of the complete protein sequence was identified. copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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1.2.5. Lack of glycosylation for Cry1A.105 protein Some eukaryotic proteins are post-translationally modified by the addition of carbohydrate moieties (Rademacher et al., 1988). These carbohydrate moieties may be complex, branched polysaccharide structures or simple oligosaccharides to monosaccharides. In contrast, prokaryotic organisms such as non-virulent E. coli strains used for cloning and expression purposes lack the necessary biochemical synthetic capacity required for protein glycosylation. Therefore, determining whether the MON 89034-produced Cry1A.105 protein is equivalent to the E. coli-produced Cry1A.105 protein requires an investigation of its glycosylation status.

To assess whether potential post-translational glycosylation of the MON 89034-produced and Cry1A.105 protein occurred, the purified protein sample was andsubjected toregime. glycosylation analysis. The E. coli-produced Cry1A.105 referenceAG standard represented a negative control. The positive controls were the transferrin and horseradish peroxidase (HRP) proteins which are known to have multiple covalently-linked carbohydrate modifications. Bayer property protectionpublishing The transferrin protein and HRP, as wellof as the purified Cry1A.105 proteincontents isolated from MON 89034 and E. coli were separated on SDS-PAGE, and glycosylationits analysis was parties.data therefore performed to detect oxidized carbohydrate moieties on the protand/oreins.or The results of these analyses are shown in Figure VI.6. Theaffiliates. positiv e controls were detected at the expected property intellectualthird may molecular weights in a concentration-dependits ent manner (Figure VI.6, lanes 2-5). No as detectable signal was observedthe of for the MONand 89034-produced and E. coli-produced is regulatory owner. Cry1A.105 protein (Figure VI.6, lanes 6-9). a document any such its reproductiondocument of These results indicate that the MONowner 89034-produced thisprotein is not glycosylated and, thus under of is equivalent to the E. colirights-producedthe Cry1A.105 referencethis standard with respect to the documentand/or rights lack of glycosylation. to of fall use of the This may distribution,and 1.2.6. Cry1A.105 functionalrights activity owner subject The biological activities of E. coli- and MONthe 89034-producedviolate Cry1A.105 proteins were be copy estimated by determining EC50 valuesof in a corn earworm (CEW) diet-incorporation bioassay. The ECdocumentvalue is defined as theand level of Cry1A.105 protein in the diet that may 50 publication,exploitation resultsIt in 50% inhibition of larval growth. this any The EC50 values for each replicatepermission bioassay are summarized in Table VI.2, and the dose prohibited response relationships for MON 89034- and E. coli-produced Cry1A.105 are illustrated the be in Figure VI.7. commercialThe ranges of the estimated EC50 values overlapped for the protein from the two host sources. The EC values for the MON 89034-produced protein ranged from Furthermore,any 50 0.0055Consequently, to 0.0089without µ g Cry1A.105/ml diet and the EC50 values for the E. coli-produced protein ranged from 0.0053 to 0.0170 µg Cry1A.105/ml diet. Figure VI.7 shows an equivalent slope for the dose-response relationship for the E. coli- and MON 89034- produced Cry1A.105 proteins in the CEW bioassay, which demonstrates that the they have equivalent functional activities.

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

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

20 regime.and 18 and 15 AG 10

Bayer property protectionpublishing of contents Figure VI.6. Glycosylation Analysis of the MON 89034-produced Cry1A.105its parties.data therefore Protein and/oror affiliates. Aliquots of the MON 89034-prpropertyoduced Cry1A.105intellectualthird protein, E. coli-producedmay Cry1A.105 its reference standard (negative control),as horseradish peroxidase (positive control) and the of and transferrin (positive control)is were separated regulatoryby SDS-PAGE (4→20%owner. gradient) and a document electrotransferred to a PVDFany membrane.such Where present, periodate-oxidizedits protein- reproductiondocument bound carbohydrate moieties reactedowner with Pro-Q Emeraldthis 488of glycoprotein stain and emitted a fluorescent signal at 488 nm. The signalof was captured using a Bio-Rad rights under this Molecular Imager and/orFX. Approximatethe molecular weights (kDa) correspond to the document to of fall of rights CandyCane glycoprotein markers loaded in lausene 1 and the Precision Dual Color markers the in lane 10.This may distribution,and rights owner subject Lane be Sample the violate Amount (ng) copy of and 1may document CandyCanepublication, glycoprotein molecular weight standards ⎯ It exploitation 2 this Horseradish Peroxidase (positive control) 48 3 any Horseradish Peroxidase (positive control) 96 4 Transferrinpermission (positive control) 48 prohibited 5 Transferrinthe (positive control) 96 6 commercial MON 89034 be -produced Cry1A.105 48 7 MON 89034-produced Cry1A.105 96 Furthermore,any Consequently,8 without E. coli -produced Cry1A.105 (negative control) 48 9 E. coli-produced Cry1A.105 (negative control) 96 10 Precision Plus Dual Color molecular weight markers ⎯

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Table VI.2. EC50 Values of E. coli- and MON 89034-produced Cry1A.105 Proteins in a Corn Earworm Diet-incorporation Bioassay

1 EC50 (µg Cry1A.105/ml diet) E. coli-produced MON 89034-produced 1 0.0150 ± 0.0025 0.0055 ± 0.0014 Replicate2 2 0.0053 ± 0.0022 0.0089 ± 0.0018 3 0.0170 ± 0.0021 0.0077 ± 0.0012 and Overall 0.0120 ± 0.0062 0.0074 ± 0.0017and regime. AG 1 EC50 (mean ± standard error) represents the concentration needed to inhibit the growth of the target insect by 50%. Bayer property protectionpublishing 2 Each bioassay replicate consisted of a seriesof of five protein levels yielding acontents dose series its ranging from 0.00048 – 0.039 µg Cry1A.105 protein/mlparties. diet withdata a 3-fold separationtherefore factor between dose levels. Insect larvae were placed on the diets with and/or16 insectsor per treatment. affiliates. The combined weight of the survivingproperty insects intellectual at eachthird dose level was recordedmay at the end of the 6-7 day incubation period. EC its was calculated with SAS software. 50 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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250

200

150

100 E. coli Plant 50 and and regime. Mean Insect Mass (mg) 0 AG Controls 2 3 4 5 LogBayer Cry1A.105 property Dose Levelprotectionpublishing of (pg/mL diet) contents its parties.data therefore and/oror affiliates. may property intellectualthird its as Figure VI.7. Functional Equivalencethe of of theand MON 89034- and E. coli-produced is regulatory owner. Cry1A.105 Proteins Againsta Corn Earwormdocument (CEW) any such its reproductiondocument Bioassay results from three replicatesowner have been combinedthis for illustrationof purpose only and are shown on a log concentration underscale. Eachof data point represents the mean of the rightsthe this three bioassaysdocument alongand/or with the standard error of the mean.of rights Equivalent slope was seen for dose-response relationshipsto offor fallthe MONuse 89034-produced and E. coli-produced the Cry1A.105This protein in the CEWmay bioassay. distribution,and The dose response curve was modeled with rights owner SAS software. subjectThe data demonstrate that the test insect body weight decreases with the increase of the Cry1A.105 dose level, indicating the greater growth inhibitory effect on be the violate the test insects at copyhigher doses. of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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1.2.7. Conclusions for characterization of the Cry1A.105 protein The Cry1A.105 protein isolated from MON 89034 was purified and characterized, and results confirmed the equivalence of the MON 89034- and E. coli-produced Cry1A.105 proteins. SDS-PAGE demonstrated that the MON 89034-produced Cry1A.105 co- migrated with the E. coli-produced Cry1A.105 protein on the gel, indicating the protein from both sources have equivalent molecular weights. On the basis of western blot analysis with a polyclonal antibody against Cry1A.105, the electrophoretic mobility and immunoreactivity of the MON 89034-produced Cry1A.105 protein were shown to be equivalent to that of the E. coli-produced Cry1A.105 reference standard. Tryptic peptide mapping by MALDI-TOF MS yielded peptide masses consistent with the expected tryptic peptides generated in silico based on the predicted trypsin cleavage sites in the and Cry1A.105 sequences. In addition, the MON 89034- andand the E. regime.coli-produced Cry1A.105 proteins were found to be equivalentAG in terms of functional activity and the lack of glycosylation. Taken together, these data provide a detailed characterization of the Cry1A.105 protein isolated from MON 89034 and establish its equivalence to the E. Bayer property protectionpublishing coli-produced Cry1A.105 protein. of contents its parties.data therefore 2.1. Identity and function of the Cry2Ab2 protein and/oror affiliates. The Cry2Ab2 protein producedproperty in MON 89034intellectual thirdis derived from the Bt subspeciesmay kurstaki its and its amino acid sequence differs fromas that of the wild-type protein by a single amino the of and acid. The Cry2Ab2 proteinis has 88% amino acidregulatory sequence identity to theowner. Cry2Aa protein, a document which is present in commercany ialsuch microbial pest control productsits such as Dipel and reproductiondocument Crymax. The Cry2Ab2 proteins producedowner in MON 89034this and Bollgardof II cotton share an under of identical amino acid sequence.rights theBollgard II cotton has beenthis on the market since 2003 and documentand/or rights there have been no concernsto reofgarding fall its safetyuse to date.of the This may distribution,and Like the Cry2Ab2 proteinrights produced in the biotechnology-derivedowner cotton Bollgard II, the Cry2Ab2 proteinsubject produced in corn MON 89034 is a variant of the wild-type Cry2Ab2 protein frombe Bt. Accumulation of the Cry2Athe b2violate protein in MON 89034 is targeted to the copy of chloroplast using a chloroplast transit peptideand (CTP), which facilitates the intracellular transportmay of proteinsdocument frompublication, the cytoplasm to the plastids (Bruce, 2000). CTPs are typically It exploitation cleaved from thethis mature protein upon translocation into the chloroplast, and then rapidly degraded. To allowany targeting of the Cry2Ab2 protein to the plastids in MON 89034, the permission DNA sequence encoding the CTPprohibited region of corn ribulose 1,5-biosphosphate carboxylase small subunit was fusedthe to thebe cry2Ab2 coding sequence. commercial TheFurthermore, Cry2Ab2any protein expressed in cotton Bollgard II is also targeted to a plastid through a CTP.Consequently, Attemptswithout to determine the N-terminal sequence of the Cry2Ab2 protein in cotton Bollgard II (after the CTP was cleaved) indicate that the N-terminus was blocked, and therefore the exact excision site of the CTP is not known. Attempts to determine the N- terminal sequence of the full-length MON 89034-produced Cry2Ab2 also indicated that the protein was also blocked at the N-terminus, and as a consequence, the cleavage site in the CTP that is processed in the chloroplast could not be determined. The CTP used in MON 89034 has a potential cleavage site (methionine), located three amino acids upstream from the start of the Cry2Ab2 protein sequence (Figure VI.8). As such, the

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three additional amino acids from the CTP were included at the N-terminus for the E. coli-produced Cry2Ab2 protein used in the safety assessment studies for MON 89034 (Figure VI.8). This resulted in the production of a Cry2Ab2 protein of 637 amino acids (634 from Cry2Ab2 and 3 from the CTP) with a theoretical molecular weight of 71 kDa.

MON 89034 M-Q-A1-M-D2- N-S-V-L-N Recombinant E. coli M-Q-A1-M-D2- N-S-V-L-N B. thuringiensis -M- - N-S-V-L-N

regime.and 1 M-Q-A – predicted amino acids from chloroplast transitand peptides (CTP) AG 2 D – an additional amino acid included for the ease of cloning

Figure VI.8. Comparison of the PutativeBayer N-terminal property Sequences protection of thepublishing Cry2Ab2 Proteins Produced in MONof 89034, Recombinant E. colicontents, and Bacillus its thuringiensis parties.data therefore and/oror affiliates. property intellectualthird may its 2.2. Characterization of the Cry2Ab2as protein the of and is regulatory owner. The Cry2Ab2 protein was purified from the graia n of MONdocument 89034 corn as well as from an any such its reproductiondocument E. coli culture. A panel of analyticalowner tests was used tothis identify, characterizeof and compare the MON 89034- and E. coli-producedunder Cry2Ab2 of proteinsthis including: (1) western blot analysis; (2) SDS-PAGE;and/or rights (3) MALDI-TOFthe MS; (4) N-terminal sequence analysis with document fall of rights Edman degradation chemistry;to of (5 ) glycosylationuse analysis; and (6) insect activity bioassay. the This may distribution,and rights owner 2.2.1. Cry2Ab2subject protein molecular weight equivalence The equivalencebe in molecular weight ofthe the violatepurified MON 89034- and the E. coli- copy of produced Cry2Ab2 protein was demonstratedand using SDS-PAGE stained with Brilliant Blue G-Colloidalmay documentstain.publication, The MON 89034-produced, full-length Cry2Ab2 protein It exploitation migrated to a positionthis comparable to that of the E. coli-produced protein standard, which was analyzed concurrentlyany (Figure VI.9, band-1). Based on the comparable permission electrophoretic mobility, the MONprohibited 89034- and E. coli-produced, full-length Cry2Ab2 proteins were determinedthe to be have equivalent molecular weight. Band-2 observed below the full-length bandcommercial is a proteolytic fragment (see Section 2.2.2 below). Furthermore,any 2.2.2.Consequently, Cry2Ab2without protein immunoreactivity Western blot analysis using a polyclonal anti-Cry2Ab2 antibody was conducted to confirm the identity and determine the relative immunoreactivity of the MON 89034- produced Cry2Ab2 protein and the E. coli-produced Cry2Ab2 reference standard. Results indicated that the anti-Cry2Ab2 antibody recognized the MON 89034-produced Cry2Ab2 protein, which migrated comparably to the E. coli-produced reference standard protein (Figure VI.10, band-1). The immunoreactive signal increased with increasing levels of the Cry2Ab2 protein. Besides the expected band, an immunoreactive band with

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lower molecular weight (Figure VI.10, band-2) was observed in the partially purified MON 89034-produced Cry2Ab2 sample, which represents a proteolytic fragment of the Cry2Ab2 protein.

These western blot results confirm the identity of Cry2Ab2 protein produced by MON 89034, and demonstrate that MON 89034- and E. coli-produced Cry2Ab2 proteins have equivalent immunoreactivity with anti-Cry2Ab2 antibody.

and and regime. AG

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1 234 5678910 MWM (kDa) MWM (kDa)

200 200 116.3 116.3 97.4 97.4

66.2 66.2 Band-1

45 45 Band-2

31 31

regime.and 21.5 and 21.5 AG 14.4 14.4 6.5 6.5 Bayer property protectionpublishing of contents its parties.data therefore Figure VI.9. SDS-PAGE of the MON 89034- and E. coli-producedand/oror Cry2Ab2 affiliates. Proteins property intellectualthird may its Samples of the partially purified MONas 89034-produced Cry2Ab2 protein, and the E. coli- the of and produced Cry2Ab2 referenceis standard were separatedregulatory by a Tris-glycineowner. 4→ 20% SDS a document polyacrylamide gel and stainedany withsuch Brilliant Blue G-Colloidal stain.its Amounts loaded reproductiondocument correspond to total protein loadedowner per lane. Approximathis te molecularof weights (kDa) correspond to the markers loaded in Lanesunder 2 and 10.of rightsthe this documentand/or rights to of fall use of Lane Sample the Amount (µg) This may distribution,and rights owner 1 Empty Lane...... subject N/A 2 MWM (molecular weight markers,the Bio-Rad, violate broad range) ...... 4.5 be copy 3 E. coli-produced Cry2Ab2 referenceof standard...... 1 and 4 may MON 89034-produceddocumentpublication, Cry2Ab2 protein ...... 1 It exploitation 5 MON 89034-producedthis Cry2Ab2 protein ...... 2 6 MON 89034-producedany Cry2Ab2 protein ...... 2 permission 7 MON 89034-produced prohibitedCry2Ab2 protein ...... 3 8 MON 89034-produced Cry2Ab2 protein ...... 3 the be 9 MON 89034-producedcommercial Cry2Ab2 protein ...... 1 Furthermore, 10 MWMany (molecular weight markers, Bio-Rad, broad range) ...... 4.5 Consequently,without

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regime.and and AG

Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its Figure VI.10. Western Blot Analysisas of MON 89034- and E. coli-produced Cry2Ab2 the of and Proteinsis regulatory owner. a document Samples of the partially purifiedany such MON 89034- and E. coli-producedits Cry2Ab2 proteins reproductiondocument were separated by SDS-PAGE (4owner→20% gradient),this electrotransferredof to a PVDF membrane. The membrane was thenunder detected usingof goat anti-Cry2Ab2 antibody and rightsthe this developed usingdocument anand/or ECL system. Amounts loaded correspondrights to subject protein after to of fall use of normalization with purity. The approximate molecularthe weights (kDa) correspond to the markers Thisloaded in Lanes 2 andmay 10. distribution,and rights owner subject Amount of Lane Samplethe violate Cry2Ab2 (ng) be copy of and 1 may Empty Lane...... documentpublication, N/A It exploitation 2 Precisionthis Plus Dual Color molecular weight markers (MWM) ...... N/A 3 E. coli-producedany Cry2Ab2 protein ...... 20 permission 4 MON 89034-produced prohibitedCry2Ab2 protein ...... 20 5 MON 89034-produced Cry2Ab2 protein ...... 20 the be 6 MON 89034-producedcommercial Cry2Ab2 protein ...... 30 Furthermore, 7 MONany 89034-produced Cry2Ab2 protein ...... 30 8Consequently, MON 89034-producedwithout Cry2Ab2 protein ...... 40 9 MON 89034-produced Cry2Ab2 protein ...... 40 10 Precision Plus Dual Color molecular weight markers (MWM) ...... N/A

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2.2.3. Analysis of the N-terminal sequence N-terminal sequencing analysis demonstrated that the MON 89034-produced, full-length Cry2Ab2 (Figure VI.10, band-1) was blocked at the N-terminus with no definitive sequence obtained in the sequencing analysis using Edman degradation chemistry. A minor portion of the protein co-migrating with the full-length protein was proteolytically degraded and the sequence was determined to start from amino acid residue 24. N- terminal sequence analysis of the lower molecular weight proteolytic fragment (Figure VI.10, band-2) revealed that this its sequence starts from amino acid residue 145. With E. coli-produced Cry2Ab2 (band-1) the N-terminus sequence was determined as MQAMDN, as expected (Figure VI.8). This result further confirms that band-1 is the full-length Cry2Ab2 protein. and and regime. In summary, the N-terminal sequencing results indicateAG that MON 89034-produced, full- length Cry2Ab2 protein is blocked at its N-terminus, but the N-terminal sequencing result of the lower molecular weight proteolytic fragment (band-2) confirms the Cry2Ab2 Bayer property protectionpublishing identity. In addition, the N-terminus ofof E. coli-produced, full-length contentsCry2Ab2 was confirmed. its parties.data therefore and/oror 2.2.4. Cry2Ab2 tryptic peptide mappingaffiliates. by MALDI-TOF MS property intellectualthird may its The MON 89034-produced, full-lengthas Cry2Ab2 (band-1, in Figure VI.10) and the the of and proteolytic fragment (band-2,is in Figure VI.10)regulatory were char acterized owner.by tryptic peptide a document mapping analysis with MALDI-TOFany such MS to further confirm their identity.its For band-1, a reproductiondocument total of 32 observed peptide massesowner matched the theoreticalthis trypticof peptide masses of Cry2Ab2 protein (Appendix B). These identifieofd peptides were used to assemble a rights under this coverage map in theand/or Cry2Ab2 theprotein sequence (Figure VI.11). The overall peptide document to of fall of rights sequence coverage was 44.4% out of the 637use amino acid residues of the full-length the Cry2Ab2This protein. For band-2,may a distribution,total andof 24 observed peptide masses matched the rights owner expected trypticsubject peptide masses of Cry2Ab2 protein, which yielded a coverage map equal to 47.7% outbe of the 493 amino acid residuesthe of violatethis proteolytic fragment (Figure VI.12). copy of and In general,may a proteindocument identificationpublication, made by peptide mapping is considered to be reliable if theIt measured coverage ofexploitation the sequence is 15% or higher with a minimum of five this matched peptides (Jensenany et al., 1997). In the current case, the detected peptide coverage was 44.4% with 32 matchedpermission peptides for the full-length Cry2Ab2 protein, and 47.7% prohibited with 24 matched peptides for the proteolytic fragment of Cry2Ab2; therefore, the identity the of the MON 89034-producedcommercial be Cry2A b2 protein is confirmed. Furthermore,any Consequently,without

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1 MQAMDNSVLN SGRTTICDAY NVAAHDPFSF QHKSLDTVQK EWTEWKKNNH SLYLDPIVGT 61 VASFLLKKVG SLVGKRILSE LRNLIFPSGS TNLMQDILRE TEKFLNQRLN TDTLARVNAE 121 LTGLQANVEE FNRQVDNFLN PNRNAVPLSI TSSVNTMQQL FLNRLPQFQM QGYQLLLLPL 181 FAQAANLHLS FIRDVILNAD EWGISAATLR TYRDYLKNYT RDYSNYCINT YQSAFKGLNT 241 RLHDMLEFRT YMFLNVFEYV SIWSLFKYQS LLVSSGANLY ASGSGPQQTQ SFTSQDWPFL 301 YSLFQVNSNY VLNGFSGARL SNTFPNIVGL PGSTTTHALL AARVNYSGGI SSGDIGASPF 361 NQNFNCSTFL PPLLTPFVRS WLDSGSDREG VATVTNWQTE SFETTLGLRS GAFTARGNSN 421 YFPDYFIRNI SGVPLVVRNE DLRRPLHYNE IRNIASPSGT PGGARAYMVS VHNRKNNIHA 481 VHENGSMIHL APNDYTGFTI SPIHATQVNN QTRTFISEKF GNQGDSLRFE QNNTTARYTL 541 RGNGNSYNLY LRVSSIGNST IRVTINGRVY TATNVNTTTN NDGVNDNGAR FSDINIGNVV 601 ASSNSDVPLD INVTLNSGTQ FDLMNIMLVP TNISPLY

Figure VI.11. Peptide Sequence Coverage Map of the MON 89034-Producedand and regime. Cry2Ab2 Protein by MALDI-TOFAG MS Analysis

Shaded regions correspond to 32 fragments of tryptic peptide masses that were identified from the band-1 (Figure VI.10.) of the isBayerolated Cry2Ab2property protei protectionn frompublishing MON 89034. of contents MQA sequence (underlined) originates from the chloroplast transit peptideits (CTP). parties.data therefore and/oror affiliates. property intellectualthird may its the as of and owner. 145 AVPLSITSSV NTMQQLFLNRis LPQFQMQGYQ LLLLPLFAQAregulatory ANLHLSFIRD VILNADEWGI a document 205 SAATLRTYRD YLKNYTRDYSany NYCINTYQSAsuch FKGLNTRLHD MLEFRTYMFLits NVFEYVSIWS reproductiondocument 265 LFKYQSLLVS SGANLYASGS GPQQTQSFTSowner QDWPFLYSLFthis QVNSNYVLNGof FSGARLSNTF 325 PNIVGLPGST TTHALLAARV NYSGGISSGDunder IGASPFNQNFof NCSTFLPPLL TPFVRSWLDS 385 GSDREGVATV TNWQTESFETrights the TLGLRSGAFT ARGNSNYFPDthis YFIRNISGVP LVVRNEDLRR documentand/or rights 445 PLHYNEIRNI ASPSGTPGGAto of RAYMVSVHNRfall useKNNIHAVHEN of GSMIHLAPND YTGFTISPIH 505 ATQVNNQTRT FISEKFGNQG DSLRFEQNNT TARYTLRGNGthe NSYNLYLRVS SIGNSTIRVT 565 INGRVYTATNThis VNTTTNNDGVmay NDNGARFSDI distribution,and NIGNVVASSN SDVPLDINVT LNSGTQFDLM rights owner 625 NIMLVPTNISsubject PLY

be the violate Figure VI.12. Peptidecopy Sequence Coverageof Map of the MON 89034-derived Cry2Ab2 Proteolytic Fragmentand by MALDI-TOF MS Analysis may documentpublication, It exploitation Shaded regionsthis correspond any to 24 tryptic peptide masses that were identified from the band-2 proteolytic fragmentpermission of Cry2Ab2 (Figure VI.10) from MON 89034. The amino acid residue number was assignedprohibited based on the respective position in the full-length the sequence of Cry2Ab2commercial protein. be N-terminal sequencing result showed that the sequence of band-2 started from amino acid residue 145. Furthermore,any Consequently,without

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2.2.5. Lack of glycosylation of MON 89034-produced Cry2Ab2 Some eukaryotic proteins are post-translationally modified with carbohydrate moieties (Rademacher et al., 1988). These carbohydrate moieties may be complex, branched polysaccharide structures or simple oligosaccharides to monosaccharides. In contrast, prokaryotic organisms such as non-virulent E. coli strains used for cloning and expression purposes lack the necessary biochemical “machinery” required for protein glycosylation. Therefore, determining whether the MON 89034-produced Cry2Ab2 protein is equivalent to the E.coli-produced Cry2Ab2 protein requires an investigation of its glycosylation status.

To determine whether post-translational glycosylation of the MON 89034-produced and Cry2Ab2 protein occurred, the isolated MON 89034-producedand Cry2Ab2regime. protein was analyzed for the presence of covalently boundAG carbohydrate moieties. The E. coli- produced Cry2Ab2 reference standard (negative control) and transferrin (positive control) were analyzed concurrently with the MON 89034-produced Cry2Ab2 protein. Bayer property protectionpublishing of contents The result showed that positive glycoprotein transferrin was detected,its as expected, in a parties.data therefore concentration-dependent manner at loadings of 0.5 and 1.0 µg/laneand/oror (Figure VI.13, lanes 3-4). No detectable signal was observedaffiliates. for the MON 89034-produced and E. coli- property intellectualthird may produced Cry2Ab2 protein at the positionsits of the expected molecular weights between 50 as to 75 kDa (Figure VI.13, lanesthe 5-8).of and is regulatory owner. a documentits These results indicate thatany thesuch M ON 89034-producedreproduction Cry2Ab2 protein is not this documentof glycosylated and, thus is equivalentowner to the E. coli-produced Cry2Ab2 reference standard under of with respect to the lack of glycosylation.rightsthe this documentand/or of rights to of fall use 2.2.6. Functional activity of the Cry2Ab2 protein the This may distribution,and rights owner The functional subjectactivities of the MON 89034- and E. coli-produced Cry2Ab2 proteins were compared by determining EC50 valuesthe in violatea CEW diet-incorporation bioassay. The be copy EC50 value is defined as the level of ofCry2Ab2 protein in the diet that results in 50% and inhibitionmay to larval growth.documentpublication, It exploitation this The EC50 values forany each replicate bioassay are summarized in Table VI.3 and the dose response relationships for permissionthe Cry2Ab2 protein from the two sources are illustrated in prohibited Figure VI.14. The mean EC50 values for the E. coli- and the MON 89034-produced the be proteins were similarcommercial and estimated to be 0.16 µg Cry2Ab2/ml diet, with standard errors ofFurthermore, 0.04 andany 0.01 µg Cry2Ab2/ml diet, respectively. Figure VI.14 shows an equivalent slopeConsequently, for the dose-responswithout e relationship for the E. coli- and MON 89034-produced Cry2Ab2 protein in the CEW bioassay. These results clearly showed that the MON 89034- and E. coli-produced Cry2Ab2 proteins have equivalent functional activities.

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12 345678910 MWM (kDa) MWM (kDa)

250 250 150 150 100 100 75 75

50 50 37 37 25 25

20 20 regime.and and 15 AG 15 10 10

Bayer property protectionpublishing of contents its Figure VI.13. Glycosylation Analysis of the MON-89034-parties.data and E.coli-producedtherefore Cry2Ab2 Proteins and/oror affiliates. property intellectualthird may its Samples of the MON 89034-producedas Cry2Ab2 protein, E. coli-produced Cry2Ab2 the of and reference standard (negativeis control) and transferrinregulatory (positive control)owner. were separated by a document a Tris-glycine 4→20% SDSany polyacrylamidesuch gel, and electrotransferredits to PVDF reproductiondocument membrane. Where present, protein-boundowner carbohydratethis moietiesof were labeled with biotin, and detected with streptavidinunder -horseradishof peroxidase and enhanced rightsthe this chemiluminescence.documentand/or Amount refers to total protein loadedrights per lane, except for the E. coli to of fall use of and the MON 89034-produced proteins whose concentrationsthe were normalized based on Cry2Ab2This purity. may distribution,and rights owner subject be the violate Lane copySample of Amount (µg) and may documentpublication, 1 It Empty Lane...... exploitation N/A 2 MWMthis (molecular any weight markers, Precision Plus Dual Color)...... N/A 3 Transferrin (positive control)1 ...... 0.5 permission 4 Transferrin (positive control)prohibited1 ...... 1 the 5 E. coli-producedcommercial Cry2Ab2 be protein (negative control)...... 0.5 6 E. coli-produced Cry2Ab2 protein (negative control)...... 1 Furthermore, 7 MONany 89034-produced Cry2Ab2 protein ...... 0.5 8Consequently, MON 89034-producedwithout Cry2Ab2 protein ...... 1 9 Empty Lane...... N/A 10 Empty Lane...... N/A

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

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Table VI.3. EC50 Values of the MON 89034- and E.coli-produced Cry2Ab2 Proteins in a Corn Earworm Diet-incorporation Bioassay

1 EC50 (µg Cry2Ab2/ml diet) E. coli-produced MON 89034-produced 1 0.13 ± 0.03 0.17 ± 0.03 Replicate2 2 0.16 ± 0.02 0.16 ± 0.03 3 0.20 ± 0.02 0.16 ± 0.02

Overall 0.16 ± 0.04 0.16 ± 0.01 and and regime. AG 1 EC50 (mean ± standard error) represents the concentration needed to inhibit the growth of the target insect by 50%. Bayer property protectionpublishing 2 Each bioassay replicate for the E. coli-producedof and MON 89034-produced Cry2Ab2contents proteins its consisted of a series of seven dilutions yielding a doseparties. seriesdata with a 2-fold separationtherefore factor ranging from 0.016 – 1.0 µg Cry2Ab2 protein/ml diet. Insect larvaeand/or wereor placed on the diets affiliates. with 16 insects per treatment. Theproperty combined intellectual weithirdght of the surviving insectsmay at each dose level was recorded at the end of the 7its day incubation period. EC50 was calculated with SAS as software. 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 FDA BNF No. 00105 / Monsanto 06-CR-161F Page 121 of 323

200

150

100 and and regime. E. coli AG 50 Plant Bayer property protectionpublishing of contents

Mean Insect Mass (mg) its 0 parties.data therefore and/oror Controls 1 affiliates. 2 3 4 property intellectualthird may its Log Cry2Ab2as Dose Level (ng/mL diet) the of and is regulatory owner. a document Figure VI.14. Functional anyEqui valencesuch of the MON 89034- and E.its coli -produced reproductiondocument Cry2Ab2 Proteins Againstowner Corn Earwormthis (CEW)of under of Bioassay results from threerights replicatesthe have been combinedthis for illustration purpose only documentand/or rights and are shown on a log toconcentration of fall scale. useEach dataof point represents the mean of the three bioassays along with the standard error of the mean.the Equivalent slope was seen for This may distribution,and dose-response relationshipsrights for the E. coli- andowner MON 89034-produced Cry2Ab2 protein in the CEW bioassay.subject The dose response curve was modeled with SAS software. The data demonstratebe that the test insect bodythe weviolateight decreases with the increase of the copy of Cry2Ab2 dose level, indicating the greaterand growth inhibitory effect on the test insects at higher doses.may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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2.2.7. Conclusions of the characterization of the Cry2Ab2 protein The Cry2Ab2 protein from MON 89034 was purified and characterized, and the results confirmed the equivalence between MON 89034- and E. coli-produced Cry2Ab2 proteins. The apparent molecular weight was estimated by SDS-PAGE. Since the MON 89034-derived Cry2Ab2 migrated comparably to the E. coli-produced protein on SDS- PAGE, the apparent molecular weight of the Cry2Ab2 protein from both MON 89034 and E. coli was determined to be equivalent. On the basis of western blot analysis, the electrophoretic mobility and immunoreactive properties of the MON 89034-produced Cry2Ab2 protein were demonstrated to be comparable to those of the E. coli-produced Cry2Ab2 reference standard. The N-terminus of the Cry2Ab2 from MON 89034 was blocked but the peptide mapping analysis by MALDI-TOF MS yielded the expected and peptide masses based on the trypsin cleavage sites in the aminoand acid sequenceregime. of the Cry2Ab2 protein, thereby confirming its identity.AG In addition, the MON 89034- and the E. coli-produced Cry2Ab2 reference standard were found to be equivalent based on functional activities and the lack of glycosylation. Taken together, these data provide a Bayer property protectionpublishing detailed characterization of the Cry2Ab2of protein isolated from MONcontents 89034 and established its equivalence to the E. coli-produced Cry2Ab2 protein. its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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SECTION 2. Levels of the Cry1A.105 and Cry2Ab2 Proteins Produced in MON 89034

The levels of the Cry1A.105 and Cry2Ab2 proteins in various tissues of MON 89034 that are relevant to the risk assessment were assessed by validated enzyme-linked immunosorbent assay (ELISA). Tissue samples for analysis were collected from five field trials conducted in the U.S. during 2005. The trial locations were in the states of Iowa, Illinois, Ohio, and Nebraska, which represent the major corn-growing region of the U.S. and provide a range of environmental conditions that would be encountered in the commercial production of corn. At each site, three replicated plots of MON 89034 and a conventional control hybrid were planted using a randomized complete block field design. Overseason leaf, overseason whole plant, overseason root, pollen, silk,and forage, and regime. forage root, grain, stover, and senescent root tissuesAG were collected from each replicated plot at all field sites. A description of the tissues collected is provided below.

Leaf. The youngest immature whorl leafBayer was collectedproperty from protection15 plantspublishing in each plot at of contents each site and combined to form the leaf sample. Overseason leaf samplesits were collected parties.data therefore as follows: and/oror affiliates. property intellectualthird may Overseason leaf (OSL) Corn developmeits nt stage Days after planting (DAP) OSL-1 theV2-V4 as 21-29 of and owner. OSL-2 is V6-V8 regulatory 28-43 a documentits OSL-3 anyV10-V12 such 41-53 reproductiondocument of OSL-4 pre-VT owner(pre-tasseling) this 56-68 under of rightsthe this documentand/or rights Whole plant. The aerialto portionof offall the plantuse without of the root was collected from four plants in each plot at each site at the V2-V4 stage and thecombined to form the whole plant This may distribution,and sample. Two plants wererights collected and combinedowner to form the whole plant samples for the later growth stages.subject Overseason whole plant samples were collected as follows: be the violate copy of Overseason whole plant (OSWP) Cornand development stage DAP may documentpublication, OSWP-1It exploitation V2-V4 21-29 OSWP-2 V6-V8 28-43 this any OSWP-3 V10-V12 41-53 permission OSWP-4 prohibited pre-VT (pre-tasseling) 56-68 the commercial be Root. Roots remaining after collection of whole plants from each plot were combined to formFurthermore, the rootany sample. Overseason root samples were collected as follows. Consequently,without Overseason root (OSR) Corn development stage DAP OSR-1 V2-V4 21-29 OSR-2 V6-V8 28-43 OSR-3 V10-V12 41-53 OSR-4 pre-VT (pre-tasseling) 56-68 Forage root early dent stage (R4-R6) 100-120 Senescent root after harvest 130-160

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Pollen. Approximately 10 ml of pollen was collected from multiple tassels in each plot at each site at pollination, approximately 60-74 days after planting.

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

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

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

Stover. Following harvest, approximatelyBayer 130-160 dapropertyys after planting,protectionpublishing two whole plants in each plot at each site were cut aboveof th e soil surface and combined to contentsform the stover its sample. parties.data therefore and/oror affiliates. All tissue samples, except grain,property were storintellectualedthird and shipped on dry mayice to Monsanto’s its facility for processing and analysis. Grainas was stored and shipped at room temperature. the of and All tissue samples wereis stored in a –80ºC freezerregulatory upon receipt. Tissueowner. samples were a document extracted and analyzed byany ELISA such according to applicable Monsantoits SOPs (standard reproductiondocument operating procedures). Further detailsowner rega rding samplethis collectionof and ELISA analysis are provided in Appendix E. under of rightsthe this documentand/or rights to of fall use of The results obtained from ELISA analysis are summarizedthe in Table VI.4 for the various tissue typesThis and in Tables VI.5may and VI.6distribution, andfor the tissues collected throughout the growing rights owner season. Cry1A.105subject and Cry2Ab2 proteins levels are provided in µg/g fresh weight tissue (fwt) and µg/g dry weight tissue (dwt).the violate be copy of and The levelsmay of the Cry1A.105documentpublication, and Cry2Ab2 proteins estimated in tissues of MON 89034 It exploitation showed trends thisthat were consistent for exposure calculations and intended uses. The mean Cry1A.105 levelsany across sites were highest in young leaf (520 µg/g dwt), followed permission by stover (50 µg/g dwt), forageprohibited (42 µg/g dwt), silk (26 µg/g dwt), pollen (12 µg/g dwt), forage root (12 µg/g dwt), senescent root (11 µg/g dwt), and grain (5.9 µg/g dwt). The the be mean Cry2Ab2 levelscommercial across sites were highest in young leaf (180 µg/g dwt), followed by silkFurthermore, (71 µg/gany dwt), stover (62 µg/g dwt), forage (38 µg/g dwt), senescent root (26 µg/g dwt),Consequently, forage rootwithout (21 µg/g dwt), and grain (1.3 µg/g dwt). In general, the levels of the two Cry proteins declined over the growing season.

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Table VI.4. Cry1A.105 and Cry2Ab2 Protein Levels in MON 89034 Tissues

Tissue Growth Cry1A.1052 Cry2Ab22 Type Stage Mean (SD) Mean (SD) [Range], n=15 [Range], n=15 µg/g fwt µg/g dwt µg/g fwt µg/g dwt

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

Pollen R1 6.4 (1.5) 12 (1.7) 0.34 (0.084) 0.64and (0.091) 3.8 – 8.8 8.5 - 16 and0.21 – 0.47regime. 0.49 - 0.79 AG Silk R1 3.0 (0.57) 26 (3.9) 8.2 (3.6) 71 (35) 2.0 – 3.8Bayer 20property - 31 protection3.3publishing - 16 33 - 160 of contents its Forage R4-R6 14 (3.6) 42parties. (9.4)data 12 (4.0) therefore38 (14) (early dent) 8.3 - 24 20 - 56 and/or6.5or - 18 15 - 55 affiliates. property intellectualthird may its Forage root R4-R6 2.2 (0.35)as 12 (3.1) 4.1 (1.4) 21 (5.9) the of and (early dent)is 1.3 – 2.7 regulatory6.2 - 16 2.2 owner.– 6.5 14 - 33 a document any such its reproductiondocument Grain R6 5.1owner (0.67) 5.9this (0.77) of1.1 (0.31) 1.3 (0.36) under of rightsthe 4.1 – 6.0 4.7 -this 7.0 0.67 – 1.8 0.77 – 2.1 documentand/or rights to of fall use of Stover R6 17 (4.4) 50 the(17) 22 (3.6) 62 (15) This may distribution,and (after harvest)rights 9.5 - 26 owner26 - 85 17 - 29 46 - 97 subject Senescent be R6 2.2 (0.36)the violate11 (1.4) 5.3 (2.0) 26 (8.8) copy of root (after harvest) 1.7 – 3.1and 9.4 - 15 2.4 – 9.1 13 - 43 may document 1 publication,exploitation YoungIt leaf = overseason leaf 1 (OSL-1); n = number of samples; SD = standard deviation; fwt this = fresh weight tissue;any dwt = dry weight tissue. 2 Limits of detection (LOD) permissionand limits of quantitation (LOQ) in the various tissues were as follows: prohibited the commercial be Cry1A.105 (µg/g fwt) Cry2Ab2 (µg/g fwt) Furthermore,Tissueany LOD LOQ LOD LOQ Consequently,Forage without 0.372 0.44 0.191 0.44 Leaf 0.568 0.66 0.081 0.44 Pollen 0.412 1.1 0.055 0.11 Root 0.254 0.33 0.056 0.22 Silk 0.275 0.44 0.040 0.22 Grain 0.262 1.1 0.123 0.22

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

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Table VI.5. Cry1A.105 Proteins Levels in Overseason Tissues of MON 89034

Over- Plant growth stages1 Season V2-V4 V6-V8 V10-V12 pre-VT R4-R6and R6 Tissue and regime. (21-29 DAP) (28-43 DAP) (41-53 DAP) AG (56-68 DAP) (100-120 DAP) (130-160 DAP) (n = 15) 2 Units µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g dwt fwt dwt fwt dwt fwt dwt fwt dwt fwt dwt fwt Bayer property protectionpublishing Tissues of contents its parties.data therefore Leaf Mean (SD) 520 (130) 85 (21) 140 (36) 28 (8.7) 72 (14) 16 (4.3) 120 (77) and/or 30 (20)or N/A N/A N/A N/A Range 380-850 56-130 80-200 12-45 47-89affiliates. 9.4-24 27-240 6.3-59 N/A N/A N/A N/A property intellectualthird may its as Whole Mean (SD) 380 (90) 40 (5.7) 260 (52) 24 (3.7)the of 100 (26) 11and (2.4) 120 (29) 17 (3.7) 42 (9.4) 14 (3.6) 50 (17) 17 (4.4) is regulatory owner. plant Range 230-570 30-52 170-350 16-31 58-160 7.0-a 15 58-170document 9.3-22 20-56 8.3-24 26-85 9.5-26 any such its reproductiondocument of Root Mean (SD) 79 (17) 8.9 (1.3) 48 (11) 5.8 (1.6) 45 (10)owner 6.4 (1.8) this 42 (10) 6.7 (0.63) 12 (3.1) 2.2 (0.35) 11 (14) 2.2 (0.36) under of Range 52-110 7.3-12 30-63 3.0-8.5rights the 26-64 4.4-10 30-63this 5.6-8.1 6.2-16 1.3-2.7 9.4-15 1.7-3.1 and/or document to of fall of rights 1 use Growth stages V2-V4, V6-V8, V10-V12 and pre-VT (tasseling) correspond to overseason tissuethe samp les –1, -2, -3 and –4, respectively (e.g., OSWP-1, etc.). The whole plant and root samples collectedThis at the R4-R6 (early dent)may and distribution, R6 (afterand harv est) stages correspond to forage plant/root and stover/senescent root, rights owner respectively. N/A - not applicable; VT-vegetativesubject stage at tasseling; DAP - days after planting; dwt - dry weight tissue; fwt - fresh weight tissue. 2 The LODs and LOQs for Cry1A.105 can be found in Table VI.4. SD = standard deviation. 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 VI.6. Cry2Ab2 Proteins Levels in Overseason Tissues of MON 89034

1 Plant growth stages and and regime. Over- V2-V4 V6-V8 V10-V12 AG pre-VT R4-R6 R6 Season (21-29 DAP) (28-43 DAP) (41-53 DAP) (56-68 DAP) (100-120 DAP) (130-160 DAP) Tissue Units2 µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g µg/g Bayer property protectionpublishing (n = 15) dwt fwt dwt fwt dwt of fwt dwt fwt dwtcontents fwt dwt fwt its parties. data therefore Leaf Mean (SD) 180 (59) 29 (6.8) 170 (34) 32 (5.3) 130 (34) 29 (5.4) 160 (44) and/or 37 (12)or N/A N/A N/A N/A affiliates. Range 94-270 19-43 110-230 23-44 property85-200 intellectual23-41third 48-210 11-56 may N/A N/A N/A N/A its as Whole Mean (SD) 130 (51) 13 (4.6) 79 (18) 7.5 (1.8)the of 40 (9.9) 4.2and (0.94) 39 (16) 5.9 (2.6) 38 (14) 12 (4.0) 62 (15) 22 (3.6) is regulatory owner. plant Range 52-230 5.2-21 45-110 4.0-9.7 22-61 2.4-5.8a 5.0-67document 0.7-11 15-55 6.5-18 46-97 17-29 any such its reproduction document owner this of Root Mean (SD) 56 (17) 6.4 (1.6) 58 (18) 7.6 (4.2) 35 (17) 5.0 (7.7) 26 (5.9) 4.2 (1.2) 21 (5.9) 4.1 (1.4) 26 (8.8) 5.3 (2.0) under of this Range 33-100 4.4-10 25-86 and/or2.5-15rights the 15-74 2.2-12 15-45 3.2-7.6 14-33 2.2-6.5 13-43 2.4-9.1 document rights to of fall use of 1 Growth stages V2-V4, V6-V8, V10-V12 and pre-VT (tasseling) correspond to overseason tissuethe samp les –1, -2, -3 and –4, respectively (e.g., OSWP-1, etc.). This may distribution,and The whole plant and root samples collected at the R4-R6rights (early dent) and R6 (afterowner harvest) stages correspond to forage plant/root and stover/senescent root, respectively. N/A - not applicable; VT-vegetativesubject stage at tasseling; DAP - days after planting; dwt - dry weight tissue; fwt - fresh weight tissue. 2 The LODs and LOQs for Cry1A.105 can be foundbe in TableVI.4. SD = standard thedeviation. 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. Dietary Exposure Assessment

A dietary safety assessment was conducted to assess the potential risks to humans and animals from dietary exposure to the Cry1A.105 and Cry2Ab2 proteins from the consumption of foods and feeds derived from MON 89034. Potential risks were evaluated by calculating a margin of exposure (MOE), which was defined as the ratio of the no observed effect level (NOEL) from the acute mouse gavage study to the estimates of the dietary intake of the respective Cry protein. The MOEs for the overall U.S. population were greater than or equal to 199,000 and 981,000 for the Cry1A.105 and Cry2Ab2 proteins, respectively. For children aged 3-5 years old, the age group with the highest corn consumption (body weight basis), the MOEs were greater than or equal to 79,400 and 390,000 for the Cry1A.105 and Cry2Ab2 proteins, respectively. Forand poultry and regime. and livestock, the MOEs ranged between 1,930AG – 13,500 and 2,160 – 47,600 for the Cry1A.105 and Cry2Ab2 proteins, respectively.

Bayer property protectionpublishing These large MOEs indicate that there areof no meaningful risks to human orcontents animal health from dietary exposure to the Cry1A.105 and Cry2Ab2 proteins. Detailsits are provided parties.data therefore below. and/oror affiliates. property intellectualthird may 3.1. Human dietary safety assessmentits as the of and is regulatory owner. An acute dietary safety assessment was conduca ted to assessdocument the potentialits human health risks from dietary exposureany to suchCry1A. 105 and reproductionCry2Ab2 from consumption of MON this documentof 89034. Details are discussed belowowner and the results of this assessment are summarized in under of Table VI.7. rightsthe this documentand/or rights to of fall use of 3.1.1. Risk assessment of the Cry1A.105 and Cry2Ab2the proteins to mammals This may distribution,and rights owner Cry proteins havesubject been used as components of topical Bt microbial pesticides for over 45 years. Numerousbe animal safety studies havethe demonstratedviolate that Bt microbial insecticides containing mixturescopy of Cry proteins areof non-toxic when fed to mammals at high does (Betz et al., 2000; EPA,document 1988, 2000). Collectiveland y, these studies demonstrate the absence may publication,exploitation of acute,It subchronic, and chronic oral toxicity in mice, rats and humans (Betz et al., this 2000). As discussedany previously (Part VI, Section 1), the Cry1A.105 and Cry2Ab2 proteins are structurally andpermission functionally related to Cry proteins that have a history of prohibited safe use as the active ingredients either in Bt microbial pesticides and/or in the biotechnology-derivedcommercial food andbe feed crops.

Furthermore,any MostConsequently, known proteinwithout toxins, including the insecticidal Cry proteins, act through acute mechanisms to exert toxicity (Sjoblad et al., 1992; Pariza and Johnson, 2001; Hammond and Fuchs, 1998). The primary exceptions to this rule consist of certain anti-nutritional proteins such as lectins and protease inhibitors, which typically require a short-term (2-4 week) feeding study to manifest toxicity (Leiner, 1994). The amino acid sequences of the Cry1A.105 and Cry2Ab2 proteins produced in MON 89034 are not similar to any of these anti-nutritional proteins or to any other known protein toxin (see Part VI, Sections 4 and 5). In addition, since Cry proteins act through acute mechanisms to control insect pests, and have no activity against non-target organisms such as mammals, the U.S. EPA

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has determined that a high dose acute test is sufficient to confirm their absence of toxicity towards mammals (McClintock et al., 1995). Therefore, an acute oral mouse toxicity study was considered appropriate and adequate to confirm the lack of mammalian toxicity of the Cry1A.105 and Cry2Ab2 proteins. Results from these tests showed that no adverse effects were observed, and that the NOEL for Cry1A.105 was equal to or greater than 2072 mg/kg bw, the highest dose tested. For Cry2Ab2, the NOEL was equal to or greater than 2198 mg/kg bw, the highest dose tested (Part VI, Section 5).

The potential for synergistic interaction between the Cry1A.105 and Cry2Ab2 proteins was also considered for human dietary safety assessment. Synergism is generally defined as an interaction that occurs when the combined effect of two substances is much greater than the sum of the effects of each substance administered alone. In order for the two regime.and Cry proteins to act synergistically, they must be capable of andproduc ing toxicity in the AG species of interest. The mode of action for Cry proteins involves binding of the Cry protein to Cry-specific receptors in the intestinal tissues of susceptible insect species. However, Cry proteins do not show bindingBayer to theproperty intestinal protection tissuespublishing of mammalian species such as mouse, rat, monkey andof hu man (Hoffmann et al., 1988; Noteborncontents et al., its 1993). This indicates that the mode of action by whichparties. Crydata proteins producetherefore toxicity in susceptible insects would be inoperative in humans and otherand/or mammalianor species, and affiliates. that no toxic response let aloneproperty a synergis intellectualticthird interaction would occur.may Furthermore, its studies have shown that the Cry1A.105as and Cry2Ab2 proteins do not act synergistically the of and even in target species is(e.g ., lepidopteran) thatregulatory have specific receptorsowner. and are thus a document susceptible to the toxic effectany s suchof Cry proteins (see Part VI, Sectionits 5.1, for further reproductiondocument discussion). Therefore it is reasonableowner to concludethis that the Cry1A.105of and Cry2Ab2 proteins would not have synergistic activityunder in mammalsof and it is appropriate to evaluate rightsthe this the toxicity ofdocument the twoand/or proteins separately. rights to of fall use of the 3.1.2. CornThis consumption may distribution,and rights owner The amount of corn-derivedsubject food consumed by humans in the U.S. that could potentially be the violate contain the Cry1A.105copy and Cry2Ab2 proteinsof from MON 89034 was estimated using the Dietary Exposure Evaluation Model (DEEM-FCIand D version 2.03, Exponent Inc.) and food may documentpublication, consumptionIt data from the exploitation1994-1996 and 1998 USDA Continuing Surveys of Food Intakes by Individualsthis any (CSFII). MON 89034 is intended for use in field corn, sweet corn and popcorn. Therefore, all three commodities were included in this assessment. permission DEEM-FCID separates field cornprohibited into six fractions: flour, meal, bran, starch, oil and the syrup. However,commercial corn oil and be corn syrup were excluded from this analysis since these food items are essentially devoid of protein. The remaining corn-derived food items were consideredFurthermore, potentialany sources of the two Cry proteins, although, because of the very low proteinConsequently, content,without any contribution from corn starch is expected to be minimal. No adjustments were made for anticipated market share; i.e., for the purposes of this assessment, all corn-derived food products consumed were assumed to be derived from MON 89034. This is a very conservative assumption since both field corn and popcorn are blended commodities. Thus, MON 89034 field corn would be mixed with other field corn, and MON 89034 popcorn would be mixed with other popcorn, before these commodities are processed and consumed.

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According to the DEEM-FCID analysis, 95% of the overall U.S. population consumes no more than 2.04 g/kg bw of corn (flour, meal, bran, starch, sweet corn and popcorn) on any one day. The highest value was for children 3-5 years of age, for whom the 95th percentile estimate of consumption was 5.12 g/kg bw.

3.1.3. Intake of the Cry1A.105 and Cry2Ab2 proteins For the purposes of this assessment, the concentration of the Cry1A.105 and Cry2Ab2 proteins in all consumed corn products was assumed to be equal to the mean level of the protein in the whole grain [5.1 µg/g fwt for Cry1A.105 and 1.1 µg/g fwt for Cry2Ab2; from Part VI, Section 2]. The dietary intake of each protein from consumption of MON 89034 can then be estimated by multiplying these levels by the estimates of corn and consumption. These estimates conservatively assume thatand there is noregime. loss of the Cry1A.105 and Cry2Ab2 proteins during storage,AG processing and/or cooking of the grain or food items. Utilizing these assumptions, the 95th percentile estimates for acute dietary intake of Cry1A.105 are 10.4 and 26.1 µg/kg bw for the overall U.S. population and Bayer property thprotectionpublishing children aged 3-5 years, respectively. of For Cry2Ab2, the 95 percentilecontents estimates for acute dietary intake are 2.24 and 5.63 µg/kg bw for the overall U.S.its population and parties.data therefore children aged 3-5 years, respectively. and/oror affiliates. property intellectualthird may 3.1.4. Margins of exposure its as the of and As discussed above, nois adverse health effectsregulatory were observed owner.when mice were a document administered a total of 2072any mg/kg such bw of the Cry1A.105 protein itsor 2198 mg/kg bw of reproductiondocument the Cry2Ab2 protein in one day. ownerTherefore, potentialthis health risksof from acute dietary intake of these proteins from consumptionunder of MONof 89034this were evaluated by calculating rightsthe th the MOEs baseddocument onand/or the acute mouse oral NOELs and therights 95 percentile estimates of to of fall use of acute dietary exposure. Utilizing these values, the MOEs for Cry1A.105 were the determinedThis to be approximatelymay greater distribution, andthan or equal to 199,000 for the overall U.S. rights owner population and subjectgreater than or equal to 79,400 for children aged 3-5 years old, an age group with thebe highest corn consumption the(body violate weight basis). The corresponding MOEs for Cry2Ab2 werecopy approximately greaterof than or equal to 981,000 and 390,000, and respectively.may Thedocument verypublication, large MOEs for both proteins indicate that there are no It exploitation meaningful risksthis to human health from dietary exposure to either Cry1A.105 or Cry2Ab2 from consumption ofany MON 89034. permission prohibited the commercial be Furthermore,any Consequently,without

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Table VI.7. Acute Dietary Intake and Margin of Exposure (MOE) for the Cry1A.105 and Cry2Ab2 Proteins from Human Consumption of MON 89034 in the U.S.

Corn Acute Dietary Intake MOEc Population Consumption (mg/kg bw x 10-3)b (g/kg bw)a Cry1A.105 Cry2Ab2 Cry1A.105 Cry2Ab2 Overall U.S. 2.04 10.4 2.24 ≥ 199,000 ≥ 981,000 population Children 5.12 26.1 5.63 ≥ 79,400 ≥ 390,000 aged 3-5 y regime.and a th and 95 percentile estimate by DEEM-FCID of acute consumptionAG of flour, meal, bran, starch, sweet corn and popcorn. b Assuming mean levels of Cry1A.105 and Cry2Ab2 in corn grain of 5.1 and 1.1 µg/g fw, respectively. Bayer property protectionpublishing c MOE = NOEL/Acute Dietary Intake; valuesof rounded to three significant figures.contents Based on its NOELs of 2072 and 2198 mg/kg for Cry1A.105 and Cry2Ab2,parties.data respectively, in thetherefore acute mouse gavage studies. and/oror affiliates. property intellectualthird may its as 3.2. Animal dietary safetythe assessment of and is regulatory owner. a document any such its Since corn is a major ingredient in the diets ofreproduction poultry anddocument livestock,of a dietary safety assessment was conducted for the Cry1A.105owner and Cry2Ab2this proteins produced in MON under of 89034. The quantity of cornrights consumedthe on a daily basisthis by poultry and livestock, as well documentand/or rights as the levels of Cry1A.105to ofand Cry2Ab2fall proteiuse nsof in corn, is necessary to derive an estimate of daily dietary intake. Livestock may consumethe both corn grain and forage (as This may distribution,and corn silage). Daily dietaryrights intake is computedowner as follows: subject be the violate Daily dietary intakecopy = daily corn consumpof tion (g) x Cry1A.105 or Cry2Ab2 protein level [µg/g dry weight (dwt)]1 and may documentpublication, It exploitation A MOE, definedthis as anythe ratio of the NOEL derived from toxicology tests to the estimate of the daily dietary intake, can then be calculated to provide a measure of potential risks to permission animals from dietary exposure toprohibited the Cry1A.105 and Cry2Ab2 proteins in MON 89034. the commercial be The exposure calculations make the conservative assumption that there is no loss of the Cry1A.105Furthermore, anyand Cry2Ab2 proteins during the processing of corn grain or forage into animalConsequently, feed. Itwithout also assumes that 100% of the corn grain or forage used in animal feed is derived from the MON 89034. This would be the case if a farmer were to feed all

1 In contrast to the human dietary safety assessment, corn consumption and protein levels are expressed on a 100% dry weight basis for the dietary safety assessment in animals. This is the convention of choice for animal nutritionists when there is variability in dry matter content among dietary ingredients.

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harvested corn to livestock on farm. However, larger livestock operations purchase commodity corn that is a blend of many different hybrids.

3.2.1. Corn consumption The daily U.S. consumption of corn grain is ~ 36 g/kg bw/day (assuming 60% dietary inclusion rate) for the young pig and ~ 22 g/kg bw/day for the finishing pig (assuming 80% dietary inclusion rate) (NRC, 1998). The four-week old broiler consumes ~51 g/kg bw/day of corn grain when the inclusion rate of corn is 60% of the diet (NRC, 1994). The lactating dairy cow consumes about 7.7 g/kg bw/day of corn grain and about 18.2 g/kg bw/day of forage (as corn silage), respectively (Ouellet et al., 2003).

and 3.2.2. Dietary intake of the Cry1A.105 and Cry2Ab2 proteinsand regime. The dietary intakes of the Cry1A.105 and Cry2Ab2AG proteins for the broiler chicken, young pig, and finishing pig were estimated using the daily corn grain consumption and the highest levels of the Cry1A.105 (7.0 µBayerg/g dwt) andproperty Cry2Ab2 protection (2.1publishing µg/g dwt) proteins in grain (Table VI.4). The highest expressionof levels were used since therecontents are situations its where farmers grow corn for feeding their livestock parties.directlydata on farm. Fortherefore the lactating dairy cow, dietary intake was estimated based on the daily cornand/or grainor and forage (corn affiliates. silage) consumption. In forage,property the highe intellectualst levelsthird of Cry1A.105 and mayCry2Ab2 were 56 its and 55 µg/g dwt, respectively (Table asVI.4). Table VI.8 provides estimates of the daily the of and dietary intakes of the Cry1A.105is and Cry2Ab2 proteinsregulatory by poultry andowner. livestock. a document any such its reproductiondocument 3.2.3. Margins of exposure owner this of under of The MOE, defined as the ratiorights ofthe the NOEL to the dailythis dietary intake, was calculated for documentand/or rights the Cry1A.105 and Cry2Ab2to of proteins fall for theuse broiler of chicken, young pig, finishing pig and lactating dairy cow (See Table VI.8). For poultrythe and livestock, the MOEs ranged This may distribution,and between 1,930 – 13,500rights and 2,160 – 47,600 ownerfor the Cry1A.105 and Cry2Ab2 proteins, respectively. Thesesubject large MOEs indicate that there is negligible risk to poultry and livestock frombe dietary exposure to animal thefeed containingviolate MON 89034. copy of and may documentpublication, It exploitation Table VI.8. Daily Dietary Intake and Margin of Exposure (MOE) for the this any Cry1A.105 and Cry2Ab2 Proteins from Consumption of MON 89034 permission by Poultry and Livestockprohibited the commercial be Acute Dietary Intake MOEa Furthermore,any (mg/kg bw x 10-3) Consequently,Animal without Cry1A.105 Cry2Ab2 Cry1A.105 Cry2Ab2 Broiler chicken 357 107 5,800 20,500 Young pig 252 76 8,220 29,100 Finishing pig 154 46 13,500 47,600 Lactating dairy cow 1073 1017 1,930 2,160 a MOE (for poultry and livestock) = NOEL/daily dietary intake; values rounded to three significant figures. Based on NOELs of 2072 mg/kg for Cry1A.105 and 2198 mg/kg for Cry2Ab2, in the acute mouse gavage studies.

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SECTION 4. Assessment of the Potential for Allergenicity of the Cry1A.105 and Cry2Ab2 Proteins Produced in MON 89034

4.1. Approach to the assessment of allergenicity This assessment of the allergenic potential of the Cry1A.105 and Cry2Ab2 proteins compares the biochemical characteristics of these proteins to characteristics of known allergens. A protein is not likely to be an allergen if:

a) The protein is from a non-allergenic source; b) The protein does not share structural similarities to known allergensregime. basedand on the amino acid sequence; and AG c) The protein is rapidly digested in simulated gastric fluid; d) The protein represents only a very small portion of the total protein in the grain. Bayer property protectionpublishing In the following sections, these four ofcharact eristics are discussed in contentsdetail for the its Cry1A.105 and Cry2Ab2 proteins produced in MONparties. 89034.data General informationtherefore on the methods used to assess the structural similarity to known and/orallergensor and stability in affiliates. simulated digestive fluids is providedproperty below. intellectual third may its as the of and 4.1.1. Rational for studyingis structural similarityregulatory to known allergensowner. a documentits In 2003, the Codex Alimentariusany such Commission publishedreproduction guidelines for the evaluation of this documentof the potential allergenicity of novel proteinsowner (Codex, 2003). The guideline is based on the under of comparison of amino acidrights sequencesthe between introducedthis proteins and known protein documentand/or rights allergens. The potential toallergenic of fallcross-reac tivityuse mayof exist if the introduced protein is found to have at least 35% amino acid identity with athe known allergen over any segment This may distribution,and of at least 80 amino acids.rights The Codex guidelineowner also recommended that a sliding window search with a scientificallysubject justified peptide size, such as eight amino acids, could be used be the violate to identify immunologicallycopy relevant peptidesof in otherwise unrelated proteins. and may documentpublication, BioinformaticIt analyses wereexploitation performed on Cry1A.105 and Cry2Ab2 proteins expressed in MON 89034this to anyassess potential similarity to allergens and identify immunologically relevant peptides. The comparisons were performed using the AD6 database, which is an permission allergen, gliadin, and glutenin sequenceprohibited da tabase assembled from sequences found on the the FARRP allergencommercial database (FARRP, be 2006).

TheFurthermore, bioinformaticsany analyses of potential simililarity of Cry1A.105 and Cry2Ab2 amino acidConsequently, sequences withoutto known allergens reveal no significant matches to known allergens.

4.1.2. Rational for studying stability in simulated digestive fluids A factor that increases the likelihood of allergic oral sensitization to proteins is the stability of the proteins 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 through the intestinal mucosa to elicit an allergenic response (Astwood et al., 1996; Metcalfe et al., 1996). Proteins that are rapidly digestible are highly correlated with a

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significantly decreased likelihod to cause sensitization or allergic reaction when consumed.

One aspect of this assessment includes analysis of the digestibility of the protein in a simulated gastric fluid (SGF) assay containing pepsin. A relationship between digestibility in SGF and the likelihood of being an allergen has been previously reported with a group of proteins consisting of both allergens and non-allergens (Astwood et al., 1996). Recently, the International Life Science Institute (ILSI) standardized the SGF assay protocol based on results obtained from an international, multi-laboratory ring study (Thomas et al., 2004). 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 Cry1A.105 and regime.and Cry2Ab2 proteins to pepsin digestion in vitro. and AG

In addition to SGF, simulated intestinal fluid (SIF) is also used for in vitro studies to assess the digestibility of food componentsBayer (Yagami propertyet al., 2000; protection Okunukipublishing et al., 2002). SIF is an in vitro digestion model whereof proteins undergo digestion at neutralcontents pH by a its mixture of enzymes known as pancreatin. The relationshipparties.data between proteintherefore allergenicity and protein stability in the in vitro SIF study is limited, becauseand/or theor protein has not been affiliates. first exposed to the acidic, denaturingproperty conditi intellectualonsthird of the stomach, as wouldmay be the case in its vivo (FAO/WHO, 2001). In vitro susceptibilityas of Cry1A.105 and Cry2Ab2 proteins to the of and pancreatin was assessedis for digestibility in SIFregulatory according to methodsowner. described in the a document United States Pharmacopeiaany (1995). such its reproductiondocument owner this of under of rightsthe this documentand/or rights 4.2. Assessment of the poto tentialof forfall all ergenicityuse ofof the Cry1A.105 protein the 4.2.1. SourceThis of the Cry1A.105may protein distribution, and rights owner Cry1A.105 is a subjectBt Cry1A chimeric protein with overall amino acid sequence identity of be the violate 93.6%, 90.0%, andcopy 76.7% to Cry1Ac, ofCry1Ab, and Cry1F proteins, respectively. Data presented in Table VI.1 shows the extremandely high homology of the various domains of may documentpublication, the Cry1A.105It protein to theexploitation respective domains in the Cry1Ab, Cry1Ac and Cry1F proteins. Domainsthis anyI and II of Cry1A.105 are 100% identical in amino acied sequence to Domains I and II of both Cry1Ab and Cry1Ac. Domain III of Cry1A.105 is 99% permission identical in amino acid sequenceprohibited to Domain III of Cry1F. The C-terminal portion of the the Cry1A.105 proteincommercial is identical be to the C-terminal portion of the Cry1Ac protein. Bt is a spore-forming, gram-positive bacterium that is found naturally in soil. Bt strains have beenFurthermore, used commerciallyany in the U.S. since 1958 to produce microbial-derived products withConsequently, insecticidalwithout activity (EPA, 1988). There are no known reports of allergies to Bt species or to the proteins produced by these species.

4.2.2. Bioinformatics analyses of sequence similarity of the Cry1A.105 protein produced in MON 89034 to allergens Using the methods described in Part VI, Section 4.1.1, bioinformatics analyses were performed to assess the potential for allergenicity of the Cry1A.105 protein sequence.

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The data generated from these analyses confirm that the Cry1A.105 protein does not share any amino acid sequence similarities with known allergens, gliadins, or glutenins.

The allergen database 6 (AD6) was used for the evaluation of sequence similarities shared between the Cry1A.105 protein and all proteins. Using the FASTA sequence alignment tool, proteins were ranked according to their degree of similarity to Cry1A.105. None of the proteins in the AD6 database met or exceeded the threshold of 35% identity over 80 amino acids. One low quality alignment between Actinidia deliciosa kiwifruit actinidin protein was identified, where a stretch of 24.2% identity over a 318 aa (amino acids) window was identified. This alignment had an E-score of 2.3. The E-score (expectation score) is a statistical measure of the likelihood that the observed similarity score could have occurred by chance in a search. A larger E-score indicates a regime.and lower degree of similarity between the query sequence andand th e sequence from the AG database. Typically, alignments between two sequences will need to have an E-score of 1×10-5 or smaller to be considered to have potentially significant homology in sequence. This E-score of 2.3 is not reflective of homoloBayergy betweenproperty Cry protection1A.105publishing and the kiwifruit protein, as E-scores of ~1 or larger are expectedof to occur for alignments betweencontents random, its non-homologous sequences (Pearson, 2000). Therefore,parties. datathis low qualitytherefore alignment is considered not relevant from an allergenic assessment perspective.and/oror Inspection of the affiliates. remaining alignments also didproperty not showintellectual anythird significant similaritiesmay between the its Cry1A.105 protein and other allergens.as the of and is regulatory owner. a document A second bioinformatics tool,any ansuch eight-ami no acid sliding windowits search, was used to reproductiondocument specifically identify short linear polypeptideowner matchesthis to known orof suspected allergens. It is possible that proteins structurally unrelatedunder to ofallergen s, gliadins, and glutenins may rightsthe this still contain smallerdocument and/orimmunol ogically significant epitopes. rights An amino acid sequence may to of fall use of be considered to have allergenic potential if it has an theexact sequence identity of at least eight linearlyThis contiguous aminomay acids distribution, withand a po tential allergen epitope (Metcalfe et al., rights owner 1996; Hileman etsubject al., 2002). Using a sliding window of less than eight amino acids can produce matches containing significant uncertainthe violatety depending on the length of the query be copy sequence (Silvanovich et al., 2006) andof are not useful to the allergy assessment process and (Thomasmay et al., 2005).document publication, It exploitation this An eight-mer searchany was performed using an algorithm (ALLERGENSEARCH) that was permission developed to identify whether prohibitedor not a linea rly contiguous match of eight amino acids existed between the Cry1A.105 sequence and amino acid sequences within AD6. This the be program comparescommercial the Cry1A.105 sequence to each protein sequence in the allergen databaseFurthermore, usingany a sliding-window of eight amino acids; that is, with a seven-amino acid overlapConsequently, relativewithout to the preceding window. No alignments of eight contiguous amino acid identities were detected when the Cry1A.105 protein sequence was compared to all sequences in the AD6 database.

Together, these data demonstrate that the Cry1A.105 protein does not share any relevant amino acid sequence similarities with known allergens, gliadins, or glutenins.

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4.2.3. Digestibility of the Cry1A.105 in simulated gastric digestive fluid Digestibility of the Cry1A.105 protein in simulated gastric fluid (SGF) was assessed by SDS-PAGE and western blot methods. The extent of digestion of the Cry1A.105 protein was evaluated by visual analysis of stained polyacrylamide gels (Figure VI.15) or by visual analysis of developed X-ray film (Figure VI.16). A separate gel or blot was performed concurrently to determine the limit of detection (LOD) of each assay (Figure VI.15, panel B; Figure VI.16, panel B). The limit of detection of the full-length Cry1A.105 protein by Colloidal Brilliant Blue G staining was 0.005 µg or approximately 0.7% of the total Cry1A.105 protein loaded (0.005 µg divided by 0.7 µg of the loaded protein in the test). The limit of detection of the full-length Cry1A.105 protein by western blotting was 1.0 ng or approximately 5% of the total Cry1A.105 protein loaded and (1 ng divided by 20 ng of the loaded protein in the test). In bothand methods, regime. in addition to the full-length Cry1A.105 protein, some bandsAG with lower molecular weight were observed. These bands represent proteolytic fragments of the Cry1A.105 protein that result from proteolysis during the purification procedure. Bayer property protectionpublishing of contents The gel used to assess the digestibility of the Cry1A.105 protein to pepsinits (Figure VI.15, parties.data therefore panel A) by Colloidal Brilliant Blue G staining was loaded withand/or 0.7or µg (based on pre- digestion concentrations) for each of theaffiliates. digest ion time points. Visual examination of the property intellectualthird may stained gel showed that the full-lengthits Cry1A.105 protein was digested below LOD as within 30 seconds of digestionthe inof SGF (Figureand VI.15, panel A, lane 5). Therefore, at least is regulatory owner. 99.3% (100% – 0.7% = 99.3%) of the full-lenga th Cry1A.105document proteinits wa s digested within 30 seconds of incubation basedany onsuch this analysis. reproductionA faint band with a molecular weight of this documentof approximately 4.5 kDa was observedowner at a very low level between the 30-second and 20- under of minute digestion time pointsrights (Figurethe VI.15, panel A, thislanes 5-9). No protein band was documentand/or of rights visible at the 30-minuteto digest of ion falltime pointuse (Figure VI.15, panel A, lane 10). Since there were only trace amounts of the ~4.5 kDa fragmentthe present in the first 20 minutes of This may distribution,and digestion and this fragmentrights was undetectable inowner the 30 minute time point and beyond, it is unlikely to posesubject a human health risk. be the violate copy of The gel used to assess the Cry1A.105 proteinand in vitro digestibility by western blot was may documentpublication, loadedIt with 20 ng total proteinexploitation (based on pre-digestion concentrations) for each of the digestion time thispoints. any Western blot analysis demonstrated that the Cry1A.105 protein was digested below the LOD within 30 seconds of incubation in SGF (Figure VI.16, permission panel A, lane 5). Based on the prohibitedwestern blot LOD for the Cry1A.105 protein in SGF and the the observation thatcommercial no full-length be protein or immunoreactive bands were observed on the western blot at the 30-second digestion time point, it was concluded that greater than 95%Furthermore, ()any =− %95%5%100 of the full-length Cry1A.105 protein was digested within 30 Consequently,without seconds of incubation with SGFs.

The results of this study demonstrated that the full-length Cry1A.105 protein was rapidly digested after incubation in SGF. The full-length Cry1A.105 protein was digested below the LOD within 30 seconds when analyzed using Colloidal Brilliant Blue G staining or by western blotting. There were no stable proteolytic degradation products, with the exception of a very faint band detected by Colloidal Brilliant Blue G staining with a MW of ~4.5 kDa; this band was not observed in the 30-minute time point or beyond.

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and and regime. AG

Bayer property protectionpublishing of contents Lane assignment for Panel A Lane assignmentits for Panel B parties.data therefore ______and/oror Lane Sample Incubationaffiliates. Lane Sample Amount property intellectualthird may Timeits (min) (µg) as 1 MW marker the of — and 1 MW marker — is regulatory owner. 2 N0, SGF only 0 a 2 document T0, protein+SGF 0.7 any such its 3 P0, protein only 0 reproduction3 documentT0, protein+SGF 0.35 owner this of 4 T0, protein+SGF 0 4 T0, protein+SGF 0.1 under of 5 T1, protein+SGFrights the 0.5 5 this T0, protein+SGF 0.05 documentand/or of rights 6 T2, protein+SGFto of fall2 use 6 T0, protein+SGF 0.02 7 T3, protein+SGF 5 7 the T0, protein+SGF 0.01 This may distribution,and 8 T4, protein+SGFrights 10 owner 8 T0, protein+SGF 0.005 9 T5, protein+SGFsubject 20 9 T0, protein+SGF 0.0025 be the violate 10 T6, protein+SGFcopy 30of 10 MW marker — 11 T7, protein+SGF 60 and may documentpublication, 12 It P7, protein only exploitation 60 13 N7, thisSGF any only 60 14 MW marker — permission prohibited the Figure VI.15. Colloidalcommercial Brilliant be Blue G Stained SDS-gels of Cry1A.105 Protein Digestion in SGF Furthermore,any PanelConsequently, A correspondswithout to Cry1A.105 protein digestion in SGF. Based on pre-digestion protein concentrations, 0.7 µg (total Cry1A.105 protein) was loaded in lanes containing Cry1A.105 protein. The incubation times are indicated. Panel B corresponds to the limit of detection (LOD) of Cry1A.105 protein. Approximate molecular weights (kDa) are shown on the left and correspond to the markers loaded in each gel.

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and and regime. AG

Bayer property protectionpublishing Lane assignment for Panel A of Lane assignment forcontents Panel B its ______parties.data therefore Lane Sample Incubation Lane Sampleand/or or Amount affiliates. Timeproperty (min) intellectual third may (ng) its 1 MW marker —as 1 MW marker — the of and 2 N0, SGF onlyis 0 regulatory 2 T0, protein+SGFowner. 7 a document 3 P0, protein only any such0 3 T0, protein+SGFits 3.5 reproductiondocument 4 T0, protein+SGF owner0 4this T0, protein+SGFof 2 5 T1, protein+SGF 0.5 of5 T0, protein+SGF 1 rights under this 6 T2, protein+SGFand/or the 2 6 T0, protein+SGF 0.5 document to of fall of rights 7 T3, protein+SGF 5 use 7 T0, protein+SGF 0.2 the 8 This T4, protein+SGF may distribution,10 and 8 T0, protein+SGF 0.1 rights owner 9 T5, protein+SGFsubject 20 9 T0, protein+SGF 0.05 10 T6, protein+SGF 30 10 MW marker — be the violate 11 T7, protein+SGFcopy 60of and 12 may P7, proteindocument onlypublication, 60 13 It N7, SGF only exploitation 60 this 14 MW markerany — permission Figure VI.16. Western blot Analysisprohibited of Cry1A.105 Protein Digestion in SGF the be Panel A correspondscommercial to Cry1A. 105 protein digestion in SGF. Based on pre-digestion proteinFurthermore, concentrations,any 20 ng (total protein) was loaded in lanes containing Cry1A.105 protein.Consequently, Panel withoutB corresponds to the limit of detection (LOD) of the Cry1A.105 protein. Approximate molecular weights (kDa) are shown on the left and correspond to the markers loaded in each gel.

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4.2.4. Digestibility of the Cry1A.105 protein in simulated intestinal fluid The digestibility of the Cry1A.105 protein in SIF, which contains a mixture of enzymes called pancreatin.

The digestion of the Cry1A.105 protein was evaluated by western blot method (Figure VI.17). A western blot to determine the LOD (Figure VI.17, panel A) of the Cry1A.105 protein was performed concurrently with the western blot used to assess the Cry1A.105 protein digestibility in SIF (Figure VI.17, panel B). The LOD was estimated to be 0.1 ng, which represented 0.5% of the total protein loaded in this experiment (0.1 ng divided by 20 ng of loaded protein).

and The gel to assess the Cry1A.105 protein SIF digestibility by westernand blot wasregime. loaded with 20 ng total protein (based on pre-digestion concentrations)AG for each of the digestion time points. Western blot analysis demonstrated that the full-length Cry1A.105 protein was digested below the LOD within 5 minutes of incubation in SIF (Figure VI.17, panel B, Bayer property protectionpublishing lane 5). Therefore, at least 99.5% (100%of - 0.5% = 99.5%) of the full-lengthcontents Cry1A.105 protein was broken down within 5 minutes. Proteolytic fragments itswith approximate parties.data therefore molecular weight of ~60, 32 and 30 kDa were observed at theand/or 5 minuteor digestion time point and were stable for the various timaffiliates.es up to 24 hours of digestion in SIF. property intellectualthird may its as The results of this study demonstratethe of that theand full-length Cry1A.105 protein was digested is regulatory owner. within 5 min of incubation in SIF, yieldiang fragmentsdocument with molecular weights of any such its approximately 60, 32, and 30 kDa. At least 99.5%reproduction of the full-lengthdocument of Cry1A.105 protein was digested in SIF within 5 minutes.owner The major proteolyticthis fragment at approximately under of 60 kDa migrated as a doublet,rights whichthe represents the trypticthis core of the Cry1A.105 protein, documentand/or rights was observed for up toto 24 ofhours fall (the longestuse timeof point tested). These results are consistent with observations for other Cry proteins withthe demonstrated safety. This may distribution,and rights owner Overall, the resultssubject for digestibility of Cry1A.105 are consistent with proteins with be the violate demonstarted safety.copy The fact that Cry1A.105of is readily digestible in simulated gastric fluid makes it unlikely be a food allergen. and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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regime.and and AG

Bayer property protectionpublishing of contents its parties.data therefore Lane assignment for Panel A Lane assignmentand/oror for Panel B affiliates. ______property intellectual third ______may its Lane Sample Amountas Lane Sample Incubation the of and is (ng) regulatory owner. Time a document 1 Molecular weight markerany such — 1 Molecular itsweight marker — reproductiondocument 2 T0, protein+SIF 10owner 2 this N0, SIFof only 0 3 T0, protein+SIF 5 under of 3 P0, protein only 0 4 T0, protein+SIF rightsthe 2 4 this T0, protein+SIF 0 documentand/or of rights 5 T0, protein+SIFto of fall 1 use 5 T1, protein+SIF 5 min 6 T0, protein+SIF 0.5 6 the T2, protein+SIF 15 min This may distribution,and 7 T0, protein+SIFrights 0.2 owner 7 T3, protein+SIF 30 min 8 T0, protein+SIFsubject 0.1 8 T4, protein+SIF 1 h 9 T0, protein+SIF 0.05 the violate 9 T5, protein+SIF 2 h be copy 10 Molecular weight marker — of 10 T6, protein+SIF 4 h document and 11 T7, protein+SIF 8 h may publication,exploitation It 12 T8, protein+SIF 12 h this any 13 T9, protein+SIF 24 h permission 14 P9, protein only 24 h prohibited 15 N9, SIF only 24 h the commercial be Figure VI.17. Western Blot Analysis of Cry1A.105 protein Digestion in SIF Furthermore,any PanelConsequently, A correspondswithout to the limit of detection of Cry1A.105 protein. Panel B corresponds to Cry1A.105 protein digestion in SIF. Based on the pre-digestion protein concentration, 20 ng (total protein) was loaded in lanes containing Cry1A.105 protein. The digestion times are indicated. Approximate molecular weights (kDa) are shown on the left and correspond to the markers loaded on each gel.

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4.2.5. Proportion of Cry1A.105 protein to the total protein in MON 89034 grain The overall mean level of Cry1A.105 protein in MON 89034 grain is 5.9 µg/g (dwt) (Part VI, Section 2). The mean % dry weight of total protein in MON 89034 grain is 12.51% (or 125,100 µg/g). The percent of Cry1A.105 protein in MON 89034 grain is calculated as follows:

(5.9 µg/g ÷ 125,100 µg/g) x 100% = 0.0047%

Therefore, the Cry1A.105 protein represents a very small portion of the total protein in MON 89034 grain. and and regime. AG 4.3. Assessment of the potential for allergenicity of the Cry2Ab2 protein Bayer property protectionpublishing 4.3.1. Source of the Cry2Ab2 protein of contents its The Cry2Ab2 protein is a variant of the wild-typeparties. Cry2Ab2data protein isolatedtherefore from Bt subsp. kurstaki, which is a spore-forming, gram-positive bacteriumand/oror found naturally in affiliates. soil. Bt strains have been propertyused commerc intellectualiallythird in the U.S. since may1958 to produce its microbial-derived products with insecticidalas activity (EPA, 1988). There are no known the of and reports of allergies to Bt isspecies or the proteins regulatoryproduced from these species.owner. a document any such its reproductiondocument 4.3.2. Bioinformatics analysis of sequenceowner similaritythis of the Cry2Ab2of protein produced in MON 89034 to allergensunder of rightsthe this documentand/or rights Using the methods describedto of in Partfall VI, Sectionuse 4.1.1,of potential structural similarities shared between the Cry2Ab2 protein and proteins in thethe allergen database were evaluated This may distribution,and using the bioinformaticrights tool. owner subject FASTA comparisionbe results showed that thethe greatestviolate similarity to the Cry2Ab2 protein copy of was to the Coprinus comatus protein Cop andc1, demonstrating only 32.7% identity over a 52 aa (aminomay acids)document windowpublication, with an E score of 0.89. This protein did not meet or It exploitation exceeded the threshold of 35% identity over 80 amino acids, and based on the low this any percent-identity and the small alignment window relative to the length of the Cry2Ab2 permission protein (637 aa), this FASTAprohibited alignment does not represent a bona fide homology (Doolittle, 1990). Therefore,the be this low quality alignment is considered not relevant from an allergenic assessmentcommercial perspective. Inspection of the remaining alignments also did not showFurthermore, any significantany similarities between the Cry2Ab2 protein and other allergens. The pair-wiseConsequently, comparisonwithout of eight-mer search results showed that no immunologically relevant sequences (eight contiguous amino acid identities) were detected when the Cry2Ab2 protein sequence was compared to the sequence database.

Together these data demonstrate that the Cry2Ab2 protein is unlikely to share structurally relevant or immunologically relevant sequence similarities with known allergens, gliadins, or glutenins.

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4.3.3. Digestibility of the Cry2Ab2 protein in simulated gastric fluid Digestibility of the Cry2Ab2 protein in SGF was assessed by SDS-PAGE and western blot methods. The extent of digestion of the Cry2Ab2 protein was evaluated by visual analysis of stained polyacrylamide gels (Figure VI.18) or by visual analysis of developed X-ray film (Figure VI.19). A separate gel or blot was performed concurrently to determine the limit of detection (LOD) of each assay (Figure VI.18, panel B; Figure VI.19, panel B). The limit of detection of the full-length Cry2Ab2 protein by Colloidal Brilliant Blue G staining was 0.005 µg or approximately 0.6% of the total Cry2Ab2 protein loaded (0.005 µg divided by 0.8 µg of the loaded protein in the test). The limit of detection of the full-length Cry2Ab2 protein by western blotting was 0.2 ng or approximately 1% of the total Cry2Ab2 protein loaded (0.2 ng divided by 20 ng of the and loaded protein in the test). and regime. AG Digestibility of the Cry2Ab2 protein in SGF assessed using stained SDS-polyacrylamide gels showed that the full-length Cry2Ab2 protein was rapidly digested. At least 99.4% of Bayer property protectionpublishing the full-length Cry2Ab2 protein was digestedof within 30 seconds when contentsanalyzed using Colloidal Brilliant Blue G stained polyacrylamide gels (Figure VI.18). its A very faint band parties.data therefore with molecular weight of ~5 kDa was observed at the 30-secondand/or digestionor time point. No Cry2Ab2 bands were visible at the 2-minuteaffiliates. dige stion time point (Figure VI.18, panel A, property intellectualthird may lane 6). its as the of and is regulatory owner. The extent of digestion of the Cry2Ab2 praotein was alsodocument evalua ted by western blot any such its method (Figure VI.19). At least 99% of the Cry2Ab2reproduction proteindocument was of digested in SGF within 30 seconds when analyzed using westernowner blot analysisthis (Figure VI.19, panel A). No stable under of proteolytic bands were observedrightsthe at any time points by westernthis blot analysis. documentand/or rights to of fall use of The results of this study demonstrated that Cry2Ab2 theprotein was rapidly digested after This may distribution,and incubation in SGF. Therights Cry2Ab2 protein ownerwas digested below the LOD within 30 seconds when analyzedsubject using SDS-PAGE by Colloidal Brilliant Blue G staining or by be the violate western blotting. copyThere were no stable ofproteolytic degradation products detected. and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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and and regime. AG

Bayer property publishing protectioncontents Lane assignment for Panel A of Lane assignment for Panel B its ______parties.data therefore and/oror Lane Sample Incubation Lane Sample Amount affiliates. Timeproperty (min) intellectual third may (µg) its 1 Molecular weight marker n/a as 1 Molecular weight marker n/a the of and 2 N0, SGF only is 0 regulatory 2 T0, protein+SGFowner. 1 a document 3 P0, protein only any such 0 3 T0, protein+SGFits 0.5 reproductiondocument 4 T0, protein+SGF 0 owner 4 this T0, protein+SGFof 0.2 5 T1, protein+SGF 0.5 5 T0, protein+SGF 0.1 under of this 6 T2, protein+SGF rightsthe 2 6 T0, protein+SGF 0.05 documentand/or of rights 7 T3, protein+SGFto of 5 fall use 7 T0, protein+SGF 0.02 8 T4, protein+SGF 10 8 the T0, protein+SGF 0.01 This may distribution,and 9 T5, protein+SGFrights 20 owner 9 T0, protein+SGF 0.005 10 T6, protein+SGFsubject 30 10 T0, protein+SGF 0.0025 the violate 11 T7, beprotein+SGF copy 60 11 Molecular weight marker n/a 12 P7, protein only 60 of and 13 N7, SGF onlydocument 60 may publication,exploitation 14It Molecular weight marker n/a this any

permission Figure VI.18. Colloidal Brilliantprohibited Blue G Stained SDS-PAGE Gels of Cry2Ab2 the Proteincommercial Digestion be in SGF PanelFurthermore, A correspondsany to Cry2Ab2 protein digestion in SGF. Based on the pre-digestion proteinConsequently, concentration,without ~0.8 µg (total protein) was loaded in lanes containing Cry2Ab2 protein. Panel B corresponds to the limit of detection (LOD) of Cry2Ab2 protein. Approximate molecular weights (kDa) are shown on the left and correspond to the markers loaded in each gel.

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and and regime. AG

Lane assignment for Panel A Lane assignment for Panel B Bayer property protectionpublishing ______of ______contents Lane Sample Incubation Lane Sample its Amount parties.data therefore Time (min) and/oror (ng) 1 Molecular weight marker n/aaffiliates. 1 Molecular weight marker n/a 2 P0, protein only property 0 intellectualthird2 T0, protein+SGFmay 10 its 3 N0, SGF only 0 as 3 T0, protein+SGF 5 the of and 4 T0, protein+SGFis 0 regulatory 4 T0, protein+SGFowner. 2 a document 5 T1, protein+SGF any such 0.5 5 T0, protein+SGFits 1 reproductiondocument 6 T2, protein+SGF 2owner 6 this T0, protein+SGFof 0.5 7 T3, protein+SGF 5 under of 7 T0, protein+SGF 0.2 rightsthe this 8 T4, protein+SGFdocumentand/or 10 8 of T0,rights protein+SGF 0.1 9 T5, protein+SGFto of fall 20 use 9 T0, protein+SGF 0.05 10 T6, protein+SGF 30 10 the Molecular weight marker n/a This may distribution,and 11 T7, protein+SGFrights 60 owner 12 P7, proteinsubject only 60 the violate 13 N7,be SGF copy only 60 14 Molecular weight marker n/a of and may documentpublication, It exploitation this Figure VI.19. Westernany Blot Analysis of Cry2Ab2 Protein Digestion in SGF permission prohibited Panel A corresponds tothe Cry2Ab2 be protein digestion in SGF. Based on the pre-digestion protein concentration,commercial 20 ng (total protein) was loaded in lanes containing Cry2Ab2 protein.Furthermore, Panelany B corresponds to the limit of detection of the Cry2Ab2 protein. ApproximateConsequently, molecularwithout weights (kDa) are shown on the left and correspond to the markers loaded in each gel.

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4.3.4. Digestibility of the Cry2Ab2 protein in Simulated Intestinal Fluid The in vitro digestibility of the Cry2Ab2 protein in SIF was assessed by western blot analysis. A western blot to determine the LOD (Figure VI.20, panel A) of the Cry2Ab2 protein was run concurrently with the western blot used to assess the Cry2Ab2 protein digestibility in SIF (Figure VI.20, panel B). The LOD was determined by the lowest amount of Cry2Ab2 protein observed on the X-ray film under visual inspection. The LOD was estimated to be 0.5 ng, which represents 2.5% of the total protein (0.5 ng divided by 20 ng of loaded protein in the test).

The gel used to assess the Cry2Ab2 protein digestibility by western blot was loaded with 20 ng total protein (based on pre-digestion concentrations) for each of the incubation time and points. Western blot analysis demonstrated that a band correspondingand to regime.the full-length Cry2Ab2 protein was digested below the LOD withinAG 15 minutes of incubation in SIF (Figure VI.20, panel B, lane 6). Therefore, at least 97.5% (100% - 2.5% = 97.5%) of the full-length Cry2Ab2 protein was digested within 15 minutes. Proteolytic bands with Bayer property protectionpublishing approximate molecular weight of 60, 55,of 50, 40, 12 and 10 kDa were observedcontents at the 5-minute time point. Several new bands with molecular weights smallerits than 50 kDa parties.data therefore were detectable beginning at the 4-hour digestion time point. and/or Theseor bands, which were transient in nature and displayed a weakaffiliates. immunoreactive signal, were detectable at the property intellectualthird may 24-hour incubation time point. its as the of and is regulatory owner. Overall, the results for digestibility of Cry2Aa b2 are consistentdocument with other Cry proteins any such its with demonstrated safety. The fact that Cry2ABb2reproduction is readilydocument of digestible in simulated gastric fluid makes it unlikely be a foodowner allergen this 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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regime.and and AG

Lane assignment for Panel A BayerLane property assignment protection forpublishing Panel B ______of ______contents Amount its thereforeIncubation Lane Sample Lane parties.Sampledata (ng) and/oror Time affiliates. 1 Molecular weight property— intellectual1 third Molecular weightmay — its marker as marker the of and 2 T0, protein + SIFis 0.5 2 regulatoryP0, protein only owner.0 a document 3 T0, protein + SIF any 1 such 3 N0, SIF only its 0 reproductiondocument 4 T0, protein + SIF 2 owner 4 this T0, protein of+ SIF 0 5 T0, protein + SIF 5 under 5 of T1, protein + SIF 5 min rightsthe this 6 T0, proteindocument and/or+ SIF 10 6 T2, proteinrights + SIF 15 min to of fall use of 7 T0, protein + SIF 20 7 T3,the protein + SIF 30 min 8 This MW marker may — distribution,and 8 T4, protein + SIF 1 h rights owner subject 9 T5, protein + SIF 2 h the 10 violate T6, protein + SIF 4 h be copy of 11 T7, protein + SIF 8 h and may documentpublication, 12 T8, protein + SIF 12 h It exploitation this 13 T9, protein + SIF 24 h any 14 P9, protein only 24 h permission prohibited 15 N9, SIF only 24 h

the be Figure VI.20. Westerncommercial Blot Analysis of Cry2Ab2 Protein Digestion in SIF PanelFurthermore, A correspondsany to the LOD of Cry2Ab2 protein (5 min exposure). Panel B correspondsConsequently, to withoutCry2Ab2 protein digestion in SIF (5 min exposure). Based on the pre- digestion concentration, 20 ng (total protein) was loaded in lanes containing Cry2Ab2 protein. The incubation times are indicated. Approximate molecular weights (kDa) are shown on the left and correspond to the markers loaded on each gel.

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4.3.5. Proportion of Cry2Ab2 protein to the total protein in MON 89034 grain The mean level of Cry2Ab2 protein in corn grain is 1.3 µg/g (dwt) (Part VI, Section 2). The mean % dry weight of total protein in MON 89034 grain is 12.51% (or 125,100 µg/g). The percent of Cry2Ab2 protein in MON 89034 grain is calculated as follows:

(1.3 µg/g ÷ 125,100 µg/g) x 100% = 0.0010 %

Therefore, the Cry2Ab2 protein represents a very small portion of the total protein in MON 89034 grain.

and 4.4. Conclusions and regime. AG The Cry1A.105 and Cry2Ab2 proteins have been assessed for their potential allergenicity according to the recommendations of Codex Alimentarius Commission. The proteins are Bayer from non-allergenic sources, lack structural similarityproperty to known protection allergens,publishing are rapidly of contents digested in simulated gastric fluid, and constitute a very small portion ofits the total protein parties.data therefore present in the grain of MON 89034. and/oror affiliates. property intellectualthird may The Cry1A.105 and Cry2Ab2 protits eins are derived from Bacillus thuringiensis, an as organism that is not a commonlthe ofy allergenicand source, and the Cry class of proteins has a is regulatory owner. long history of safe use. Bioinformatics analysesa demonstratdocumented that the proteins do not any such its share structurally or immunologically relevant reproductionamino acidocumentd sequenceof similarities with known allergens. Thus, the Cry1A.105owner and Cry2Ab2this proteins are highly unlikely to under of contain immunologically cross-rerightsthe active allergenic epitopes.this Digestive fate experiments documentand/or rights conducted with Cry1A.105to orof Cry2Ab2fall proteinsuse demonstratedof that both proteins are rapidly digested in simulated gastric fluid, a characteristithe c shared among many proteins This may distribution,and with a history of saferights consumption. Finally,owner the Cry1A.105 and Cry2Ab2 proteins represent no moresubject than 0.005% and 0.001% of the total protein in the grain of MON the 89034, respectively.be Taken together these data violatelead to the conclusion that the Cry1A.105 copy of and Cry2Ab2 proteins are unlikely to beand allergenic, and MON 89034 is as safe as may documentpublication, conventionalIt corn regarding thexploitatione risk for allergenicity. this any permission prohibited the commercial be Furthermore,any Consequently,without

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SECTION 5. Assessment of the Potential for Toxicity of the Cry1A.105 and Cry2Ab2 Proteins

5.1. Approach to the assessment of toxicity The previous section described an assessment of the potential for allergenicity of the Cry1A.105 and Cry2Ab2 proteins in MON 89034 based on the sources of the proteins, a comparison of their sequence to known allergens, their digestibility in simulated digestive fluids, and finally their proportion to the total protein in MON 89034. In this section, an assessment of a potential for toxicity of the Cry1A.105 and Cry2Ab2 proteins is conducted. This assessment is based on the established premise that a regime.proteinand is not likely to have a toxic effect if: and AG

a) The protein has a demonstrated history of safe use; b) The protein has no structural similarityBayer to knownproperty toxins protection orpublishing other biologically active proteins that could cause adveof rse effects in humans or animals;contents its c) The protein does not exert any acute toxic effectsparties. todata mammals. therefore and/oror affiliates. In addition, the low concentrationproperty of the intrintellectualoducedthird proteins in tissues maythat are consumed its and the rapid digestibility in simulatedas diges tive fluids provide further assurance for their the of and safety. is regulatory owner. a document any such its reproductiondocument The potential for synergistic or antagonistowneric effects betweenthis the Cry1A.105of and Cry2Ab2 under of proteins was considered forrights assessmentthe of potential toxicity.this To date, there have been only a few compellingdocumentand/or examples of interactive effects betweenrights Cry proteins that have to of fall use of either decreased (antagonism) or increased (synergism)the activity towards target pests when combinedThis (Tabashnik 1992, Schnepfmay distribution, et al.and 1998). Demonstrating the lack of interaction rights owner between the twosubject Cry proteins allows for each of them to be tested independently in safety assessment studies. The principle of indepethe ndentviolate assessment has been used for many be copy years for food additives and microbialof ri sk assessments (EPA, 2004). The Cry1A.105 and and Cry2Ab2may proteinsdocument publication,were tested alone and in combination against two sensitive It exploitation lepidopteran speciesthis – the European corn borer (ECB) and corn earworm (CEW). Two species were testedany to demonstrate reproducibility thereby strengthening the validity of permission the study. The insects were exposedprohibited to purified Cry1A.105 and Cry2Ab2 proteins in diet-incorporation bioassays. Results demonstrated that when tested in combination, the the be insecticidal activitiescommercial of the two proteins were additive - no synergistic or antagonistic effectsFurthermore, wereany observed. These results are consistent with the results from a study that demonstratedConsequently, nowithout interaction between the Cry1Ac and Cry2Ab2 proteins (Greenplate et al, 2003). Therefore, it is appropriate that the safety of each protein be assessed independently in the safety assessments.

5.2. Safety of the donor organism: Bacillus thuringiensis Bacillus thuringiensis is a spore-forming, gram-positive bacterium that is found naturally in soil. Bt strains have been used commercially in the U.S. since 1958 to produce microbial-derived products with insecticidal activity (EPA, 1988). Many Bt microbial

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strains have been shown to produce protein crystals or inclusion bodies that are selectively toxic to certain orders and species of insect pests. The current nomenclature, based on amino acid identity, allows closely related proteins to be ranked together. Each protein is assigned a unique name incorporating four hierarchical ranks consisting (in order) of an Arabic numeral, upper case letter, lower case letter, and an Arabic numeral (e.g., Cry2Ab2 protein). Thus, proteins with identity of:

a) <45% differ in a primary rank (e.g., Cry2, Cry3, etc.), 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., Cry2Ab1, Cry2Ab2) and are consideredand allelic variants. and regime. AG

In general, the primary rank of the Cry proteins denotes its specific insecticidal activity; for example, Cry1, Cry2, Cry3, and BayerCry4 proteinsproperty are toxicpublishing to lepidopteran, protectioncontents lepidopteran/dipteran, coleopteran, and ofdipteran pests, respectively (Bravo, 1997; Höfte its and Whitely, 1989). It has been established that Cryparties. proteinsdata have a narrowtherefore range of and/oror insecticidal activity against one or, less commonly, two orders of insects (Crickmore et affiliates. al., 1998). property intellectualthird may its as the of and In the U.S., the FDA grantedis an exemption fromregulatory the requirement of owner.a tolerance for the a document first microbial Bt product inany 1960 such after an extensive toxicity and itsinfectivity evaluation reproductiondocument program. The testing program consistedowner of acute, subchronic,this andof chronic studies, which under of resembled the testing requiredrights the for conventional chemicalthis pesticides. In 1971, EPA documentand/or rights assumed responsibility tofor allof pesticidefall tolerausence exemptions.of Since then, a variety of naturally occurring and genetically modified microbialthe Bt products have been registered and includedThis under this tolerancemay exemption.distribution,and EPA has established separate tolerance rights owner exemptions by subjectamendment for various Cry proteins (e.g., Cry1Ab, Cry1Ac, Cry2Ab2, Cry3Bb1) expressedbe in biotechnology-detherived violate crops (EPA 1996, 1997, 2001). The copy of conclusion of reasonable certainty of no harmand and the resultant tolerance exemptions for this widemay array of Btdocument mixturespublication, and Cry proteins in food or feed were based on the lack of It exploitation adverse effectsthis to mammals in numerous toxicological studies. This conclusion is supported by a historyany of safe use in agriculture for over 40 years (McClintock et al., permission 1995). There are no adverse prohibitedeffects known to have occurred in humans during this prolonged period of usethe (EPA, be 1998). An extensive review of the safety of the various Cry proteins thatcommercial have been expressed in biotechnology-derived plants is available (Betz etFurthermore, al., 2000).any Consequently,without 5.3. Assessment of the potential for toxicity of the Cry1A.105 protein In this section the potential toxicity of the Cry1A.105 protein is discussed. The assessment includes comparing the similarity of Cry1A.105 to proteins with an established history of safe use and consumption, bioinformatics analyses of structural similarity of the Cry1A.105 protein to known toxins and other proteins that may adversely affect animal or human health, and testing of acute oral toxicity.

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5.4. Similarity of Cry1A.105 to Cry1A proteins with a history of safe use and consumption

5.4.1. Structural similarity of the Cry1A.105 protein with Cry1A class of Bt proteins As previously described (Part VI, Section 4.2.1), Cry1A.105 is a chimeric protein with overall amino acid sequence identity of Cry1A.105 to the Cry1Ac, Cry1Ab, and Cry1F proteins is 93.6%, 90.0%, and 76.7%, respectively. To better understand the structural relatedness of the Cry1A.105 protein to other Cry proteins, a phylogenetic tree (Figure VI.20) was produced using the Cry1A.105 amino acid sequence and the methods employed to define the Bt protein nomenclature (Crickmore, 2004; Crickmoreand et al., and regime. 1998). The analysis demonstrates that Cry1A.105AG belongs to the Cry1A class of Bt proteins and is most closely related (93.6% identical) to the Cry1Ac protein.

Recombinant DNA techniques have beenBayer used to propertygenerate Bt strainspublishing with enhanced protectioncontents insecticidal activity for use as biopesticidesof (Baum, 1998 and 1999). For example, the its microbial pesticide Lepinox that contains a Cry1Ac/Cry1Fparties.data chimeric proteintherefore has been and/oror approved and used for control of lepidopteran pests since 1997 (Baum, 1998; Baum et al., affiliates. 1999). property intellectualthird may its as the of and Cry1A proteins are widelyis used as topical bioinsecticidesregulatory and in biotechnology-derivedowner. a document crops. The US FDA completedany such consultations for the safety assessmentits of corn that reproductiondocument produces the Cry1Ab (YieldGard owner– MON 810 andthis Bt11) andof Cry1F (Herculex® I) under of ® proteins, as well as cotton producingrightsthe the Cry1Ac (Bollgardthis and Bollgard II, WideStrike ) documentand/or rights and Cry1F (WideStrike)to proteins. of Thefall U.S. EPuseA, whichof has responsibility of assessment the safety of pesticidal proteins, issued exemptions fromthe the requirement of a tolerance This may distribution,and for the Cry1Ab proteinrights in corn (YieldGard – MONowner 810 and Bt11), the Cry1Ac protein in cotton Bollgardsubject and WideStrike, and the Cry1F protein in corn Herculex I and cotton WideStrike. be the violate copy of and The detailedmay humandocument andpublication, animal safety assessments conducted on the Cry1Ab, Cry1Ac, It exploitation and Cry1F proteins, combined with a history of safe use, confirm their safety. Due to the this any chimeric nature of Cry1A.105, which was derived from domains of Cry1Ab, Cry1Ac, permission and Cry1F, a similar safety profilprohibitede is expected for Cry1A.105. the be commercial Furthermore,any Consequently,without

®Herculex and Widestrike are registered trademarks of Dow AgroSciences LLC.

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regime.and and AG

} Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and Figure VI.21. Phylogramis Establishing that theregulatory Cry1A.105 and Cry1Acowner. Proteins are a document Located in anythe Samesuch Cluster Based on Amino Acidits Sequence Identity reproductiondocument owner this of under of rightsthe this 5.4.2. Functionaldocument similarityand/or of the Cry1A.105 Protein torights the Cry1A class of Bt to of fall use of proteins the This may distribution,and Having established therights high structural similarityowner between the Cry1A.105 protein and the Cry1A class of subjectproteins, it is important also to establish the functional similarity. To be the violate assess the functioncopy of the Cry1A.105 protein,of its biological activity was compared to that of the related proteins Cry1Ab, Cry1Ac, aandnd Cry1F. Amino acid sequence identity for may documentpublication, insecticidalIt proteins predict exploitationsimilarity in biological function, i.e., activity towards a similar spectrumthis of anyinsects.

permission It has been established that Cryprohibited proteins have a defined spectrum of insecticidal activity within a particular insectthe order (Crickmore et al., 1998; De Maagd et al., 2001). This commercial be high degree of specificity is governed by four levels of selectivity, which collectively leadFurthermore, to intoxicationany (Federici, 2002). The levels of selectivity include: 1) the route by whichConsequently, the insectwithout is exposed to Cry proteins (i.e., ingestion of plant tissues); 2) protein toxin activation by specific proteolytic enzymes (determined by differences in gut physiology between insects); 3) toxin binding to available specific midgut receptors; and 4) changes in the protein configuration. The reconfigured protein then is able to enter the midgut membrane and form channels. This activity affects larval ability to feed and develop, and eventually leads to death of the susceptible insect.

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As a consequence, only insects with specific receptors are affected and no toxicity is observed in species that lack these receptors. For example, the Cry1Ab, Cry1Ac and Cry1F proteins are active against lepidopteran but not coleopteran insects, and the Cry3Bb1 protein is active against coleopteran insects such as corn rootworms but not against lepidopteran insects.

Studies were conducted to evaluate the insecticidal activity of the Cry1A.105 protein against a variety of agronomically important insects from three major taxa. Insect species tested included four representative lepidopterans: black cutworm, corn earworm, fall armyworm, and European corn borer; two representative coleopterans: boll weevil (Anthonomus grandis grandis) and southern corn rootworm (Diabrotica undecimpunctata howardi); and two representative hemipterans: western tarnished plant bug (Lygus regime.and hesperus) and green peach aphid (Myzus persicae). The insectsand were exposed to high AG concentrations of Cry1A.105 protein (up to 80 or 100 µg of protein per ml of insect diet in diet-incorporation bioassays). The results showed that Cry1A.105 had activities against all four representative lepidopteranBayer insects. Howepropertyver, thereprotection waspublishing no indication of activity by Cry1A.105 against the twoof cole opteran or two hemipteran contentsrepresentatives. its This activity spectrum of Cry1A.105 is similar to theparties. activitiesdata of the Cry1Ac,therefore Cry1Ab, and Cry1F proteins described in the literature (De Maagdand/or et al.,or 2001 and 2003). affiliates. Similarity in biochemical and functionalproperty charactintellectualthirderistics between proteinsmay with established its safety is a good indication of the generaasl safety of that particular class. the of and is regulatory owner. a document 5.4.3. Structural similarityany of Cry1A.105such to known toxins or otherits biologically reproductiondocument active proteins owner this of under of The safety assessment of rightsproteinsthe expressed in biotechnology-derivedthis crops evaluates documentand/or rights potential health effects throughto of a comprehensfall iveuse approach, of which includes bioinformatic analysis of the amino acid sequence of the introduced theprotein to ensure the protein is not This may distribution,and similar to proteins that rightsare known to cause adverseowner health effects. subject The FASTA sequence alignment tool wasthe used violate to assess structural similarity. Although be copy the FASTA program directly comparesof amino acid sequences (i.e., primary protein and structure),may the alignmentdocumentpublication, data may be used to infer secondary and tertiary protein It exploitation structural similarities.this Proteins that share a high degree of similarity throughout their entire lengths are oftenany homologous. Homologous proteins share secondary structure and permission common three-dimensional configuration.prohibited Structural similarities between the Cry1A.105 protein sequence and the toxin (TOXIN5) and public domain (ALLPEPTIDES) database the be sequences werecommercial examined. The extent of each similarity was evaluated by visual inspectionFurthermore, ofany the alignment, the calculated percent identity, and the E score value for that alignment.Consequently, without

The results showed that no biologically relevant structural similarities to human and animal toxins were observed for the Cry1A.105 protein sequence.

5.4.4. Acute oral toxicity study with the Cry1A.105 protein An oral acute toxicity study was conducted with E. coli-produced Cry1A.105 protein. The E. coli-produced Cry1A.105 protein was shown to be equivalent to the MON 89034-

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produced Cry1A.105 (see Part VI, Section 1.2). Acute administration was considered appropriate to assess the potential toxicity of Cry1A.105 protein because toxic proteins generally act via acute mechanisms (Pariza and Foster, 1983; Sjoblad et al., 1992; Pariza and Johnson, 2001).

Two groups of ten male and ten female young adult CD1 mice received an acute high dose of the Cry1A.105 protein by oral gavage. The target dose of 2072 mg/kg body weight was based on the maximum attainable Cry1A.105 concentration of the dosing solution (estimated at 34.1 mg/mL) and a total dose volume of 66.6 mL/kg body weight. The limited solubility of the Cry1A.105 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 2072 mg/kg body weight. On the day of dosing regime.and (Day 0) the two individual doses of 33.3 mg/mL body weightand were administered about AG four hours apart. A separate group of ten male and ten female animals served as protein control group and received bovine serum albumin (BSA) at a dose of 1998 mg/kg body weight. In addition, the vehicle control Bayer(buffer) groupsproperty of tenprotection malespublishing and ten females received the dosing vehicle substance only.of contents its parties.data therefore Following dosing, all mice were observed daily, and body weightsand/or andor food consumption affiliates. were measured weekly. A grossproperty necropsy intellectual examthirdination was performedmay on all animals at its the time of death or the end of the studyas (day 14). the of and is regulatory owner. a document No test article related mortalityany suchor clinical observations were recorded.its There were no reproductiondocument significant differences in body weight,owner cu mulative thisbody weight,of or food consumption between the vehicle or bovine serum albuminunder proteinof control groups and the Cry1A.105 rightsthe this treated group.document No and/ortreatment–related gross pathologicalrights findings were observed at to of fall use of necropsy. the This may distribution,and rights owner Under the conditionssubject of this test, no test article related mortality or other toxicity was observed in the Cry1A.105 treated group.the violateTherefore, the acute oral LD of the be copy 50 Cry1A.105 protein in mice is greaterof than 2072 mg/kg body weight, which was and determinedmay to be NOELdocumentpublication, for this protein. It exploitation this 5.4.5. Exposure toany the Cry1A.105 protein permission Cry1A.105 is present at low levelsprohibited of approximately 0.005% of the total protein in the the grain of MON 89043commercial (Part VI, be Section 4.2.5). A dietary safety assessment based on these levels indicates that the MOEs for Cry1A.105 are equal to or greater than 199,000 for the overallFurthermore, U.S.any population and 79,400 for children aged 3-5 years old, an age group with the highestConsequently, corn consumptionwithout on a body weight basis (see Part VI, Section 3). Also, greater than 95% of Cry1A.105 is digested after 30 seconds incubation in SGF (Part VI, Section 4.2.3). These results suggest that there are no meaningful risks to human and animal health from the dietary exposure to the Cry1A.105 protein.

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5.5. Assessment of the potential for toxicity of the Cry2Ab2 protein The safety assessment of the Cry2Ab2 protein includes an assessment of the similarity of the Cry2Ab2 protein to proteins with an established history of safe use and consumption, bioinformatics analyses of structural similarity of the Cry2Ab2 to known toxins and other proteins that may adversely affect animal or human health, and acute oral toxicity.

5.5.1. Similarity of the Cry2Ab2 protein with proteins with a history of safe use and consumption The Cry2Ab2 protein produced in MON 89034 is derived from the Bt subspecies kurstaki and its amino acid sequence differs from that of the wild-type protein by a single amino acid. The Cry2Ab2 protein has 88% amino acid sequence identity to the Cry2Aa andprotein, and regime. which is present in commercial microbial pestAG control products such as Dipel and Crymax. The Cry2Ab2 proteins produced in MON 89034 and Bollgard II cotton share an identical amino acid sequence. Bollgard II cotton has been on the market since 2003 and there have been no reports of adverse effectsBayer on human property or animal health.publishing protectioncontents of its 5.5.2. Structural similarity of the Cry2Ab2 to knownparties. toxinsdata or other therefore and/oror biologically active proteins affiliates. property intellectualthird may The safety assessment of proteinsits produced in genetically modified crops evaluates as potential health effects throughthe ofa comprehensandive approach, which includes bioinformatic is regulatory owner. analysis of the amino acid sequence of the newlya expresseddocument protein to ensure that the any such its protein is not similar to toxic proteins that are knownreproduction to causedocument adverse health effects. The owner this of comparison of the Cry2Ab2 sequence was performed with the toxin (TOXIN5), and under of this public domain (ALLPEPTIDES)rightsthe database sequences using bioinformatic tools. documentand/or of rights Sequence identity and aminoto of acid similarityfall touse allergens, toxins, and other proteins was assessed using the sequence alignment tool FASTA.the Identified proteins were ranked This may distribution,and according to their degreerights of similarity. The highestowner similarity observed was to pesticidial subject crystal protein Cry2Ab, demonstrating 100% identity over 632 amino acids with and an E be the violate score of zero. Allcopy remaini ng alignmentsof with significant E scores are to Cry protein homologues derived from B. thuringiensisand, Paenibacillus popilliae or Paenibacillus may documentpublication, lentimorbusIt . Based on theseexploitation data, the Cry2Ab2 protein does not share structural congruence withthis any any proteins that may cause adverse effects in humans and animals. permission 5.5.3. Acute oral toxicity studyprohibited with Cry2Ab2 protein the be An acute oral toxicitycommercial assessment was conducted to evaluate potential adverse effects on miceFurthermore, as a resultany from exposure to E. coli-produced Cry2Ab2 protein. The Cry2Ab2 proteinConsequently, producedwithout in E. coli was shown to be equivalent to the MON 89034-produced Cry2Ab2.

Two groups of ten male and ten female CD1 mice received an acute high dose of the Cry2Ab2 protein by gavage. The target dose 2,198 mg/kg body weight was based on the maximum attainable Cry2Ab2 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 the Cry2Ab2 protein precluded its administration as a single dose. Therefore, dosing was

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subdivided into two doses of 33.3 mL/kg body weight (66.6 mL/kg total) to achieve the target dose of 2,198 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 control group of ten male and ten female animals received BSA at a dose of 2,442 mg/kg.

Following dosing, all mice were observed daily, and body weights and food consumption were measured weekly. A gross necropsy examination was performed on all animals at the time of death or the end of the study (day 14).

No mortality or treatment-related clinical observations were observed during the study. Likewise, there were no statistically significant differences in food consumption, body weight, or body weight changes. No gross pathological findings related to consumption regime.and of Cry2Ab2 protein were observed at necropsy. and AG

Under the conditions of this test, no mortality or other evidence of toxicity was observed following Cry2Ab2 administration at the maximumBayer attainableproperty dose. Thpublishingerefore, the acute protectioncontents oral LD50 of the Cry2Ab2 protein in ofmice is greater than 2,198 mg/kg body weight, its which was was determined to be the NOEL for this protein.parties.data therefore and/oror affiliates. 5.5.4. Exposure to the Cry2Ab2property protein intellectual third may its as Cry2Ab2 is present at levelsthe of ofapproximately and 0.001% of the total protein in the grain of is regulatory owner. MON 89043 (Part VI, Section 4.3.5). A dietarya safety assessmentdocument based on these levels any such its indicates that the MOEs for Cry2Ab2 are greaterreproduction than or eqdocumentual toof 981,000 for the overall U.S. population and greater than or ownerequal to 390,000this for children aged 3-5 years old, an under of age group with the highest rightscorn theconsumption on a bodythis weight basis (see Part VI, Section documentand/or rights 3). Also, greater than 99%to ofof Cry2Ab2fall is diusegested ofafter 30 seconds incubation in SGF (Part VI, Section 4.3.3). These results suggest that thethere are no meaningful risks to This may distribution,and human and animal healthrights from the dietary exposureowner to the Cry2Ab2 protein. subject be the violate 5.6. Conclusionscopy of and The Cry1A.105may anddocument Cry2Ab2publication, proteins have been assessed for their potential toxicity It exploitation according to thethis recommendations of Codex. The proteins have a long history of safe use, lack structural anysimilarity to known toxins or biologically active proteins known to permission have adverse effects on mammals,prohibited do not show acute oral toxicity in mice, and constitute a very small portion ofthe the betotal protein present in feed and food derived from MON 89034. commercial Furthermore,any TheConsequently, Cry1A.105without and Cry2Ab2 proteins are from Bt, an organism that has been used commercially in the U.S. since 1958 to produce microbial-derived products with insecticidal activity and whose safety has been demonstrated by over 45 years of use. Bioinformatics analyses demonstrated that the proteins do not share structural or sequence similarities to known toxins or biologically active proteins that are known to cause adverse health effects in humans or animals. Results from acute oral toxicity studies with mice demonstrated that the Cry1A.105 and Cry2Ab2 proteins are not acutely toxic and do not cause any adverse effects even at maximum attainable dose levels.

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Finally, the Cry1A.105 and Cry2Ab2 proteins represent no more than 0.005% and 0.001% of the total protein in the grain of MON 89034, respectively. Taken together these data lead to the conclusion that the Cry1A.105 and Cry2Ab2 proteins are unlikely to have any toxic effect on animals or humans.

References

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Astwood, J.D., J.N. Leach, and R.L. Fuchs. 1996. Stability of food allergens to digestion in vitro. Nat. Biotechnol. 14:1269-1273. and and regime. AG Baum, J.A. 1998. Transgenic Bacillus thuringiensis. Phytoprotection 79(S):127-130.

Baum, J.A., T.B. Johnson, and B.C. Carlton.Bayer 1999. propertyBacillus thuringiensisprotectionpublishing : Natural and of contents Recombinant Biopesticide Products. Pp 189-209. In Methods itsin Biotechnology: parties.data therefore Biopesticides: Use and Delivery, Vol. 5. Hall, F.R. and J.J.and/or Mennor (eds.). Humana Press Inc., Totowa, New Jersey. affiliates. property intellectualthird may its Betz, F.S., B.G. Hammond,the and r.J. Fuchs.as 2000. Safety and advantages of Bacillus of and owner. thuringiensis protectedis plants to control insectregulatory pests. Regul. Toxicol. Pharmacol. a documentits 32:156-173. any such reproductiondocument of owner this under of Bravo, A. 1997. Phylogeneticrights relationshipsthe of Bacillusthis thuringiensis ∆-endotoxin family documentand/or rights proteins and their functionalto of domaifall ns. J. Bacteriol.use of 179(9):2793-2801. the This may distribution,and Brown, J.L. and W.K. Roberts.rights 1976. Evidenceowner that approximately eighty percent of the soluble proteinssubject from Ehrlich ascites cells are N-acetylated. J. Biol. Chem. 251:1009- 1014. be the violate copy of and may documentpublication, Bruce,It B.D. 2000. Chloroplastexploitation transit peptide: structure, function and evolution. Trends Cell Biol. 10:440-447. this any

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De Maagd, R.A., A. Bravo, C. Berry, N. Crickmore, and H.E. Schnepf. 2003. Structure, diversity and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu. Rev. Genet. 37:409-433.

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Federici, B.A. 2002. Case study: Bt crops — a novel mode of insect resistance. Pp 154-200. In Safety Assessment of Genetically Engineered Crops. Atherton, K.A. (ed.). Taylor and Francis, Ltd., London.

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Gao, Y., K.J. Fencil, X. Xu, D.A. Schwedler, J.R. Gilbert, and R.A. Herman. 2006. Purification and characterization of a chimeric Cry1F δ-endotoxin expressed in transgenic cotton plants. J. Agric. Food Chem 54:829-835.

Gill, S.S., E.A. Cowles, and P.V. Pietrantonio. 1992. The mode of action of Bacillus thuringiensis endotoxins. Annu. Rev. Entomol. 37:615-636.

Greenplate, J.T., J.W. Mullins, S.R. Penn, A. Dahm, B.J. Reich, J.A. Osborn, P.R. Rahn, L. Ruschke, and Z.W. Shappley. 2003. Partial characterization of cotton plants expressing two toxin proteins from Bacillus thuringiensis: relative toxin contribution, toxin interaction, and resistance management. J. Appl. Entomol. regime.and 127:340-347. and AG

Hammond, B. and R.L. Fuchs. 1998. Safety evaluation for new varieties of food crops developed through biotechnology. BayerPp 61-79. property In Biotechnology protectionpublishing and Safety Assessment, Vol. 2. Thomas, ofJ.A. (ed.). Taylor & Francis,contents Philadelphia, its Pennsylvania. parties.data therefore and/oror affiliates. Hileman, R.E., A. Silvanovich,property R.E. Goodman, intellectualthird E.A. Rice, G. Holleschak,may J.D. Astwood, its and S.L. Hefle. 2002. Bioinformaticas methods for allergenicity assessment using a the of and comprehensive allergenis database. Int. Arch.regulatory Allergy Immunol. 28:280-291.owner. a document any such its reproductiondocument Hofmann, C., P. Luthy, R. Hutter, andowner V. Pliska. 1988.this Binding ofof the ∆-endotoxin from Bacillus thuringiensis to brush-borderunder membrane of vesicles of the cabbage butterfly rightsthe this (Pieris brassicaedocumentand/or). Eur. J. Biochem. 173:85-91. rights to of fall use of the Höfte, H.This and H.R. Whitely. 1989.may Insecticidaldistribution,and crystal proteins of Bacillus thuringiensis. rights owner Microbiol. Rev.subject 53:242-255. the violate be copy Jensen, O.L., A.V. Podtelejnikov, and M.of Mann. 1997. Identification of the components and of simplemay proteindocument mixturespublication, by high-accuracy peptide mass mapping and database It exploitation searching. thisAnal. Chem. 69:4741-4750. any permission Leiner, I.E. 1994. Implicationsprohibited of antinutritional components in soybean foods. Crit. Rev. Food Sci. Nutr. 34(1):31-67. the be commercial Li,Furthermore, J., J. Carroll,any and D.J. Ellar. 1991. Crystal structure of insecticidal delta-endotoxin fromConsequently, Bacilluswithout thuringiensis at 2.5 Å resolution. Nature 353:815-821.

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McClintock, J.T., C.R. Schaffer, and R.D. Sjoblad. 1995. A comparative review of the mammalian toxidcity of Bacillus thuringiensis-based pesticides. Pest. Sci. 45:95-105.

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Metcalfe, D.D., J.D. Astwood, R. Townsend, H.A. Sampson, S.L. Taylor, and R.L. Fuchs. 1996. Assessment of the allergenic potential of foods derived from genetically engineered crop plants. Crit. Rev. Food Sci. Nutr. 36:S165-S186.

Noteborn, H.P.J.M., M.E. Rienenmann-Ploum, J.H.J. van den Berg, G.M. Alink, L. Zolla, and H.A. Kuiper. 1993. Food safety of transgenic tomatoes expressing the insecticidal crystal protein Cry1Ab from Bacillus thuringiensis and the marker enzyme APH(3’)II. Med Fac. Landbouww. Univ. Gent. 58/4b.

NRC. 1998. Nutrient requirements of swine, 10th edition. National Research Council. National Academy Press, Washington, D.C. regime.and and AG NRC. 1994. Nutrient requirements of poultry, 9th edition. National Research Council. National Academy Press, Washington, D.C. Bayer property protectionpublishing Okunuki, H., R. Techima, T. Shigeta, J.of Sa kushima, H. Akiyama, Y. Goda,contents M. Toyoda, its and J. Sawada. 2002. Increased digestibility of twoparties. productsdata in geneticallytherefore modified food (CP4-EPSPS and Cry1Ab) after preheating. J. Food Hyg.and/or Soc.or Japan 43:68-73. affiliates. property intellectualthird may its Ouellet, D.R., H. Lapierre, and J. Chiquette.as 2003. Effects of corn silage processing and the of and amino acid supplementationis on the performanceregulatory of lactating dairy cows.owner. J. Dairy Sci. a document 86:3675-3684. any such its reproductiondocument owner this of Pariza, M.W. and E.A. Johnson. 2001.under Evaluatingof the safety of icrobial enzyme rightsthe this preparationsdocument usedand/or in food processing: Update for a newrights century. Regul. Toxicol. to of fall use of Pharmacol. 33(2):173-186. the This may distribution,and rights owner Pariza, M.W. andsubject E.M. Foster. 1983. Determining the safety of microbial enzymes used in food processing. J. Food Protec. 46:453-468.the violate be copy of and Pearson,may W.R. 2000.document Flexiblepublication, sequence similarity searching with the FASTA3 program It exploitation package. Methodsthis Mol. Biol. 132:185-219. any permission Rademacher, T.W., R.B. Parekh,prohibited and R.A. Dwek. 1988. Glycobiology. Annu. Rev. biochem. 57:785-838. the be commercial Schnepf,Furthermore, E.,any N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Zeigler, andConsequently, D.H. Dean.without 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:775-806.

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Tabashnik, B.E. 1992. Evaluation of synergism among Bacillus thuringiensis toxins. Appl. Environ. Micobiol. 58(1):3343-3346.

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Thomas, K., G.A. Bannon, S. Hefle, C. Herouet,Bayer M. Holsapplproperty e, protectionG. Ladics,publishing S. MacIntosh, and L. Privalle. 2005. In silico methodsof for evaluating human allergenicitycontents to novel its proteins: International bioinformatics workshopparties. meetingdata report, Februarytherefore 23-24, 2005. Toxicol. Sci. 88:307-310. and/oror affiliates. property intellectualthird may its U.S. Pharmacopeia. 1995. Volume XXIIas (Rockville, MD: United States Pharmacopeia the of and Convention, Inc. is regulatory owner. a document any such its reproductiondocument Yagami, T., Y. Haishima, A. Nakamura,owner H. Osuna, andthis Z. Ikezawa.of 2000. Digestibillity of allergens extracted from natural underrubber latexof and vegetable foods. J. Allergy Clin. rightsthe this Immunol.document 106:752-762.and/or rights to of fall use of the Zhuang, ThisM. and S.S. Gill. 2003.may Modedistribution, andof action of Bacillus thuringiensis toxins. Pp rights owner 213-236. Insubject Chemistry of Crop Protection, Progress and Prospects in Science and Regulation. Boss, G and G. Ramos (eds.)the . violateWiley-VCH Verlag GmbH & Co., KGaA, be copy Weinheim, Germany. of document and may publication,exploitation It this any permission prohibited

the commercial be

Furthermore,any Consequently,without

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Part VII. FOOD AND FEED SAFETY AND NUTRITIONAL ASSESSMENT OF MON 89034

SECTION 1. Corn as the Comparable Food and Feed

Corn is widely used for a variety of food and feed purposes, and it is intended that MON 89034 will be utilized in the same manner and for the same uses as conventional corn. Corn grain and its processed products are consumed in a multitude of human food and animal feed products. Corn forage (as silage) is extensively consumed as an animal feed by ruminants. Further discussion of the food and feed uses of corn are provided in and Section 2 below. and regime. AG SECTION 2. Historical Uses of Corn Bayer property protectionpublishing Corn is the largest crop grown in the U.S.of in terms of acreage planted andcontents net value. In 2005, corn was planted on 81.8 million acres and grain harvested from its75.1 million acres parties.data therefore (NCGA, 2006). The corn grain harvested had an average yieldand/or of 148or bushels per acre, affiliates. with a total production of 11 propertybillion bushels intellectual valuedthird at $21 billion (NCGA,may 2006). In 2005, more than 42 million acresits (52% of the total) were planted with biotechnology- as derived corn seed (USDA-NASS,the of 2006a). and is regulatory owner. a document any such its reproductiondocument The low price and ready availabilityowner of corn has resultedthis in theof development of large volume food, feed and industrial uses. under In 2005, theof major uses of corn grain in the U.S. rightsthe this were: feed/residualdocument (56%);and/or food, seed, industrial (27%);of and,rights export (17%; NCGA, 2006). The food, seed and industrialto of usesfall include usethe production of ethanol (14.6%), high the fructose Thiscorn syrup (4.9%), andmay other distribution, foodand ingredients (8%) including starch, sweetners, and cereals. Only a smallrights proportion (<0.2%)owner of the overall corn produced is used as subject seed. be the violate copy of 2.1. History and utilization of corn and may documentpublication, It exploitation In addition to thethis descriptionany below regarding the history and uses of corn as food and feed, OECD consensus document on corn compositional considerations (OECD, 2002) permission provides an overview of whole andprohibited processed corn fractions for food and feed uses. the commercial be Corn (Zea mays L.) originated in Mexico and was grown as a food crop as early as 2700 B.C.Furthermore, (Salvador,any 1997). The history of corn has been studied extensively and multiple hypothesesConsequently, for withoutits origin and parentage have been advanced (Mangelsdorf, 1974). The preponderance of evidence supports the hypothesis that corn descended from teosinte (Galinat, 1988). The history and development of corn has been discussed previously (see Part IV, Section 1). Corn has been a staple in the human diet for centuries. The native peoples of the Americas domesticated and improved corn after realizing its potential for food, feed, and fuel. By the time European settlers came to America, corn was the main cultivated crop (Kastner, 1980). The settlers rapidly adopted corn as the main staple

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crop, and by the late 19th century, nearly 90% of Americans depended on mainly on corn for their survival (Hardeman, 1981).

Today, the high yield of corn makes it one of the most economical sources of metabolizable energy for food, feed and industrial products. Little whole kernel corn is consumed by humans when compared to corn-based food ingredients, in spite of its great value as a source of energy (OECD, 2002). Therefore, indirect consumption is much greater than direct consumption by humans. Approximately two-thirds of the corn produced in the U.S. is fed to livestock, either as silage, grain, or by the use of processed feeds in the animal diet. Corn is valued for feed and food uses as it does not produce significant quantities of toxins or anti-nutritional factors that warrant analytical or toxicological tests (Watson, 1982; White and Pollak, 1995). regime.and and AG 2.2. Corn as a food source Corn is the leading cereal in the U.S. Most of the human consumption is in the form of Bayer property protectionpublishing corn-based ingredients produced by theof wet mill process including highcontents fructose corn syrup, starch, sweetners, cereals, oil and alcohol. Other food-basedits ingredients are parties.data therefore derived from the dry mill process, and include, corn meal, flour,and/or gritsor and oil. Corn grain affiliates. is also used for the production propertyof tortillas intellectualand otherthird ethnic Mexican preparedmay foods by the alkaline cooking process. Fresh-cookedits corn provides macronutrients, vitamins and as minerals in the human diet.the Cornof is a siandgnificant source of the nutritionally essential is regulatory owner. sulfur-containing amino acids, methionine anda cystine. Corndocument contains colored pigments any such its reproductiondocument called carotenoids, which are primarilyowner responsi ble thisfor the yellowof color of corn grain. The carotenes are precursors for theunder produc tionof of thisvitamin A and also function as antioxidants. Corn and/orgrain rights is a significantthe source of Vitamin E (tocopherol), which also document to fall of rights serves as an antioxidant (Whiteof and Webber, 2003).use the This may distribution,and rights owner Corn is an excellentsubject raw material for the manufacture of starch, not only because of price and availability, but also because the starch is easily recovered in high yield and purity. be the violate Approximately 6.7copy billion pounds of starchof were produced in the U.S. and sold into food and industrial markets in 2004 (CRA, 2005).and Starch can be converted to a variety of may documentpublication, sweetenerIt and fermentation exploitationproducts including high fructose corn syrup and ethanol. Starch is used thisas a anyfood ingredient in: dairy and ice cream; batters and breading; baked goods; soups, sauces and permissiongravies; salad dressings; meat, poultry, and fish analogues; confections; and, in drinks. prohibited the commercial be Corn oil, commercially processed from the germ, is another important food ingredient derivedFurthermore, fromany corn grain. In 2004, approximately one billion pounds of corn oil were Consequently,without produced in the U.S. (CRA, 2005). Although a minor component of overall vegetable oil market, corn oil’s high polyunsaturated fatty acid content has important nutritional and health benefits. Corn oil is considered a premium vegetable oil because of its flavor, color, stability, and clarity at refrigerator temperatures. The nutritional benefits, primarily related to its linoleic fatty acid and vitamin E content, have made it a premium oil for the consumer and for the production of margarine. About 50% of the refined oil is used for frying and salad oil, 25% is used in margarine production, and 25% is used for other purposes (Orthoefer et al., 2003).

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2.3. Corn as a feed source Animal feeding represents the largest use of corn in the U.S. In 2005, approximately 55% of the grain harvested (or 6 million bushels) was used as animal feed (NCGA, 2006). In addition, corn silage harvested from 5.9 million acres (approx. 7% of total acres planted) was fed to livestock (USDA-NASS, 2006b). Corn gluten meal, corn gluten feed, and distillers dried grains, derived as co-products by wet and dry milling, are also important components of livestock feed. In 2004, approximately 10 and 2.6 billion pounds of corn gluten feed and meal, respectively, were produced for the animal feed industry in the U.S. (CRA, 2005).

and Corn grain contains the most metabolizable energy of all the grainsand used inregime. livestock feed (Ensminger et al., 1990). The corn kernel containsAG about 83% carbohydrate in the form of starch, pentosans, dextrins, sugars, cellulose, and hemicellulose. Starch is the biggest component in the carbohydrate fraction and provides most of the energy. The fiber Bayer property protectionpublishing portion includes cellulose and hemicellulose,of which are generally availablecontents to ruminants but not to nonruminants. Corn grain contains approximately 4% (w/w)its oil (White and parties.data therefore Weber, 2003), which has a high content of 18:2 linoleic acid,and/or orone of the essential polyunsaturated fatty acids needed byaffiliates. swine and poultry. Although corn grain has a property intellectualthird may relatively low protein content (10%its dw; ILSI-CCD, 2006) compared to other cereal as grains, it is a major sourthece ofof essentialand amino acids due to the high percentage is regulatory owner. incorporated in animal diets. Corn grain isa a good sourdocumentce of methionine, but a poor any such its source for lysine and tryptophan. Methionine andreproduction lysine aredocument the two most limiting amino owner this of acids for poultry, swine and other livestockunder fed corn-basedof diets (NRC, 2001). rightsthe this documentand/or of rights Calcium and phosphorusto are ofimportant fall mineralsuse in animal nutrition. Corn grain has low levels of calcium, and thus, is not a big contributor to thecalciu m in the animal diet. On the This may distribution,and other hand, corn grainrights is a source of phospownerhorus in the animal diet. However, the subject phosphorus is bound up in the form of phytic acid, which reduces its bioavailability to be the violate nonruminants suchcopy as swine and poultryof (Ensminger et al., 1990). Many producers now add the enzyme phytase to the diet to reandlease the phosphorus bound in phytic acid. may documentpublication, NutritionistsIt incorporate supplementalexploitation sources of calcium, phosphorus, sodium, magnesium, iron,this zinc,any copper, manganese, iodine, and selenium as needed to balance animal diets. Corn grain permissionis a source of a number of vitamins in animal feed, which include: vitamins A, B1 (thiamin),prohibited B2 (riboflavin), B6 (pyridioxine), C (ascorbic acid), E, the folate, niacin andcommercial pantothenic be acid. While the content of niacin in corn grain is relatively high, it exists in a bound form (niacytin) that is not biologically available to monogastric animals.Furthermore, Nutritionistsany supplement animal diets with vitamins, since their levels in corn grainConsequently, are insufficientwithout to meet dietary needs.

Corn silage is a major forage ingredient for feedlot and dairy cattle due its importance as a palatable energy source (Newcomb, 1995). Corn gluten feed and meal are byproducts of the wet milling process and are incorporated into animal diets. Gluten meal contains high levels of protein (~60%) and is an important source of carotenoids. It is commonly used in feed for cattle, fish, poultry, pets, and other animals but primarily in poultry diets. Corn gluten feed (wet or dry) is an excellent feed that is a significant source of protein

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(~20%), low in starch, high in digestible fiber, and low in oil and is used mainly in dairy and beef cattle diets. In addition, with the increasing use of U.S. corn in dry mill plants to produce ethanol, the distillers dried grains co-product will be in greater supply and is expected to replace small amounts of corn grain in livestock and poultry diets.

SECTION 3. Comparison of the Composition and Characteristics of MON 89034 to Conventional Corn

Analyses of the composition of crops produced by methods of biotechnology, and their comparison to conventional varieties, are an integral part of the nutritional and safety assessment of these products. These assessments are conducted according to the principles of comparative risk assessment or substantial equivalence, which hasand been and regime. adopted by a number of international organizationsAG including the World Health Organization, United Nations Food and Agricultural Organization, and the OECD. According to this principle, the food or feed from a biotechnology-derived crop are compared with its conventional counterpartBayer as an initialproperty step protectionin thepublishing safety assessment of contents process. The goal is to determine whether the nutrient and anti-nutrientits levels in grain parties.data therefore and forage tissues from biotechnology-derived corn are and/orcomparableor to those of conventional corn. Further safety assessmentaffiliates. of these components, if necessary, should property intellectualthird may focus only on those components ofits the biotechnology-derived crop that are shown to be as significantly and biologicallythe different of to thoseand of conventional control. is regulatory owner. a document To assess whether the introductionany such or expression of the insect protectionits traits in MON reproductiondocument of 89034 caused any unintended effects,owner compositional analysesthis were conducted on the corn under of grain and forage of MON rights89034.the These tissues were producedthis in replicated field trials, documentand/or rights conducted at five sites toin theof U.S. fall during theuse 2004 ofgrowing season. The levels of the various analytes in MON 89034 were compared to thosethe of conventional control corn, This may distribution,and which has the backgroundrights genetics representativeowner of MON 89034 but does not contain the cry1A.105 andsubject cry2Ab2 genes or produce the Cry1A.105 and Cry2Ab2 proteins. Additionally,be the grain and forage from the15 conventionalviolate corn hybrids produced in the copy of same field trials alongside MON 89034and and control corn, were also subjected to may documentpublication, compositionalIt analyses. Valuesexploitation derived from these conventional hybrids were used as references to generate a 99% tolerance interval for each of the analytes for conventional this any corn. MON 89034 was found to be compositionally equivalent to conventional corn, and permission thus is considered as nutritiousprohibited as conven tional corn for food and feed uses. Further details of this assessmentthe are provided below. commercial be

Furthermore,any 3.1. Consequently, Assessmentwithout of significant nutrients, antinutrients, and key secondary metabolites in corn forage and grain Compositional analyses were conducted, following OECD recommendations, to assess whether the nutrient, antinutrient and secondary metabolite levels in the grain and forage tissues derived from MON 89034 are comparable to those in the conventional control, LH198 x LH172. The conventional control has background genetics similar to MON 89034, but does not contain the cry1A.105 and cry2Ab2 genes. Additional conventional

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corn hybrids currently in the marketplace were also included in the analysis to establish a range of natural variability for each analyte, which is defined by a 99% tolerance interval for that particular analyte (Ridley et al., 2002). Results of the comparisons indicate that MON 89034 is compositionally and nutritionally equivalent to conventional corn hybrids currently in commerce.

Grain and forage tissues of MON 89034 and control corn were harvested from plants grown at each of five field sites during 2004. The field sites were located in regions of the U.S. that are conducive to the growth of corn, and representative of commercial corn production. Seed was planted in a randomized complete block design with three replicates per block. In addition, 15 conventional, commercial corn hybrids were also included as references by growing three different hybrids at each of five sites for a total regime.and of 15 references. The 15 conventional corn reference hybridsand were included to provide AG data for the development of a 99% tolerance interval for each component analyzed. For each compositional component, a 99% tolerance interval was calculated. This interval is expected to contain, with 95% confidence,Bayer 99% propertyof the valuesprotection publishingobtained from the population of commercial references. of It is important to establish the contents99% tolerance its interval from representative conventional corn hybridsparties. fordata each of the analytes,therefore because such data illustrate the compositional variability that naturallyand/or occursor in commercially affiliates. grown varieties. By comparisonproperty to theintellectual 99%third tolerance interval,may any statistically its significant difference between MON 89034as and the control may be put into perspective, the of and and can be assessed foris biological relevance inregulatory the context of the naturalowner. variability in a document corn. any such its reproductiondocument owner this of The compositional analyses were conductedunder on a totalof of 77 components - nine in forage rightsthe this and 68 in grain.document Componentsand/or were selected based on recommendationsrights of the OECD to of fall use of (OECD, 2002). Compositional analyses of the foragethe samples included proximates (protein,This fat, ash, and moisture),may aciddistribution, deandtergent fiber (ADF), neutral detergent fiber rights owner (NDF), mineralssubject (calcium and phosphorus), and carbohydrates by calculation. Compositional analyses of the grain samplesthe includedviolate proximates (protein, fat, ash, and be copy moisture), ADF, NDF, total dietary fiberof (TDF), amino acids, fatty acids (C8-C22), and vitaminsmay (B1, B2, B6,document E,publication, niacin, and folic acid), antinutrients (phytic acid and raffinose), It exploitation secondary metabolitesthis (furfural, ferulic acid, and p-coumaric acid), minerals (calcium, copper, iron, magnesium,any manganese, phosphorus, potassium, sodium, and zinc), and permission carbohydrates by calculation. prohibited Methods for analysis were based on internationally recognized procedures and literature publications (see Appendix F). the be commercial StatisticalFurthermore, analysesany of the compositional data were conducted using a mixed model analysisConsequently, of variance.without Each individual analyte for MON 89034 was compared to that of the conventional control, for the combination of all five sites (i.e., the combined-site) and for each individual site. The statistical significance was defined at the level of p<0.05. Of the 77 components analyzed, 16 components had greater than 50% of the analytical values that were below the limit of quantitation, and therefore, were not included in the statistical analyses.

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Statistical analyses of the remaining 61 components (77 minus the 16) were conducted for comparison of MON 89034 with control corn. The overall data set was examined for evidence of biologically relevant changes. Based on this evaluation and the results of statistical analyses, analytes for which the levels were not statistically different were deemed to be present at equivalent levels between MON 89034 and the control. Analyses using data from the combined sites indicated that there were no statistical differences in the levels of 95% of the analytes (58 of the 61). Statistical analyses for the combined-site data are presented in Table VII.1 for forage and Tables VII.2 - 7 for grain. Analyses using data from the five single sites indicated that there were no statistically significant differences in analytes for 261 of 305 comparisons made between MON 89034 and the control. Table VII.8 summarizes the statistical differences observed between MON 89034 and the control for the combined-site and individual sites. regime.and Appendix G provides compostion data from the individual sitesand and Appendix H provides AG supplementary composition data, where the amino acid and fatty acid values are expressed in alternative units. Bayer property protectionpublishing 3.2. Levels of nutrients in corn forageof and grain contents its parties.data therefore For the combined-site analyses, statistical differences betweenand/or MONor 89034 and control affiliates. corn were observed for three propertyanalytes, whintellectualich thirdincluded phosphorus inmay forage, and 18:0 stearic and 20:0 arachidic acids inits grain (Table VII.8). The differences observed are as generally small (3.4 – 19.2%),the consideringof and the range of natural variability, and the mean is regulatory owner. levels and ranges of MON 89034 are wella within thedocument 99% tolerance intervals for any such its reproductiondocument commercial corn. The mean levels ownerand range s of phosphorusthis inof forage, and 18:0 stearic and 20:0 arachidic acids in grain, wereunder also withinof thethis ranges in the International Life Sciences Institute Cropand/or Compositionrights the Database (ILSI-CCD, 2006), as well as within document to fall of rights published literature ranges. Therefore,of it is concludeduse that MON 89034 and control corn the are compositionallyThis equivalentmay based distribution, onand analyses of the combined-site data. The rights owner reported ILSI andsubject published literature ranges for the analytical components present in corn are summarized in Table VII.9. be the violate copy of The reproducibility and trends across sites andwere also examined, and comparisons made to may documentpublication, conventionalIt corn hybrids usingexploitation the 99% tolerance intervals. Of the 44 statistical differences observedthis any in the individual site analyses, 33 were only observed at one site. There were no consistent trendspermission and, except in two cases, the mean and ranges of the analytes in MON 89034 were prohibitedwell within the 99% tolerance interval for conventional the corn. The meancommercial levels and be ranges for calcium and methionine in grain were slightly outside the 99% tolerance interval but within the ILSI database. It is concluded that these differencesFurthermore, anyare not biologically significant. Of the remaining 11 differences observed at Consequently,without more than one site, there were no analytes that were consistently and statistically different across five or four sites. In addition, there were no analytes that showed statistically significant differences in three sites that had not been previously observed (i.e., 20:0 arachidic acid) to be different in the combined-site analysis. Statistically significant differences were observed in as many as two sites for three analytes (carbohydrates, copper, iron), which were not previously found to be different (i.e., 18:0 stearic acid) in the combined-site analysis. For carbohydrates and iron, the observed differences from the control were small and lower at one site and higher at the other site (Table VII.8). As

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there is no evidence of any reproducibility across sites, it is concluded that the statistical differences are not biologically relevant for carbohydrates and iron. For copper, the values for MON 89034 were higher than the control at both sites (Table VII.8). The observed differences are small in magnitude, and the mean levels and ranges of MON 89034 are well within the 99% tolerance interval. Therefore, it is concluded that the statistical differences for copper are not biologically relevant.

Based on the data and information presented above, it was concluded that corn grain and forage derived from MON 89034 are compositionally and nutritionally equivalent to those of conventional corn. The few statistical differences between MON 89034 and control corn likely reflect the natural variability of the components since the mean levels of analytes for MON 89034 are well within the 99% tolerance intervals for conventional regime.and corn, and/or within the ranges in the ILSI database (ILSI-CCD,and 2006) and the scientific AG literature.

3.3. Levels of key anti-nutrients and secondaryBayer metabolitesproperty inprotection cornpublishing forage and grain of contents its parties.data therefore A description of the anti-nutrients and key secondary metabolitesand/oror present in corn is affiliates. provided in the OECD consensusproperty document intellectual onthird compositional considerationsmay (OECD, 2002). The anti-nutrients includeits phytic acid, 2,4-dihydroxy-7-methoxy-2H-1,4- as benzoxazin-3(4H)-one (DIMBOA)the of , raffinose,and and trypsin and chymotrypsin inhibitors. is regulatory owner. The secondary metabolites include furfural,a ferulic acid,document and p-c oumaric acid. The any such its reproductiondocument OECD does not recommend analysisowner of DIMBOA duethis to the variableof levels found across corn hybrids. Similarly, analysis ofunder trypsin ofand thischymotrypsin inhibitors is not recommended becauseand/or theyrights occurthe at low levels in corn and are not considered document to fall of rights nutritionally significant. Accordingof to the useOECD (2002), “in considering the anti- the nutrientsThis and natural toxins inmay maize, distribution, onlyand phytic acid is significant to the animal feed”. rights owner The anti-nutrientssubject phytic acid and raffinose, and the secondary metabolites furfural, ferulic acid and p-coumaric acid were analyzed in the grain of MON 89034 and be the violate compared to thosecopy of control corn. Furfurof al was excluded from further consideration since the levels present in MON 89034 andand cont rol corn were at or below the limit of may documentpublication, quantitationIt of the assay. exploitation this any The overall data set was examinedpermission for evidence of biologically relevant changes. In addition, statistical analyses wereprohibited applied to assess significant differences of each analyte the at p<0.05. As commercialdescribed in be Section 3.1, 58 of 61 comparisons in the combined-site analysis and 261 of 305 comparisons in the single site analysis showed no statistically significantFurthermore, anydifferences between MON 89034 and control corn. The few observed Consequently,without statistical differences were not considered biologically relevant since these differences were not reproducible across sites and no consistent trends were observed. Furthermore, the mean levels and ranges of MON 89034 analytes were within the 99% tolerance intervals, and/or within the ILSI database and literature ranges. The statistical summaries for the anti-nutrients are described under the grain analyses, where the combined-site data are presented in Table VII.7. The analytes that are statistically different between MON 89034 and control corn are presented in Table VII.8. The ILSI and literature ranges for corn are provided in Table VII.9.

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Phytic acid is considered an important anti-nutrient for animals, especially nonruminants, since it can significantly reduce the bioavailability of phosphorus in corn tissues. Feed formulators add the enzyme phytase to swine and poultry diets to improve the utilization of phosphorus. Ruminants are more efficient in utilizing phytic acid since microbes in the rumen produce phytase that breaks down phytate and releases phosphorus (Ensminger et al., 1990). Compositional analyses of the grain indicated that phytic acid was present at similar levels in MON 89034 and control corn, and no statistical differences were observed for all comparisons.

Raffinose is a low molecular weight carbohydrate present in corn grain that is considered an anti-nutrient due to the gas production and resulting flatulence caused by consumption regime.and (Maynard et al., 1979). Composition analyses of the grain indicatedand that raffinose was AG present at similar levels in MON 89034 and control corn, and no statistical differences were observed for all comparisons. Bayer property protectionpublishing The phenolic acids, ferulic acid and ofp-coum aric acid, are structural contentsand functional its components of plant cells (Kroon and Williamson, 1999).parties. data They are found thereforein vegetables, fruit and cereals and act as natural pesticides against and/orinsector and fungal pests. affiliates. Composition analyses of the grainproperty indicated intellectual thatthird ferulic acid was presentmay at similar levels its in MON 89034 and control corn, andas no statistical differences were observed for all the of and comparisons. A statisticalis significant differenceregulatory was observed for p-coumaricowner. acid at one a document site but not at the other sites.any suchSince there was no trend, the statisticallyits significant reproductiondocument difference for p-coumaric acid is notowner consid ered reproduciblethis andof hence not biologically significant. under of rightsthe this documentand/or rights to of fall use of Based on these results, it is concluded that the levels ofthe anti-nutri ents and key secondary metabolitesThis (as defined by themay OECD) distribution, inand MO N 89034 are comparable to those found in rights owner conventional corn.subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VII.1. Comparison of Proximates, Fiber, and Mineral Content in Forage from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites Difference (Test minus Control) Control Mean ± Commercial 1 1 and Test Mean ± S.E. S.E. Mean ± S.E. 95% CI and regime. (Range) Analytical Component1 (Range) (Range) (Range) AG(Lower,Upper) p-Value [99% Tolerance Int.2] Fiber Acid Detergent Fiber (% DW) 28.95 ± 1.69 27.26 ± 1.69 1.69 ± 1.18 -0.81,4.19 0.170 (26.72 - 38.94) (22.60 - 35.85) (19.93 - 35.59) (-6.22 - Bayer10.45) property protectionpublishing [16.76,43.76] of contents Neutral Detergent Fiber (% DW) 39.69 ± 1.32 37.60 ± 1.32 2.09 ± 1.40 -0.88,5.05 its 0.155 (33.70 - 46.74) parties.data therefore (33.99 - 46.82) (31.44 - 43.96) (-3.47 - 7.47) and/oror [25.94,55.67] Mineral affiliates. Calcium (% DW) 0.20 ± 0.019 0.19 ± 0.019 property 0.0066 intellectual ± 0.011third -0.017,0.031 may 0.569 (0.11 - 0.29) its (0.16 - 0.24) (0.13 - 0.28) (-0.036as - 0.063) [0.016,0.38] the of and is regulatory owner. Phosphorus (% DW) 0.25 ± 0.011 0.21 ± 0.011 0.040 ± 0.014a 0.011,0.069document 0.010 (0.14 - 0.25) any such its (0.22 - 0.32) (0.15 - 0.25) (-0.0019 - 0.13) reproduction document [0.071,0.32] owner this of Proximate under of rightsthe this Ash (% DW) 3.70 ± 0.27 documentand/or 3.90 ± 0.27 -0.20 ± 0.21 of -0.65,0.25rights 0.356 (3.40 - 5.45) (2.51 - 4.67) (2.59to - 5.10)of fall (-1.72 - 0.97)use [1.93,6.31] the This may distribution,and Carbohydrates (% DW) 86.90 ± 0.43 86.69rights ± 0.43 0.21 ± 0.53owner -0.91,1.33 0.697 (84.88 - 88.39) (84.93 - 89.13) subject (84.36 - 89.57) (-4.23 - 4.41) [83.05,90.74] be the violate Moisture (% FW) 72.20 ± 1.35 copy 71.53 ± 1.35 0.67of ± 0.52 -0.44,1.77 0.220 (64.90 - 77.40) (68.50 - 75.40) (65.90 - 76.80) (-3.50 -and 4.20) [57.62,86.45] may documentpublication, It exploitation Protein (% DW) 7.82 ± 0.27 this 7.70 ± 0.27 0.13 ± 0.26 -0.43,0.68 0.635 (6.58 - 8.82) (6.34 - 8.98) any (6.06 - 8.87) (-2.32 - 2.35) [4.78,10.38] permission Total Fat (% DW) 1.57 ± 0.24 1.71 ± 0.24 prohibited -0.13 ± 0.23 -0.59,0.32 0.558 (0.58 - 3.11) the (0.63 - 3.17) commercial (0.77 - 2.91) be (-2.28 - 1.95) [0,4.54] 1 DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval. 2 With 95% confidence, interval Furthermore,contains 99%any of the values expressed in the population of commercial lines. Negative limits were set to zero. Consequently,without

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Table VII.2. Comparison of the Amino Acid Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites

Difference (Test minus Control) and Control Mean ± and regime. Commercial Analytical Test Mean ± S.E. 1 S.E. Mean ± S.E. 95% CI1 (Range) 1 AG Component (Range) (Range) (Range) (Lower,Upper) p-Value [99% Tolerance Int.2] Amino Acid (% DW) Alanine (% DW) 0.77 ± 0.039 0.78 ± 0.039 -0.0070 ± 0.019 -0.046,0.032 0.709 (0.67 - 0.96) Bayer property protectionpublishing (0.64 - 0.89) (0.67 - 0.89) (-0.13of - 0.089) contents [0.48,1.08] its parties.data therefore Arginine (% DW) 0.48 ± 0.013 0.47 ± 0.013 0.011 ± 0.012 -0.014,0.036and/or or 0.361 (0.37 - 0.49) (0.38 - 0.52) (0.41 - 0.51) (-0.090 - 0.062) [0.33,0.56] affiliates. property intellectual third may Aspartic acid (% DW) 0.68 ± 0.029 0.67 ± 0.029 its 0.0038 ± 0.015 -0.028,0.036 0.804 (0.57 - 0.77) as (0.56 - 0.78) (0.60 - 0.76)the of (-0.11 - 0.078)and [0.43,0.90] is regulatory owner. a document Cystine (% DW) 0.23 ± 0.0057 0.23 ± 0.0057any such 0.0023 ± 0.0038 -0.0057,0.010 its 0.554 (0.20 - 0.24) reproductiondocument (0.20 - 0.26) (0.21 - 0.25) (-0.022owner - 0.023) this of [0.18,0.27] under of rightsthe this Glutamic acid (% DW) 1.97 ± 0.097 document 1.99and/or ± 0.097 -0.012 ± 0.049 of -0.11,0.091rights 0.809 (1.71 - 2.41) (1.63 - 2.29) (1.70 -to 2.26) of fall (-0.33 - 0.24)use [1.25,2.75] the Glycine (% DW) 0.38 ± 0.0087This 0.38 ± 0.0087 may 0.0042distribution, ±and 0.0071 -0.011,0.019 0.566 (0.32 - 0.40) rights owner (0.32 - 0.41) subject (0.36 - 0.41) (-0.067 - 0.035) [0.28,0.46]

be the violate Histidine (% DW) 0.31 ± 0.011 copy 0.31 ± 0.011 0.0027of ± 0.0055 -0.0090,0.014 0.632 (0.26 - 0.33) (0.25 - 0.35) (0.28document - 0.34) (-0.050 - and0.030) [0.22,0.38] may publication, It exploitation Isoleucine (% DW) 0.36 ± 0.018 this 0.36 ± 0.018 -0.00003 ± 0.0088 -0.019,0.019 0.997 (0.32 - 0.45) (0.30 - 0.43) (0.30any - 0.42) (-0.056 - 0.041) [0.23,0.51] permission prohibited the be commercial Furthermore,any Consequently,without

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Table VII.2 (cont). Comparison of the Amino Acid Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites

Difference (Test minus Control) Test Mean ± regime.and 1 Control Mean ± and Commercial Analytical S.E. S.E. Mean ± S.E. AG 95% CI1 (Range) 1 Component (Range) (Range) (Range) (Lower,Upper) p-Value [99% Tolerance Int.2] Amino Acid (% DW) Leucine (% DW) 1.31 ± 0.077 1.32 ± 0.077 -0.014 ±Bayer 0.036 property -0.089,0.062 protection publishing 0.700 (1.14 - 1.68) (1.09 - 1.57) (1.08 - 1.55) (-0.21of - 0.16) contents [0.77,1.92] its parties.data therefore Lysine (% DW) 0.33 ± 0.0097 0.32 ± 0.0097 0.0088 ± 0.0078 -0.0077,0.025and/or or 0.273 (0.24 - 0.34) (0.26 - 0.36) (0.29 - 0.36) (-0.056affiliates. - 0.033) may [0.20,0.40] property intellectual third its Methionine (% DW) 0.23 ± 0.0064 0.22 ± 0.0064 0.0038as ± 0.0047 -0.0061,0.014 0.427 (0.17 - 0.22) the of and (0.20 - 0.27) (0.20 - is0.24) (-0.017 - 0.028)regulatory owner. [0.14,0.25] a document any such its Phenylalanine (% DW) 0.51 ± 0.028 0.52 ± 0.028 -0.0012 ± 0.013 reproduction -0.029,0.026document of 0.925 (0.45 - 0.65) (0.43 - 0.61) (0.43 - 0.60) owner(-0.080 - 0.067) this [0.32,0.73] under of this and/orrights the Proline (% DW) 0.93 ± 0.030 document 0.93 ± to 0.030 of fall 0.0034 ± 0.019 of -0.037,0.044rights 0.861 (0.83 - 1.11) (0.79 - 1.05) (0.83 - 1.01) (-0.15 - 0.10)use [0.68,1.21] the This may distribution,and Serine (% DW) 0.52 ± 0.022 0.52 ±rights 0.022 -0.0046 ± 0.012owner -0.030,0.021 0.703 (0.45 - 0.62) (0.44 - 0.61) subject (0.46 - 0.60) (-0.087 - 0.058) [0.34,0.71] the violate be copy Threonine (% DW) 0.33 ± 0.010 0.33 ± 0.010 0.00063of ± 0.0074 -0.015,0.016 0.933 (0.29 - 0.37) (0.27 - 0.37) (0.29 - 0.36) (-0.052 - and0.039) [0.24,0.41] may documentpublication, It exploitation Tryptophan (% DW) 0.056 ± 0.0018 this 0.056 any ± 0.0018 0.00031 ± 0.0013 -0.0025,0.0031 0.817 (0.043 - 0.059) (0.048 - 0.064) (0.045 - 0.063) (-0.0055 - 0.0072) [0.032,0.072] permission prohibited

the commercial be Furthermore,any Consequently,without

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Table VII.2 (cont). Comparison of the Amino Acid Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites

Difference (Test minus Control) and Test Mean ± and regime. 1 Control Mean ± Commercial Analytical S.E. S.E. Mean ± S.E.AG 95% CI1 (Range) 1 Component (Range) (Range) (Range) (Lower,Upper) p-Value [99% Tolerance Int.2] Amino Acid (% DW) Bayer property publishing Tyrosine (% DW) 0.37 ± 0.015 0.36 ± 0.015 0.0088 ± 0.016 -0.026,0.043protection contents 0.596 (0.25 - 0.40) (0.22 - 0.43) (0.24 - 0.42) (-0.21of - 0.14) [0.17,0.52] its parties.data therefore Valine (% DW) 0.49 ± 0.020 0.49 ± 0.020 0.0034 ± 0.010 -0.019,0.026and/or or 0.748 (0.42 - 0.55) (0.40 - 0.55) (0.43 - 0.55) affiliates.(-0.084 - 0.055) [0.35,0.62] property intellectualthird may its 1 DW = dry weight; S.E. = standard error; CI = confidence theinterval. as of and owner. 2 With 95% confidence, interval contains 99% of the valuesis expressed in the populationregulatory of commercial lines. Negative limits were set to zero. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table VII.3. Comparison of the Fatty Acid Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites Difference (Test minus Control) Control Mean ± Commercial Test Mean ± S.E. 1 S.E. Mean ± S.E. 95% CI1 regime.and (Range) 1 and 2 Analytical Component (Range) (Range) (Range) AG (Lower,Upper) p-Value [99% Tolerance Int. ] Fatty Acid (% Total FA) 16:0 Palmitic (% Total FA) 9.19 ± 0.060 9.12 ± 0.060 0.071 ± 0.049 -0.034,0.18 0.171 (9.10 - 12.55) (8.98 - 9.46) (8.91 - 9.34) (-0.14 - 0.33) [6.12,15.67] Bayer property protectionpublishing of contents 16:1 Palmitoleic (% Total FA) 0.13 ± 0.0058 0.12 ± 0.0058 0.0022 ± 0.0054 -0.0093,0.014 0.696 (0.050 - 0.19) its (0.11 - 0.14) (0.048 - 0.14) (-0.012 - 0.079) parties.data therefore [0,0.28] and/oror affiliates. 18:0 Stearic (% Total FA) 1.89 ± 0.021 1.82 ± 0.021property 0.072intellectual ± 0.021third 0.028,0.12 may 0.002 (1.57 - 2.45) (1.79 - 2.03) (1.76 - 1.87) its (-0.055 - 0.18) [0.86,2.98] the as of and owner. 18:1 Oleic (% Total FA) 24.96 ± 0.34 24.84is ± 0.34 0.12 ± 0.20regulatory -0.32,0.55 0.574 (21.17 - 35.33) a document (23.38 - 25.75) (23.62 -any 26.66) such (-1.48 - 1.15) its [7.51,46.46] reproductiondocument owner this of 18:2 Linoleic (% Total FA) 61.82 ± 0.40 62.07 ± 0.40 under -0.25 ± 0.23 of -0.73,0.24 0.292 (50.33 - 63.59) (60.85 - 63.61) (60.51rights - 63.41)the (-1.62 - 1.24) this [39.41,76.74] documentand/or rights to of fall use of 18:3 Linolenic (% Total FA) 1.19 ± 0.027 1.22 ± 0.027 -0.028 ± 0.016 the -0.063,0.0061 0.099 (0.93 - 1.52) (1.12This - 1.23) (1.15 - 1.43)may distribution, (-0.23and - 0.036) [0.63,1.77] rights owner subject 20:0 Arachidic (% Total FA) 0.39 ± 0.0062 0.38 ± 0.0062 0.013 ± 0.0031 0.0063,0.019 <0.001 (0.32 - 0.47) be the violate (0.36 - 0.42) copy (0.36 - 0.40) (-0.019of - 0.032) [0.23,0.54]

document and 20:1 Eicosenoic (% Total FA) 0.28may ± 0.0040 0.28publication, ± 0.0040exploitation 0 ± 0.0024 -0.0051,0.0051 0.999 (0.23 - 0.32) It(0.26 - 0.29) (0.25 - 0.29) (-0.014 - 0.011) [0.15,0.39] this any 22:0 Behenic (% Total FA) 0.16 ± 0.0050 0.15 ± 0.0050 0.0027 ± 0.0062 -0.010,0.016 0.665 (0.12 - 0.19) permission (0.13 - 0.20) (0.13 - 0.18)prohibited (-0.019 - 0.029) [0.081,0.23] the be 1 FA = fatty acid; S.E. = standard error; CI = confidencecommercial interval. 2 With 95% confidence, intervalFurthermore, contains any99% of the values expressed in the population of commercial lines. Negative limits were set to zero. Consequently,without

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Table VII.4. Comparison of the Mineral Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites

Difference (Test minus Control) Control Mean ± regime.and Commercial 1 and1 Test Mean ± S.E. S.E. Mean ± S.E.AG 95% CI (Range) Analytical Component1 (Range) (Range) (Range) (Lower,Upper) p-Value [99% Tolerance Int.2]

Mineral Bayer property protectionpublishing Calcium (% DW) 0.0050 ± 0.00034 0.0049 ± 0.00034 0.of00016 ± 0.00011 -0.00008,0.00040 contents 0.180 (0.0031 - 0.0049) (0.0038 - 0.0066) (0.0040 - 0.0059) (-0.00027 - 0.00090) its [0.0016,0.0059] parties.data therefore and/oror Copper (mg/kg DW) 1.74 ± 0.38 2.07 ± 0.37 -0.33 ± 0.53 -1.45,0.79 0.547 (1.15 - 3.56) affiliates. (1.33 - 2.38) (1.26 - 4.54)property (-2.96intellectual - 0.78)third may [0,4.20] its as Iron (mg/kg DW) 21.40 ± 1.00 22.20the ± 0.99of -0.and80 ± 0.67 -2.22,0.62 0.250 (18.04 - 29.22) is regulatory owner. (19.23 - 25.23) (19.03 - 28.26) (-6.50 -a 5.90) document [8.88,34.51] any such its reproductiondocument Magnesium (% DW) 0.12 ± 0.0043 0.12 ± 0.0043 owner -0.00028 ± 0.0021 this -0.0047,0.0041of 0.893 (0.099 - 0.14) (0.10 - 0.14) (0.11 - 0.14) under(-0.018 - 0.011)of [0.075,0.17] rightsthe this documentand/or of rights Manganese (mg/kg DW) 6.79 ± 0.29 to 6.51 ± of0.29 fall 0.28 ±use 0.21 -0.18,0.73 0.213 (5.56 - 8.64) (5.43 - 9.32) (5.57 - 8.00) (-1.54 - 2.36) the [3.17,9.99] This may distribution,and rights owner Phosphorus (% DW) 0.33 ± 0.0095subject 0.33 ± 0.0095 0.00039 ± 0.0043 -0.0087,0.0095 0.929 (0.25 - 0.37) (0.27 - 0.36) (0.29 - 0.36) (-0.038 - 0.026) [0.18,0.45] be the violate copy of Potassium (% DW) 0.36 ± 0.0065 document 0.36 ± 0.0065 0.0032and ± 0.0042 -0.0052,0.012 0.450 (0.32 - 0.40) (0.32may - 0.40) publication, (0.34 - 0.40) (-0.030 - 0.035) [0.26,0.46] It exploitation this Zinc (mg/kg DW) 22.05 ± 1.14 any 21.91 ± 1.14 0.14 ± 0.51 -0.94,1.22 0.788 (16.72 - 34.04) (18.91 - 26.89) (18.81permission - 26.04) (-3.37 - 3.19) [7.16,38.55] prohibited 1 the DW = dry weight; S.E. = standard error; CI =commercial confidence interval. be 2 With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero. Furthermore,any Consequently,without

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Table VII.5. Comparison of the Proximates and Fiber Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites

Difference (Test minus Control) Test Mean ± Control Mean ± regime.and Commercial 1 and1 S.E. S.E. Mean ± S.E. AG 95% CI (Range) Analytical Component1 (Range) (Range) (Range) (Lower,Upper) p-Value [99% Tolerance Int.2] Ash (% DW) 1.41 ± 0.036 1.39 ± 0.036 0.014 ± 0.041 -0.072,0.10 0.734 (1.12 - 1.62) (1.25 - 1.56) (1.28 - 1.51) (-0.11 - 0.13) [0.74,1.96] Bayer property protectionpublishing of contents Carbohydrates (% DW) 84.85 ± 0.42 84.96 ± 0.42 -0.11 ± 0.18 -0.50,0.28 its 0.562 (82.91 - 86.78) (83.29 - 86.52) (83.58 - 86.22) (-1.42 - 0.84) parties.data therefore [81.08,88.80] and/or or affiliates. Moisture (% FW) 9.52 ± 0.77 9.50 ± 0.77 property0.021 intellectual ± 0.22 third -0.44,0.48 may0.923 (7.60 - 15.30) (7.89 - 12.80) (7.86 - 13.10) its (-1.00 - 0.87) [0.45,19.52] as the of and is regulatory owner. Protein (% DW) 10.43 ± 0.42 10.36 ± 0.42 0.070 ± 0.19a -0.34,0.48document 0.725 (9.33 - 11.82) (8.54 - 11.98) (9.22 - 11.52)any such (-1 .26 - 1.28) its [7.54,13.13] reproductiondocument owner this of Total Fat (% DW) 3.32 ± 0.069 3.29 ± 0.069 0.025under ± 0.089 of this -0.16,0.21 0.784 (2.66 - 3.71) (3.05 - 3.89) (3.05 - 3.75)rights the (-0.50 - 0.29) [2.20,4.55] documentand/or of rights to of fall use Fiber the Acid Detergent Fiber (% DW) 5.48This ± 0.19 5.27 ± 0.19 may distribution, 0.21 and± 0.25 -0.30,0.72 0.410 (4.11 - 6.33) rights owner (3.82 - 7.24) subject (4.17 - 7.00) (-3.18 - 3.07) [2.77,7.56]

be the violate Neutral Detergent Fiber (% DW) 10.06 ± 0.37 copy 9.75 ± 0.37 0.31of ± 0.34 -0.41,1.03 0.370 (8.20 - 11.30) (8.59 - 12.08) (8.48 - 11.75) (-2.26 -and 2.05) [5.93,13.63] may documentpublication, It exploitation Total Dietary Fiber (% DW) 15.17 ± 0.47 this 14.67 ± 0.47 0.50 ± 0.54 -0.66,1.65 0.375 (12.99 - 18.03) (13.39 - 17.02) (12.82any - 17.62) (-3.61 - 4.20) [9.20,20.27] permission 1 DW = dry weight; FW = fresh weight; S.E. = standard error; CIprohibited = confidence interval. 2 the With 95% confidence, interval contains 99% commercialof the values expressed be in the population of commercial lines. Negative limits were set to zero. Furthermore,any Consequently,without

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Table VII.6. Comparison of the Vitamin Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites

Difference (Test minus Control) Control Mean ± regime.and Commercial 1 and1 Analytical Test Mean ± S.E. S.E. Mean ± S.E. AG 95% CI (Range) Component1 (Range) (Range) (Range) (Lower,Upper) p-Value [99% Tolerance Int.2]

Vitamin Bayer property protectionpublishing Folic Acid (mg/kg DW) 0.35 ± 0.037 0.36 ± 0.037 -0.0080of ± 0.022 -0.054,0.038 contents 0.717 (0.13 - 0.45) (0.26 - 0.48) (0.23 - 0.53) (-0.11 - 0.11) its [0.012,0.69] parties.data therefore and/oror Niacin (mg/kg DW) 30.08 ± 1.11 29.59 ± 1.11 0.48 ± 0.65 -0.82,1.79 0.461 (16.17 - 29.19) affiliates. (25.72 - 34.84) (24.93 - 35.75) property (-4 .44intellectual - 5.64)third may [6.97,37.83] its as Vitamin B1 (mg/kg DW) 3.07 ± 0.13 2.94 ± 0.the13 of 0.13 ±and 0.17 -0.24,0.49 0.474 (2.19 - 5.60) is regulatory owner. (2.39 - 3.44) (2.39 - 3.36) (-0.66 - 0.68)a document [0.37,6.35] any such its reproductiondocument Vitamin B2 (mg/kg DW) 1.42 ± 0.046 1.42 ± 0.046 owner 0.0015 ± 0.050 this -0.099,0.10 of 0.976 (1.34 - 1.91) (1.24 - 1.65) (1.16 - 1.61) (-0.30under - 0.45) of [0.91,2.30] rightsthe this documentand/or of rights Vitamin B6 (mg/kg DW) 6.22 ± 0.23 6.26 to± 0.23 of fall-0.036 ± 0.18use -0.41,0.34 0.838 (5.08 - 7.47) (5.28 - 6.99) (5.37 - 6.80) (-0.72 - 1.10) the [3.12,9.30] This may distribution,and rights owner Vitamin E (mg/kg DW) 6.77 ± 0.42 subject 6.63 ± 0.42 0.14 ± 0.36 -0.64,0.91 0.714 (2.71 - 13.94) (5.55 - 8.62) (2.72 - 9.02) (-2.35 - 3.83) [0,20.49] be the violate copy of 1 DW = dry weight; S.E. = standard error; CI = confidencedocument interval. and 2 may publication, With 95% confidence, interval containsIt 99% of the values expressedexploitation in the population of commercial lines. Negative limits were set to zero. this any

permission prohibited the commercial be Furthermore,any Consequently,without

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Table VII.7. Comparison of the Anti-nutrients and Secondary Metabolites Content in Grain from Test (MON 89034) and Conventional Control (LH198 x LH172) Corn for Combined Sites

Antinutrient Bayer property protectionpublishing Phytic Acid (% DW) 0.75 ± 0.050 0.73 ± 0.050 0.016of ± 0.027 -0.037,0.069 contents 0.537 (0.50 - 0.94) (0.53 - 0.87) (0.56 - 0.88) (-0.15 - 0.18) its [0.21,1.22] parties.data therefore and/or Secondary Metabolite or affiliates. Ferulic Acid (µg/g DW) 2131.38 ± 108.09 2148.05 ± 108.09property -16.67 intellectual ± 50.08third -116.98,83.65 may 0.740 (1412.68 - 2297.36) (1790.25 - 2525.31) (1878.66 - 2669.85) its (-330.17 - 264.79) [1136.69,2806.24] as the of and is regulatory owner. p-Coumaric Acid (µg/g DW) 194.25 ± 7.12 183.96 ± 7.12 10.28 ± 7.08a -4.73,25document.30 0.165 (99.30 - 285.75) (166.11 - 253.04) (167.76 - any210.13) such (-24.37 - 70.84) its [0,378.57] reproductiondocument owner this of 1 DW = dry weight; S.E. = standard error; CI = confidence interval. under of this 2 With 95% confidence, interval contains 99% of theand/or valuesrights expressedthe in the population of commercial lines. Negative limits were set to zero. document to fall of rights of 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 FDA BNF No. 00105 / Monsanto 06-CR-161F Page 178 of 323

Table VII.8. Summary of Differences (p<0.05) for the Comparison of Analyte Levels for MON 89034 vs. the Conventional Control (LH198 x LH172) and Commercial References Mean Difference and (MON 89034and minus Control)regime. MON 89034 Control AG % of Signif. MON 89034 Commercial Analytical Component (Units)1 Mean Mean Control (p-Value) (Range) Tolerance Int.2 Statistical Differences Observed in Combined Site Analyses Mineral Bayer property protectionpublishing Forage Phosphorus (% DW) 0.25 of 0.21 19.24 0.010 contents (0.22 - 0.32) [0.071,0.32] its Fatty Acid parties.data therefore Grain 18:0 Stearic (% Total FA) 1.89 1.82 3.97 and/oror0.002 (1.79 - 2.03) [0.86,2.98] affiliates. property intellectualthird may Grain 20:0 Arachidic (% Total FA) 0.39 its 0.38 3.43 <0.001 (0.36 - 0.42) [0.23,0.54] the as of and owner. Statistical Differences in More Than One Site is regulatory a document Proximate any such its reproductiondocument Site IA Grain Carbohydrates (% DW) 83.38 owner84.52 this-1.34 of 0.008 (83.29 - 83.55) [81.08,88.80] under of this Site OH Grain Carbohydrates (% DW) 84.26rights the 83.80 0.55 0.009 (83.99 - 84.59) [81.08,88.80] documentand/or rights to of fall use of Mineral the Site IL-1 Grain Copper (mg/kg DW) This 1.76 may distribution,1.36and 29.35 0.023 (1.51 - 2.21) [0,4.20] rights owner subject Site NE Grain Copper (mg/kg DW) 2.15 1.67 28.66 0.023 (1.92 - 2.38) [0,4.20] be the violate copy of Site IL-1 Grain Iron (mg/kg DW) 20.86 19.and48 7.11 0.048 (19.23 - 21.79) [8.88,34.51] may documentpublication, It exploitation Site OH Grain Iron (mg/kg DW) this 21.37 25.74 -17.00 0.006 (20.59 - 21.76) [8.88,34.51] any Fatty Acid permission Site IL-1 Grain 18:0 Stearic (% Total FA) 1.96 prohibited1.82 7.94 <0.001 (1.89 - 2.02) [0.86,2.98] the be Site IL-2 Grain 18:0 Stearic (% Total FA) commercial1.98 1.82 9.05 <0.001 (1.93 - 2.03) [0.86,2.98]

Site IL-1 Grain 20:0 ArachidicFurthermore, (% Total FA) any 0.41 0.39 5.23 0.007 (0.40 - 0.42) [0.23,0.54] Consequently,without Fatty Acid (cont)

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Site OH Grain 20:0 Arachidic (% Total FA) 0.38 0.37 3.12 0.035 (0.38 - 0.39) [0.23,0.54] Bayer property publishing protectioncontents Statistical Differences Observed in One Site Only of its Amino Acid parties.data therefore Site IA Grain Alanine (% DW) 0.88 0.81 7.83 and/oror 0.030 (0.87 - 0.88) [0.48,1.08] affiliates. property intellectualthird may Site IA Grain Arginine (% DW) 0.51 its 0.46 10.83 0.005 (0.50 - 0.52) [0.33,0.56] as the of and Site IA Grain Aspartic acid (% DW) is 0.77 0.71 regulatory8.66 0.003owner. (0.77 - 0.78) [0.43,0.90] a document any such its reproductiondocument Site IA Grain Cystine (% DW) 0.25 owner0.23 this7.54 of 0.014 (0.24 - 0.26) [0.18,0.27]

under of this Site IA Grain Glutamic acid (% DW) 2.27rights the 2.09 8.66 0.011 (2.26 - 2.28) [1.25,2.75] documentand/or of rights to of fall use Site IA Grain Glycine (% DW) 0.41 0.38 the6.94 0.020 (0.40 - 0.41) [0.28,0.46] This may distribution,and rights owner Site IA Grain Histidine (% DW) subject 0.34 0.32 7.16 0.022 (0.34 - 0.34) [0.22,0.38] the violate be copy Site IA Grain Leucine (% DW) 1.49 of1. 37 8.96 0.032 (1.48 - 1.51) [0.77,1.92] and document may publication,exploitation Site IA Grain Lysine (% DW) It 0.35 0.32 6.66 0.028 (0.33 - 0.36) [0.20,0.40] this any Site IA Grain Methionine (% DW) 0.25permission 0.23 11.20 0.003 (0.25 - 0.27) [0.14,0.25] prohibited the Site IA Grain Phenylalanine (% DW) commercial0.58 be 0.53 9.45 0.028 (0.57 - 0.59) [0.32,0.73]

Furthermore,any Amino Acid (cont) Consequently,without Site IA Grain Proline (% DW) 1.05 0.98 7.29 0.028 (1.04 - 1.05) [0.68,1.21]

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 180 of 323

Bayer property protectionpublishing Site IA Grain Threonine (% DW) 0.37 of0. 34 8.45 0.004 contents (0.37 - 0.37) [0.24,0.41] its parties.data therefore Site IA Grain Tyrosine (% DW) 0.43 0.36 17.50 and/oror0.006 (0.42 - 0.43) [0.17,0.52] affiliates. property intellectualthird may its as the of and is regulatory owner. Proximate a document Site IA Grain Protein (% DW) 11.89any such 10.85 9.59 0.005its (11.73 - 11.98) [7.54,13.13] reproductiondocument owner this of Site IL-1 Forage Moisture (% FW) 69.03 under66.53 of 3.76 0.031 (68.50 - 69.40) [57.62,86.45] rightsthe this documentand/or of rights Site NE Forage Ash (% DW) to 3.20of fall 4.39 use -27.12 0.021 (2.93 - 3.38) [1.93,6.31] the Site NE Forage Carbohydrates (% DW) This 88.16 may distribution,84.98and 3.74 0.004 (86.86 - 88.84) [83.05,90.74] rights owner subject Fiber be the violate Site NE Grain Neutral Detergent Fiber (% DW) copy 10.52 of9.05 16.27 0.028 (10.43 - 10.69) [5.93,13.63] document and Site OH Forage Acid Detergent Fiber (%may DW) publication,31.31 23.58 32.78 0.012 (26.92 - 34.93) [16.76,43.76] It exploitation this Site OH Forage Neutral Detergent Fiber (% DW) any 43.21 37.87 14.11 0.027 (40.07 - 46.82) [25.94,55.67] permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 181 of 323

Site IL-1 Grain 16:1 Palmitoleic (% Total FA) 0.13 0.14 -6.87 0.012 (0.12 - 0.13) [0,0.28] Bayer property protectionpublishing of contents Site IL-2 Grain 18:1 Oleic (% Total FA) 24.75 23.82 3.93 0.003 its (24.14 - 25.25) [7.51,46.46] parties.data therefore and/oror Site IL-2 Grain 18:2 Linoleic (% Total FA) 61.87 63.17 -2.07 0.001 (61.19 - 62.42) [39.41,76.74] affiliates. property intellectualthird may its Site NE Grain 20:1 Eicosenoic (% Total FA) 0.28 0.29as -1.50 0.030 (0.28 - 0.28) [0.15,0.39] the of and is regulatory owner. Mineral a document any such its Site IA Grain Calcium (% DW) 0.0064 0.0058 reproduction 10.96 document 0.012 (0.0062 - 0.0066) [0.0016,0.0059] owner this of Site IA Grain Manganese (mg/kg DW) 8.34 6.99under 19.32of 0.017 (7.62 - 9.32) [3.17,9.99] rightsthe this documentand/or of rights Site IA Forage Calcium (% DW) 0.24to of fall 0.26 use -8.77 0.033 (0.24 - 0.24) [0.016,0.38] the This may distribution,and Site NE Forage Phosphorus (% DW) 0.25rights 0.17 owner 46.95 0.036 (0.23 - 0.28) [0.071,0.32] subject Vitamin be the violate Site IL-2 Grain Folic Acid (mg/kg DW) copy 0.37 0.32of 13.81 <0.001 (0.35 - 0.38) [0.012,0.69] and may documentpublication, Secondary Metabolite It exploitation Site OH Grain p-Coumaric Acid (µg/g DW) this 218.38 185.63 17.64 0.032 (187.79 - 253.04) [0,378.57] any 1 DW = dry weight; FW = fresh weight; FA = fatty acid; Combinedpermission Site = analyses of the combined data from each of the five replicated field trials. 2With 95% confidence, interval contains 99% of the values expressed in the populaprohibitedtion of commercial lines. Negative limits were set to zero. the commercial be

Furthermore,any Consequently,without

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 182 of 323

Table VII.9. Literature and ILSI Database Ranges of Components of Corn Forage and Grain

Tissue/ Literature ILSI Component1 Range2 Range3

Forage Proximates (% dw) Ash 2.43-9.64a; 2-6.6b 1.527 – 9.638 Carbohydrates 83.2-91.6b; 76.5-87.3a 76.4 – 92.1 Fat, total 0.35-3.62b; 1.42-4.57a 0.296 – 4.570 a b Moisture (% fw) 56.5-80.4 ;55.3-75.3 49.1 – 81.3 and Protein 4.98-11.56a and 3.14 – regime.11.57 AG Fiber (% dw) b a Acid detergent fiber (ADF) 18.3-41.0Bayer ; 17.5-38.3property protection16.13publishing – 47.39 Neutral detergent fiber (NDF) 26.4-54.5of b; 27.9-54.8a 20.29 contents– 63.71 its parties.data therefore Minerals (% dw) and/oror affiliates. b Calcium property intellectual0.0969-0.3184third 0.0714may – 0.5768 its b Phosphorous as 0.1367-0.2914 0.0936 – 0.3704 the of and is regulatory owner. a document Grain any such its reproductiondocument Proximates (% dw) owner this of under d of b Ash rightsthe 1.1-3.9 ; 0.89-6.28this 0.616 – 6.282 documentand/or b rightsa Carbohydrates to of fall 77.4-87.2use; 82.2-88.1of 77.4 – 89.5 Fat, total 3.1-5.7d; 2.48-4.81the b 1.742 – 5.823 This may distribution,and d b Moisture (% fw) rights 7-23owner; 8.18-26.2 6.1 – 40.5 Protein subject 6-12d; 9.7-16.1c 6.15 – 17.26 be the violate copy of Fiber (% dw) and may documentpublication, d a,b AcidIt detergent fiber (ADF)exploitation 3.3-4.3 ; 2.46-11.34 1.82 – 11.34 d b Neutral detergentthis any fiber (NDF) 8.3-11.9 ; 7.58-15.91 5.59 – 22.64 h Total dietary fiber (TDF)permission 10.99-11.41 8.82 – 35.31 prohibited the Minerals commercial be Calcium (% dw) 0.01-0.1d 0.00127 – 0.02084 Furthermore,Copper any(mg/kg dw) 0.9-10d 0.73 – 18.50 Consequently,without Iron (mg/kg dw) 1-100d 10.42 – 49.07 Magnesium (% dw) 0.09-1d 0.0594 – 0.194 Manganese (mg/kg dw) 0.7-54d 1.69 – 14.30 Phosphorous (% dw) 0.26-0.75d 0.147 – 0.533 Potassium (% dw) 0.32-0.72d 0.181 – 0.603 Zinc (mg/kg dw) 12-30d 6.5 – 37.2

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Table VII.9 (cont.). Literature and ILSI Database Ranges of Components of Corn Forage and Grain Tissue/ Literature ILSI Component1 Range2 Range3

Grain Amino Acids (% dw) Alanine n.a. 0.439 – 1.393 Arginine n.a. 0.119 – 0.639 Aspartic acid n.a. 0.335 – 1.208 Cystine n.a. 0.125 – 0.514 Glutamic acid n.a. 0.965 – 3.536 and and regime. Glycine n.a. AG 0.184 – 0.539 Histidine n.a. 0.137 – 0.434 Isoleucine n.a. 0.179 – 0.692 Bayer property protectionpublishing Leucine ofn.a. 0.642 – 2.492contents Lysine n.a. 0.172 –its 0.668 parties.data therefore Methionine n.a. and/or0.124or – 0.468 Phenylalanine affiliates.n.a. 0.244 – 0.930 property intellectualthird may Proline its n.a. 0.462 – 1.632 as Serine the of n.a.and 0.235 – 0.769 is regulatory owner. Threonine n.a. a document0.224 – 0.666 any such its Tryptophan n.a. reproductiondocument 0.0271 – 0.215 owner this of Tyrosine n.a. 0.103 – 0.642 under of this Valine and/orrights the n.a. 0.266 – 0.855 document to fall of rights of use the FattyThis Acids may distribution,(% totaland fat) (% total fatty acid) rights eowner 16:0 Palmiticsubject 7-19 7.94 – 20.71 e 16:1 Palmitoleicbe the1 violate 0.095 – 0.447 18:0 Stearic copy of 1-3e 1.02 – 3.40 ande 18:1may Oleic documentpublication, 20-46 17.4 – 40.2 It exploitation e 18:2 Linoleicthis 35-70 36.2 – 66.5 18:3 Linolenic any 0.8-2e 0.57 – 2.25 permission e 20:0 Arachidic prohibited0.1-2 0.279 – 0.965 20:1 Eicosenoic n.a. 0.170 – 1.917 the be 22:0 Beheniccommercial n.a. 0.110 – 0.349 Furthermore, any Consequently,Vitamins (mg/kgwithout dw) Folic acid 0.3d 0.147 – 1.464 Niacin 9.3-70d 10.37 – 46.94 e Vitamin B1 3-8.6 1.26 – 40.00 e Vitamin B2 0.25-5.6 0.50 – 2.36 d e Vitamin B6 5.3 ; 9.6 3.68 – 11.32 Vitamin E 3-12.1e; 17-47d 1.5 – 68.7

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Table VII.9 (cont.). Literature and ILSI Ranges of Components of Corn Forage and Grain

Tissue/ Literature ILSI Component1 Range2 Range3

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

regime.and Secondary Metabolites and AG (µg/g dw) Ferulic acid 113-1194f; 3000g 291.9 – 3885.8 p-Coumaric acid 22-75Bayerf property 53.4 publishing– 576.2 protectioncontents of 1 its fw=fresh weight; dw=dry weight; Niacin =Vitamin B3parties.; Vitamindata B1 =Thiamine;therefore Vitamin B2 and/oror =Riboflavin; Vitamin B6 =Pyridoxine ; n.a. = not available as percent dry wt. 2 a affiliates. b c Literature range references: Ridleyproperty et al.,intellectual 2002.third Sidhu et al., 2000. mayJugenheimer, 1976. d e f its g h Watson, 1987. Watson, 1982. Classen etas al. , 1990. Dowd and Vega, 1996. Choi et al., 1999. 3ILSI range is from ILSI Cropthe Composition of Database,and 2006. is regulatory owner. a document any such its 4 3 reproductiondocument Conversions: % dw x 10 = µg/g dw; mg/gowner dw x 10 = mg/kgthis dw; mg/100gof dw x 10 = mg/kg dw under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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SECTION 4. Other Information Relevant to the Safety and Nutritional Assessment of MON 89034

The long history of safe consumption of corn as food and feed, combined with the demonstration of the compositional equivalence of MON 89034 to conventional corn, is considered sufficient to support the safety and nutritional assessment of MON 89034.

SECTION 5. Substantial Equivalence of MON 89034 to Conventional Corn and A detailed compositional assessment of corn grain and forage andwas presentedregime. in Part VII, Section 3, where the levels of key nutrients, anti-nutrientsAG and other components in MON 89034 were examined and compared to that of the conventional control, LH198 x LH172. Additionally, tolerance intervals representing 99% of the values of each analyte from a Bayer property protectionpublishing conventional corn population were established.of Results demonstrate thatcontents the levels of key nutrients, anti-nutrients and other components in the forage and grainits of MON 89034 parties.data therefore are equivalent to those of conventional corn. Based on and/orthe dataor and information presented, Monsanto concludes thataffiliates. MON 89034 is substantially equivalent to property intellectualthird may conventional corn, and this conclusionits extends to the foods and feeds produced from as MON 89034. the of and is regulatory owner. a document any such its Collectively, these data and a history of safe use ofreproduction the host documentorga nism, corn, as a common owner this of source of human food and animal feed, support a conclusion of “no concerns” for every under of this criterion specified in therights flowchartsthe outlined in the FDA’s Food Policy document documentand/or of rights (FigureVII.1; FDA, 1992).to Cornof MONfall 89034use is not materially different in composition, safety or agronomic characteristics from conventional thecorn other than its protection from This may distribution,and lepidopteran feeding damage.rights Sales and coownernsumption of corn grain and processed subject products derived from MON 89034 would be fully consistent with the FDA’s Food be the violate Policy, the Federalcopy Food, Drug andof Cosmetic Act, and current practices for the development and introduction of new corn andvarieties. may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 186 of 323

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

Do characteristics of the host species, regime.and related species, or and No * No progenitor linesAG warrant analytical or toxicological tests?

YesBayer property protectionpublishing of contents Do test results provide its parties.data therefore evidence that toxicant and/orNewor variety not levels in the new plant No affiliates. acceptable varietyproperty do notintellectual presentthird may a safetyits concern? as the of and is regulatory owner. Yes a document any such its reproductiondocument owner this of under of rightsIsthe the concentration this documentand/or and bioavailability of rights to ofimportant fall nutrientsuse inof No concerns the new variety within No Consult Yes the FDA This maythe rangedistribution,and ordinarily rights seen in the ownerhost subject species? be the violate copy of and may documentpublication, It exploitation

this any permission prohibited Figure VII.1. Safety Assessment of New Varieties: the Host Plant the be commercial Furthermore,any Consequently,without

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References

Choi, I.H., J.H. Son, and K.H. Nahm. 1999. Dietary fiber fraction for grains containing high levels of water-soluble non-starch polysaccharides. Japan Poultry Sci. 36(4):269-274.

Classen, D., J.T. Arnason, J.A. Serratos, J.D.H. Lambert, C. Nozzolillo, and B.J.R. Philogene. 1990. Correlation of phenolics acid content of maize to resistance to Sitophilus zeamais, the maize weevil, in CIMMYT’s collections. J. Chem. Ecol. 16(2):301-315. and and regime. CRA. 2005. Corn Part of our Daily Lives. CornAG Refiners Association Annual Report. Corn Refiners Association, Washington, D.C.

Dowd, P.F., and F.E. Vega. 1996. EnzymaticBayer oxidation property products protection of allelochemicalspublishing as a of contents basis for resistance against insects: Effects on the corn leafhopperits Dalbulus maidis. parties.data therefore Natural Toxins 4:85-91. and/oror affiliates. property intellectualthird may nd Ensminger, M.E., J.E. Oldfield, andits W.W. Heineman. 1990. Feeds and Nutrition, 2 as Edition. the of and is regulatory owner. a document FDA. 1992. Statement of anyPolicy: such Foods Derived From New Plantits Varieties. Fed. Reg. reproductiondocument of 57:22984-23005. owner this under of rightsthe this documentand/or rights Galinat, W.C. 1988. Theto origof in fallof corn. Ppuse 1-31. of In Corn and Corn Improvement. Sprague, G.F. and J.W. Dudley (eds.). American Societythe of Agronomy, Crop Science This may distribution,and Society of America,rights and Soil Science Societyowner of America, Madison, Wisconsin. subject Hardeman, N.P.be 1981. Shucks, Shocks,the a ndviolate Hominy Blocks. Pp 3. Louisiana State copy of University Press, Baton Rouge, Louisiana.and may documentpublication, It exploitation ILSI-CCD. 2006. International Life Science Institute Crop Composition Database, this any Version 3.0. http://www.cropcomposition.org (27 Aug 2006). permission prohibited Jugenheimer, R.W. 1976.the Corns for Special Purposes and Uses. Pp 2-45. Corn commercial be Improvement, Seed Prodution, and Uses. John Wiley & Sons, Inc., New York. Furthermore,any Kastner,Consequently, J. 1980.without The conundrum of corn. American Heritage Magazine 31(5):14.

Kroon, P.A., and G. Williamson. 1999. Hydroxycinnamates in plants and food: Current and future perspectives. J. Sci. Food Agric. 79:355-361.

Mangelsdorf, P.C. 1974. Corn—Its Origin, Evolution, and Improvement. Harvard University Press, Cambridge, Massachusetts.

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Maynard, L.A., J.K. Loosli, H.F. Hintz, and R.G. Warner. 1979. Pp. 82. Animal Nutrition, 7th edition. McGraw Hill Book Co., New York.

NCGA. 2006. 2006 World of Corn. National Corn Growers Association. www.ncga.com/WorldOfCorn/main/index.asp (10 Oct 2006).

Newcomb, M.D. 1995. Corn and animal nutrition in the United States, U.S. Food and Drug Administration, Washington, D.C.

OECD. 2002. Consensus document on compositional considerations for new varieties of maize (Zea mays): Key food and feed nutrients, anti-nutrients and secondary plant metabolites. Organization for Economic Co-operation and Development. Series of regime.and the Safety of Novel Foods and Feeds, No. 6. ENV/JM/MONO(2002)25.and AG http://www.olis.oecd.org/olis/2002doc.nsf/43bb6130e5e86e5fc12569fa005d004c/ef5a cb7d10c9cd66c1256c1b004323e8/$FILE/JT00130429.PDF (26 Sep 2006). Bayer property protectionpublishing Orthoefer, F., J. Eastman, and G. List.of 2003. Corn oil: composition, processing,contents and nd its utilization. Pp 686. In Corn: Chemistry and Technology,parties.data 2 edition.therefore White, P.J. and L.A. Johnson (eds.). American Association of Cerealand/or orChemists, St. Paul, affiliates. Minnesota. property intellectualthird may its as the of and th NRC. 2001. Nutrientis requi rements of dairyregulatory cattle, 7 edition. owner.National Research a document Council. National Academyany Press,such Washington, D.C. its reproductiondocument owner this of Ridley, W.P., R.S. Sidhu, P.D. Pyla, underM.A. Nemeth,of M.L. Breeze, and J.D. Astwood. rightsthe this 2002. Comparisondocumentand/or of the nutritional profile of glyphosate-tolerantrights corn event NK603 to of fall use of with that of conventional corn (Zea mays L.). J.the Agric. Food Chem. 50(25):7235- 7243.This may distribution,and rights owner subject Salvador, R.J. 1997. Maize. In The Encyclopediathe violate of Mexico: History, Society, and be copy Culture. Fitzroy Dearborn Publishers,of Chicago, Illinois. and may documentpublication, It exploitation Sidhu, R.S., B.G.this Hammond, R.L. Fuchs, J-N Mutz, L.R. Holden, B. George, and T. Olson. 2000. anyGlyphosate-tolerant corn: The compositional and feeding value of permission grain from glyphosate-tolerantprohibited corn is equivalent to that of conventional corn (Zea mays L.). J. Agric. Food Chem. 48(6):2305-2312. the be commercial USDA-NASS.Furthermore,any 2006a. National Agricultural Statistics Service, United States Department ofConsequently, Agriculture.without Acreage Report, Cr Pr2-5 (6-06) a. June 30, 2006. http://usda.mannlib.cornell.edu/usda/nass/Acre/2000s/2006/Acre-06-30-2006.pdf

USDA-NASS (United States Department of Agriculture—National Agricultural Statistics Service). 2006b. Crop Production 2005 Summary, January 12, 2006. http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1047

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 189 of 323

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

Watson, S.A. 1987. Structure and composition. Pp 53-82. In Corn: Chemistry and Technology. Watson, S.A. and P.E. Ramstad (eds.). American Association of Cereal Chemists, St. Paul, Minnesota.

White, P.J. and L.M. Pollack. 1995. Corn as a food source in the Untied States: part II. Processes, Products, Composition, and Nutritive Values. Cereal Foods World 40:756- 762.

regime.and White, P.J. and E.J. Weber. 2003. Lipids of the kernel.and Pp 355-405. In Corn: AG Chemistry and Technology, 2nd edition. White, P.J. and L.A. Johnson (eds.). American Association of Cereal Chemists, St. Paul, Minnesota. Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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APPENDICES

and and regime. AG

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APPENDIX A: Materials and Methods for the Molecular Analysis of MON 89034

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

The test substance was grain, leaf and seedBayer from corn propertyMON 89034. protection publishing of contents its parties.data therefore 2. Control Substance and/oror affiliates. property intellectualthird may The control substance was conventionalits corn with genetic background similar to MON 89034. the as of and owner. is regulatory a documentits 3. Reference Substances any such reproductiondocument of owner this under of PV-ZMIR245 was used asrights a positivethe hybridization controlthis for Southern blots and as a documentand/or rights template control for PCRto analyses.of fall The plasmiduse wasof isolated and its identity confirmed by restriction enzyme digestion. the This may distribution,and rights owner The 1 kb DNAsubject extension ladder and λ DNA/Hind III fragments from Invitrogen (Carlsbad, CA)be were used for size estimatithe onsviolate on Southern blots and agarose gels. copy of Additionally, low DNA mass ladder and andhigh DNA mass ladder from Invitrogen were may documentpublication, used forIt size estimations and exploitationquantitative estimations on agarose gels.

this any 4. Characterization of Test, Control and Reference Substances permission prohibited Event specific PCR assaysthe were used to confirm the identity of these test substances and commercial be the absence of MON 89034 for control substances. The test and control substances were consideredFurthermore, anystable during storage if they did not appear visibly degraded on ethidium- stainedConsequently, gels and/orwithout yielded interpretable signals on the Southern blot.

5. Genomic DNA Isolation for Southern Blot Analyses

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

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Genomic DNA was extracted from leaf tissue using a Sarkosyl DNA extraction method (Fulton T. M. et al, 1995). For two Sarkosyl DNA extractions DNA was hooked out rather than using centrifugation. Extracted DNA solutions were stored in a 4°C refrigerator and/or a -20°C freezer.

For purification of plasmid DNA Qiagen QIA filter Plasmid Mini Kit was used.

6. Quantification of Genomic DNA

Extracted DNA was quantified using Hoefer’s DyNA Quant 200 Fluorometer. Molecular size marker IX (Roche, Indianapolis, IN) was used as the calibration standard. and and regime. AG 7. Restriction Enzyme Digestion of Genomic DNA

Approximately 10 or 20 µg of genomicBayer DNA extractedproperty from thepublishing test and control protectioncontents substances were digested overnight in aof total volume of approximately 500 µl of buffer its using 100 units of the restriction enzyme. Forparties. positivedata hybridizationtherefore controls, and/oror approximately one or one-half genomic equivalent of PV-ZMIR245 was spiked into affiliates. conventional corn DNA priorproperty to digesti intellectualon.third For positive hybridizationmay controls, its as approximately one or one-halfthe of genomic eandquivalent of PV-ZMIR245 was spiked into conventional corn DNA isprior to digestion. regulatory owner. a document any such its reproductiondocument 8. Agarose Gel Electrophoresis owner this of under of rightsthe this documentand/or rights Approximately 10 µg ofto digestedof fallDNA wereuse separated of using 0.8% agarose gels. For insert number, copy number, and cassette intactness experiments,the a ‘long run’ and ‘short This may distribution,and run’ were performed duringrights the gel electrophoresis.owner Approximately 20 µg of digested test and control subjectsubstance DNA was divided in half to load approximately 10 µg on the long run andbe approximately 10 µg on the shortviolate run. The long run enabled greater copy of separation of the higher molecular weightand restriction fragments while the short run allowedmay the smallerdocument molecularpublication, weight restriction fragments to be retained on the gel. It exploitation

this any 10. Probe preparation permission prohibited Approximately 12.5-27the ng ofbe each probe template were prepared by PCR amplification and radiolabeledcommercial with 32P-dCTP (6000 Ci/mmol) using the random priming method (exceptFurthermore, probeany 10 which was labeled by PCR). Consequently,without 11. Southern blot analyses

Digested genomic DNA isolated from test and control materials was evaluated using Southern blot analyses (Southern, 1975) with modifications.

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12. PCR and sequence analyses

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

The PCR analyses were conducted using 50 ng of genomic DNA template in a 50 µl reaction volume containing a final concentration of 2 mM MgSO4, 0.2 µM of each primer, 0.2 mM each dNTP, and 1 unit of DNA polymerase mix. The specific DNA polymerase mix used to amplify the products was Accuprime Taq (Invitrogen). The amplification of Product A was performed under the following cycling conditions:and 94 °C and regime. for 3 minutes; 30 cycles at 94°C for 15 seconds, 57AG°C for 30 seconds, 68°C for 3 minutes and 30 seconds; 1 cycle at 68°C for 5 minutes. The amplification of Product B was performed under the following cycling conditions: 94°C for 3 minutes; 35 cycles at 94°C Bayer property protectionpublishing for 15 seconds, 66°C for 30 seconds, 68of°C for 3 minutes and 30 seconds; contents1 cycle at 68°C for 5 minutes. The amplification of Products C, D, and E was performedits under the parties.data therefore following cycling conditions: 94°C for 3 minutes; 30 cyclesand/or at 94or ° C for 15 seconds, 60°C for 30 seconds, 68°C for 3 affiliates.minutes; 1 cycle at 68°C for 5 minutes. The property intellectualthird may amplification of Products F and G itswas performed under the following cycling conditions: as 94°C for 3 minutes; 30 cyclesthe atof 94°C for and15 seconds, 60°C for 30 seconds, 68°C for 1 is regulatory owner. a document minute and 30 seconds; 1 cycleany atsuch 68°C for 5 minutes. its reproductiondocument owner this of Aliquots of each PCR product were separatedunder on 1.0of %this (w/v) agarose gels and visualized by ethidium bromideand/or stainingrights to the verify that the products were of the expected size prior to document fall of rights sequencing. The PCR toproducts of were sequenceduse with multiple primers used for PCR the amplificationThis in addition to thosemay distribution,designedand internal to the amplified sequences. All rights owner sequencing wassubject performed by the Monsanto Genomics Sequencing Center using dye- terminator chemistry. be the violate copy of MON 89034 DNA generated the expected andsize PCR products of approximately 2.5 kb for may documentpublication, ProductIt A (lane 4); approximatelyexploitation 3.3 kb for Product B (lane 7); approximately 2.6 kb for Product C (lanethis 10); any approximately 2.6 kb for Product D (lane 14); approximately 3.2 kb for Product E (lane 18), approximatelypermission 1.1 kb for Product F (lane 22) and approximately 0.8 kb for Product G (lane 25). prohibited The generati on of the predicted size PCR products from the MON 89034 establishescommercial that be the arrangement or linkage of elements in the insert are the same as those in plasmid PV-ZMIR245 and that the elements within each gene cassette areFurthermore, arrangedany as depicted in the schematic of the insert in Figure A.1. Consequently,without

13. References

Fulton, T.M., J. Chunwongse, and S.D. Tanksley. 1995. Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol. Biol. Rep. 13 (3):207-209.

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Rogers, S.O. and A.J. Bendich. 1985. Extraction of DNA from milligram amounts of fresh herbarium and mummified plant tissue. Plant Mol Biol Sci: 69-76.

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

and and regime. AG

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

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

7.1 4.0 3.0 2.0 1.6 1.0 0.5

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

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

affiliates. - nos Left Border Left Hsp17 Cab FMV e35S may -Left Border Hsp70 - Ract1 property intellectualthird B T CS- P- TS I- CS- T- B- P- L- I- its as the of and Figure A.1. Overlappingis PCR analysis acrossregulatory the insert in MON 89034owner. a document any such its PCR analyses demonstrating the linkage of the individualreproduction geneticdocument elementsof within the insert in MON 89034 were performed on MON owner89034 genomic DNAthis extracted from grain (Lanes 4, 7, 10, under of 14, 18, 22, and 25). Lanes 3,rights 6, 9,the 13, 17, 21, and 24 containthis reactions with conventional corn documentand/or rights control DNA while lanes to2, 5, 8,of 12, 16,fall 20, and use23 are ofreactions containing no template DNA. Lanes 11, 15, and 19 contain reactions with PV-ZMIR245the control DNA. Lane 1 contains InvitrogenThis 1 kb DNA ladder. Lanesmay aredistribution, markedand to show which product has been loaded and is rights owner visualized on the subjectagarose gel. The gel side arrow symbols denote the sizes of DNA (kb) based on the reference DNAbe size markers on the ethidiumthe brviolateomide stained gel. The expected product size for each amplicon copyis provided in the illustrationof of the insert in MON 89034 that appears below the image. Analyses of MON 89034 insertand sequence and genomic DNA flanking the insert may documentpublication, revealedIt that the e35S promoterexploitation that regulates expression of the cry1A.105 gene has been modified and thatthis the any Right Border sequence was replaced by a Left Border sequence. This modification is likely the result of a crossover recombination which occurred prior to the DNA permission being inserted into the genome. prohibited Lane 1: Invitrogen 1 kbthe DNA be ladder 13: Conventional corn control DNA 2: No templatecommercial DNA control 14: MON 89034 genomic DNA Furthermore, 3: Conventional corn control DNA 15: PV-ZMIR245 plasmid 4: MONany 89034 genomic DNA 16: No template DNA control without Consequently,5: No template DNA control 17: Conventional corn control DNA 6: Conventional corn control DNA 18: MON 89034 genomic DNA 7: MON 89034 genomic DNA 19: PV-ZMIR245 plasmid 8: No template DNA control 20: No template DNA control 9: Conventional corn control DNA 21: Conventional corn control DNA 10: MON 89034 genomic DNA 22: MON 89034 genomic DNA 11: PV-ZMIR245 plasmid 23: No template DNA control 12: No template DNA control 24: Conventional corn control DNA 25: MON 89034 genomic DNA

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APPENDIX B: Materials and Methods for the Characterization of the Cry1A.105 and Cry2Ab2 Proteins in MON 89034

1. Materials and Methods used to Characterize the Cry1A.105 Protein

1.1. Plant-Produced Cry1A.105 Protein

The plant-produced Cry1A.105 protein was purified from grain of MON 89034. The identity of the grain containing MON 89034 was confirmed by event-specific PCR. The purified plant-produced Cry1A.105 protein was stored in a 4 ºC refrigerator in anda buffer solution containing 50 mM CAPS, 1 mM PMSF, 2 mM benzamidine-HCl,and regime. 1 mM EDTA, 0.8 M NaCl, and 30% (v/v) ethylene glycol, pH 10.0.AG

1.2. E. coli-Produced Cry1A.105 Protein Bayer property protectionpublishing of contents E. coli-produced Cry1A.105 protein was used as a reference standardits for determination parties.data therefore of protein concentration and immunoblot analysis using N-terminaland/oror peptide antibody. affiliates. This protein was also used as aproperty reference stanintellectualdardthird to evaluate equivalencemay between plant- and E. coli-produced Cry1A.105 proteinsits for the molecular weight and functional activity as assay, as a reference and athe negative of controland in glycosylation analysis, and as a reference is regulatory owner. a document and a positive control in immunoblotany such analysis using anti-Cry1A.105its antibody. The E. reproductiondocument coli-produced Cry1A.105 referenceowner standard wasthis previously of characterized. The Cry1A.105 protein was stored in a -80under °C freezer ofin a thisbuffer solution (25 mM CAPS, 1 mM benzamidine-HCl,and/or 0.1rights mMthe EDTA, and 0.2 mM DTT, pH ~10.3) at a total protein document to fall of rights concentration of 1.2 mg/ml byof amino acid analysis.use the This may distribution,and rights owner 1.3. Assay Controlssubject

be the violate Protein molecularcopy weight standards (Bio-Rad,of Hercules, CA) were used to calibrate SDS- PAGE gels and verify protein transfer toand PVDF membranes. The E. coli-produced may documentpublication, Cry1A.105It reference standardexploitation was used to create a standard curve for the quantitative western blot. Thethis Cry1A.105any trypsin-resistant core was used as a negative control for the western blot analysis using permissionN-terminal peptide antibody. A peptide mixture (Sequazyme Peptide Mass Standards kit, Appliedprohibited Biosys tems) was used to calibrate the MALDI-TOF the mass spectrometercommercial for tryptic be mass analysis. Transferrin (Amersham Biosciences, Piscataway, NJ) and horseradish peroxidase (Sigma, St. Louis, MO) were used as positiveFurthermore, controlsany for glycosylation analysis. CandyCane™ glycoprotein molecular Consequently,without weight standards were used as molecular weight markers and positive and negative controls for glycosylation analysis.

1.4. Protein Purification

The plant-produced Cry1A.105 protein was purified from the corn grain of MON 89034. The purification procedure was not performed under a GLP study or plan; however, all procedures were documented on worksheets and, where applicable, SOPs were followed.

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The Cry1A.105 protein was purified at 4°C from an extract of ground grain using a combination of ammonium sulfate fractionation, anion exchange chromatography, and immunoaffinity chromatography.

The ground grain (10 kg) was mixed in PBS extraction buffer [1× PBS: 1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, and 2.7 mM KCl, pH 7.4] for 2.5 hours at approximately a 1:10 sample weight to buffer volume ratio. The slurry was filtered using an Ertel Alsop filter press (Kingston, NY) and the PBS washed filter cakes were retained. The filter cakes were resuspended in ~100 L of CAPS extraction buffer [50 mM CAPS, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, 2 mM benzamidine- HCl, and 1% PVPP (w/v), pH 10.8] and stirred for 2 hours. To remove lipids from the extract, CelPure P65 Diatomaceous Earth (Advanced Minerals Corp, Goleta, CA) was added to the slurry at regime.and 7.5% (w/v) and allowed to mix for ~10 minutes. The extractand was clarified by filtration AG using the Ertel Alsop filter press and concentrated using a Hollow Fiber Cartridge (Amersham Biosciences, Piscataway, NJ). The contaminant DNA was removed by a combination of benzonase treatment Bayerand precipitationproperty with publishingpolyethyleneimine. protectioncontents Benzonase was added to a final concentrationof of ~7 U/ml in the presence of 5 mM MgCl2 its and allowed to mix overnight. A polyethyleneimineparties. solutiondata [10% (w/v)] thereforewas added to the concentrated extract to a final concentration of 0.05% (w/v)and/or orand the extract was affiliates. clarified by centrifugation to removeproperty the intellectualremainingthird contaminant DNA.may An ammonium its sulfate pellet (0% – 35% saturation) wasas prepared by the addition of ammonium sulfate the of and salt to the clarified extractis and was allowed to regulatorydissolve overnight. Afterowner. centrifugation, a document the ammonium sulfate pelletany was such dissolved over the weekend in ~15its L of resuspension reproductiondocument buffer [50 mM CAPS, 1 mM EDTA,owner 10 mM thisDTT, 1 mMof PMSF, and 2 mM benzamidine-HCl, pH 10.8]. The resuspeundernded sampleof was clarified by centrifugation, rightsthe this diafiltrated againstdocument freshand/or buffer (same as the resuspension rightsbuffer) to remove any residual to of fall use of ammonium sulfate and concentrated to final volume ofthe ~8 L and CHAPS was added to a final concentrationThis of 0.5 mM.may distribution,and rights owner subject The concentrated sample was loaded ontothe a 2.1violate L (6.7 cm x 20 cm) Q Sepharose Fast be copy Flow anion exchange resin column, whichof was equilibrated with AEX buffer A [50 mM and CAPS, may1 mM EDTA,document 10publication, mM DTT, 1 mM PMSF, 2 mM benzamidine-HCl, and 0.5 mM It exploitation CHAPS, pH 10.8].this The bound Cry1A.105 protein was eluted with a linear salt gradient that increased fromany 0 M to 0.20 M sodium chloride over ~12 L and then maintained a permission 0.20 M sodium chloride concentrationprohibited for ~4 L. Next, the salt gradient increased to 0.65 M sodium chloride over ~21 L, and finally stepped up to 1.0 M sodium chloride instantly. the be Fractions containingcommercial the Cry1A.105 protein (based on stained SDS-PAGE gel and westernFurthermore, blotany analysis of all fractions) were pooled to a final volume of ~10 L. Salt was removedConsequently, from thesewithout pooled fractions by diafiltration with fresh buffer [50 mM EPPS, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, and 2 mM benzamidine-HCl, pH 7.5].

The buffer exchanged sample was loaded onto a 2.1 L (13.7 cm x 14.0 cm) Q Sepharose XL anion exchange resin column, which was equilibrated with AEX buffer A [50 mM EPPS, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, 2 mM benzamidine-HCl, and 0.5 mM CHAPS, pH 7.5]. The bound Cry1A.105 protein was eluted with a linear salt gradient that increased from 0 M to 0.25 M sodium chloride over ~ 4 L and then held at 0.25 M

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sodium chloride for ~ 10 L. Next, the salt gradient increased to 0.65 M sodium chloride over ~21 L and finally increased to 1.0 M sodium chloride over ~ 4 L and held constant for ~8L. The fractions containing Cry1A.105 protein (based on stained SDS-PAGE gel and western blot analysis of all fractions) were pooled to a final volume of ~10 L. These fractions were diafiltrated into fresh buffer [50 mM EPPS, 1 mM EDTA, 10 mM DTT, 1mM PMSF, and 2 mM benzamidine-HCl, pH 7.5] to remove salt and concentrated to ~2.0 L using a hollow fiber cartridge.

Prior to the affinity purification step, the sample was diafiltrated into fresh buffer [50 mM EPPS, 1mM EDTA, 1 mM PMSF, 2 mM benzamidine-HCl, and 150 mM NaCl, pH 7.6] and concentrated down to ~1L. The solution containing Cry1A.105 protein was re- circulated over the 9.3 ml (1.75 cm x 2.6 cm) protein A agarose column (Sigma) regime.and conjugated with monoclonal anti-Cry1Ac antibody (Strategic Biandosolutions, Newark DE). AG Bound Cry1A.105 protein was eluted using 50 mM CAPS, 1 mM EDTA, 1 mM PMSF, 2 mM benzamidine-HCl, 0.8 M NaCl, and 30% (v/v) ethylene glycol, pH 10.0. After analysis of fractions by lateral flow strips,Bayer stained propertySDS-PAGE protection gelpublishing and western blot, fraction 25 was determined to contain theof majority of the full-length Cry1A.105.contents its parties.data therefore 1.5. Determination of the Total Protein Concentration and/oror affiliates. property intellectualthird may its The concentration of the full-length (~130as kDa) plant-produced Cry1A.105 protein was the of and estimated using quantitativeis immunoblot analysis.regulatory The E. coli-producedowner. Cry1A.105 a document protein (amounts ranging anyfrom such1 to 6 ng purity corrected for theits full-length protein, reproductiondocument which was 82%) was used to generateowner a standa rd curve.this Aliquotsof of the plant-produced Cry1A.105 protein and reference standardunder wereof diluted in deionized water and 5x rightsthe this Laemmli bufferdocument (5xand/or LB), heated at 95.5 °C for 5 min, andrights applied to a pre-cast tris- to of fall use of glycine 4→20% polyacrylamide gradient 12-well gel.the Three different amounts of the plant-producedThis protein were lomayaded distribution, in duplicate.and Electrophoresis was performed at a rights owner constant voltagesubject of 125 V for 15 min followed by a constant voltage of 170 V for 75 min. Pre-stained molecular weight markers (Bio-Rthe violatead Precision Plus Dual Color, Hercules, be copy CA) were used to verify electrotransferof of protein to the membrane and estimate the size and of the immunoreactivemay document publication,bands observe d. Electrotransfer to a 0.45 µm PVDF membrane It exploitation (Invitrogen, Carlsbad,this CA) was performed for 90 min at a constant voltage of 25 V. any permission The membrane was blocked forprohibited 1 hour with 5% (w/v) NFDM in 1× PBST. The membrane was probedthe with be a 1:1000 dilution of rabbit anti-Cry1A.105 antibody (lot 070705JL) in 1%commercial (w/v) NFDM in PBST for 60 min. Excess antibody was removed using fourFurthermore, 5 min anywashes with PBST. Finally, the membrane was probed with horseradish peroxidase-conjugatedConsequently,without goat anti-rabbit IgG (Sigma, St. Louis, MO) at a dilution of 1:7500 in 1% (w/v) NFDM in PBST for 60 minutes. Excess HRP-conjugate was removed using three 5 min washes with PBST. All incubations were performed at room temperature. Immunoreactive bands were visualized using the ECL detection system (Amersham Biosciences, Piscataway, NJ) and exposed (5 min, 10 min, 20 min) to Hyperfilm ECL high performance chemiluminescence film (Amersham Biosciences). Films were developed using a Konica SRX-101A automated film processor (Tokyo, Japan).

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The immunoreactive band of the plant-produced Cry1A.105 protein in each lane migrating at the same level as the full-length reference standard protein was quantitated relative to the standard curve. Quantitation was performed using the volume tool and the linear regression method in the Quantity One software (version 4.4.0) after scanning on the Bio-Rad GS-800 densitometer. The concentration of the plant-produced Cry1A.105 was determined by dividing the amount of protein in each lane by the volume of protein loaded in that lane [Microsoft Excel 2002 (10.6730.6718) SP3]. Throughout this summary, all load quantities of plant-produced Cry1A.105 refer to the amount of the full- length protein.

1.6. Western Blot Analysis regime.and and AG Western blot analyses were performed to confirm the identity of the Cry1A.105 protein, the intactness of its N-terminus, and compare immunoreactivity of the plant- and E. coli- produced proteins. Bayer property protectionpublishing of contents its The western blot described above (section 1.5) was parties.also useddata to establish thetherefore identity of the plant-produced protein and to compare the immunoreactivityand/or ofor the plant-produced affiliates. and E. coli-produced Cry1A.105property proteins. intellectual Equivalencethird was demonstratedmay for bands its representing full-length (~130 kDa) proteinsas that were identified by the anti-Cry1A.105 the of and antibody and showed similaris electrophoretic mobility.regulatory owner. a document any such its reproductiondocument 1.7. Analysis of the N-terminal Sequenceowner using westernthis blot of under of rightsthe this A western blotdocument analysisand/or using the N-terminal peptide antibodyrights was performed to confirm to of fall use of the intactness of the N-terminus of the plant-producedthe Cry1A.105 protein. The N- terminalThis peptide antibodies weremay produced distribution,and against a synthetic peptide consisting of the rights owner first 14 amino acidssubject of the Cry1A.105 protein N-terminus. The trypsin resistant core was used as a negative control because its N-tetherminus violate was removed by trypsin. The plant- be copy produced Cry1A.105 protein, the E.of coli-produced Cry1A.105 reference standard and (correctedmay for the puritydocumentpublication, of the full-length protein), and the Cry1A.105 trypsin-resistant It exploitation core (~56 kDa)this were each loaded on gels at 20 ng and 40 ng per lane. Each protein was mixed with 5× LB,any heated at 96.0 °C for 5 min, and applied to a pre-cast tris-glycine permission 4→20% polyacrylamide gradientprohibited 10-well gel. Electrophoresis was performed at a constant voltage of 150 V for 5 min followed by a constant voltage of 200 V for 60 min. the be Pre-stained molecularcommercial weight markers (Bio-Rad Precision Plus Dual Color,) were used to verifyFurthermore, electrotransferany of protein to the membrane and estimate the size of the immunoreactiveConsequently,without bands. Electrotransfer to a 0.45 µm PVDF membrane (Invitrogen) was performed for 90 min at a constant voltage of 25 V.

The membrane was blocked overnight with 5% (w/v) NFDM in 1× PBST. The membrane was probed with a 1:33.33 dilution of rabbit N-terminal peptide antibody (lot 42005MG) in 1% (w/v) NFDM in PBST for 60 min. Excess antibody was removed using three 5 min washes with PBST. Finally, the membrane was probed with HRP-conjugated anti-rabbit IgG (Sigma) at a dilution of 1:7500 in 1% (w/v) NFDM in PBST for

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60 minutes. Excess HRP-conjugate was removed using three 5 min washes with PBST. The blocking step was performed at 4 °C. All other incubations were performed at room temperature. Immunoreactive bands were visualized using the ECL detection system (Amersham Biosciences) and exposed (1 min, 2 min, 5 min, 10 min) to Hyperfilm ECL high performance chemiluminescence film (Amersham Biosciences). Films were developed using a Konica SRX-101A automated film processor.

1.8. MALDI-TOF MS Tryptic Mass Analysis

MALDI-TOF mass spectrometry was used to confirm the identity of the plant-produced Cry1A.105 protein. regime.and 1.8.1. Concentration of Protein and AG

The plant-produced protein (4.5 ml) was diluted to 9.0 ml with Cry1A.105 concentration buffer [25 mM CAPS, 0.02% (w/v) sodiumBayer dode cyl sulfate]property followed publishingby reduction of the protectioncontents volume to 64 µl using Amicon Ultrafreeof CL concentrators (Millipore Corporation, its Bedford, MA). Sixty microliters of 5× LB was usedparties. to washdata th e concentratortherefore walls and and/oror membranes. The membranes were heated with 5× LB for 5 minutes at 95.1°C. Sixteen affiliates. microliters of this 5× LB was propertythen used asintellectual the thirdloading buffer for the mayCry1A.105 sample its for SDS-PAGE separation. as the of and is regulatory owner. a document 1.8.2. SDS-PAGE Separationany ofsuch Proteins its reproductiondocument owner this of under of The concentrated plant–producedrightsthe Cry1A.105 protein wasthis subjected to electrophoresis on documentand/or rights an SDS-polyacrylamideto gel. ofThe proteinfall sampusele wasof loaded in two lanes. Broad Range molecular weight markers (Bio-Rad) were used to estimatethe molecular weights. Plant- producedThis Cry1A.105 protein andmay molecular distribution,and weight markers were heated at 95.3 °C for 5 rights owner min and then appliedsubject to a pre-cast tris-glycine 4→20% polyacrylamide gradient 10-well the violate mini-gel. Electrophoresisbe copy was performed at constant voltage (125 V for 10 min followed by 170 V for 70 min). Proteins were ofstaine d with Bio-Rad Coomassie 1 × stain for 2 and hours, andmay destained document bypublication, wash ing with Coomassie R-250 1× destain solution (Bio-Rad) It exploitation for 2 hours withthis one change of the destain solution. any permission 1.8.3. In-gel Trypsin Digestionprohibited and Sample Preparation the be In-gel protein digestioncommercial TheFurthermore, bands representingany full-length plant-produced Cry1A.105 protein (~130 kDa) were excisedConsequently, from twowithout gel lanes, destained, reduced, alkylated, and subjected to an in-gel trypsin digestion (Williams et al., 1997). Briefly, each gel band was individually destained for 30 min by incubation in 100 µl of 40% (v/v) methanol and 10% (v/v) glacial acetic acid in a microfuge tube. This was repeated two additional times. Following destaining, the gel bands were incubated in 100 µl of 100 mM ammonium bicarbonate buffer for 30 min at room temperature. The protein was reduced in 100 µl of 10 mM dithiothreitol solution for 2 hours at 37 °C. The protein was alkylated by the addition of 100 µl of 200 mM iodoacetic acid. The alkylation reaction was allowed to

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proceed at room temperature for 20 min in the dark. The gel bands were incubated in 100 µL of 100 mM ammonium bicarbonate buffer for 30 min at room temperature at which time 100 µl of acetonitrile was added and the incubation was continued for an additional 30 minutes. The ammonium bicarbonate/acetonitrile incubations were repeated two additional times to remove the reducing and alkylating reagents and other salts from the gel. The gel bands were dried in a SpeedVac concentrator, rehydrated with 40 µl 25 mM ammonium bicarbonate containing 33 µg/ml trypsin, and the protein contained in the gel band was digested for 16 hours at 37.1 °C. Digested peptides were extracted for 60-80 min at room temperature with 50 µl 70% (v/v) acetonitrile containing 0.1% (v/v) TFA per gel band. Both extraction supernatants were combined into a single tube and dried in a SpeedVac concentrator. This process of extracting the peptides was repeated two more times. The final dried materials were reconstituted in 5 µl of 0.1% (v/v) TFA. and and regime. AG Sample preparation An aliquot (4 µl) of the digested sample was desalted (Bagshaw et al., 2000) using Millipore (Bedford, MA) ZipTip C18 pipetteBayer tips. Priorproperty to desalting, thepublishing tips were wetted protectioncontents with methanol and equilibrated with 0.1%of (v/v) TFA. The sample was applied to a its ZipTip and eluted with 4 µl of Wash 1 [0.1% (v/v) parties.TFA],data followed by 4 µthereforeL of Wash 2 and/oror [20% (v/v) acetonitrile containing 0.1% (v/v) TFA], followed by 4 µl of Wash 3 [50% affiliates. may (v/v) acetonitrile containing 0.1%property (v/v) TFA],intellectual andthird finally with 4 µl of Wash 4 [90% (v/v) its as acetonitrile containing 0.1%the (v/v) of TFA]. and is regulatory owner. a document 1.8.4. MALDI-TOF Instrumentationany such and Mass Analysis its reproductiondocument of owner this under of Mass spectral analyses wererights performedthe as follows. Massthis calibration of the instrument documentand/or rights was performed using anto externalof fallpeptid e mixtureuse of from a Sequazyme Peptide Mass Standards kit (Applied Biosystems). Samples (0.5 µl) fromthe each of the desalting steps, as This may distribution,and well as a sample of solutionrights taken prior to ownerdesalting, were co-crystallized with 0.8 µl α-cyano-4-hydroxysubject cinnamic acid on the analysis plate. The sample was analyzed in the 500 to 5000 beDalton range using 100 shotsthe at a violatelaser intensity setting of 2781 (a unit-less copy of MALDI-TOF instrument specific value).and Protonated (MH+) peptide masses were observedmay monoisotopically documentpublication, in reflector mode (Aebersold, 1993; Billeci and Stults, 1993). It exploitation GPMAW32 software (Applied Biosystems, version 4.23) was used to generate a this any theoretical trypsin digest of the expected Cry1A.105 (plant) protein sequence based upon permission the nucleotide sequence. Massesprohibited were calculated for each theoretical peptide and compared to the raw massthe data.be Experimental masses (MH+) were assigned to peaks in the 500 to 1000 commercialDa range if there were two or more isotopically resolved peaks, and in theFurthermore, 1000 to any5000 Da range if there were three or more isotopically resolved peaks in the spectra.Consequently, Peaks withoutwere not assessed if the peak heights were less than approximately twice the baseline noise, or when a mass could not be assigned due to overlap with a stronger signal ±2 Daltons from the mass analyzed. Known autocatalytic fragments from trypsin digestion were identified in the raw data. The tryptic mass map coverage was considered acceptable if ≥ 40 % of the protein sequence was identified by matching experimental masses observed for the tryptic peptide fragments to the expected masses for the fragments.

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1.9. Determination of Molecular Weight using SDS-PAGE

Aliquots of the E. coli- and plant-produced Cry1A.105 proteins were mixed with 5× LB to a final protein concentration of 10 ng/µl and 2.4 ng/µl, respectively. The plant- produced protein was analyzed in duplicate at 48, 72, and 96 ng of total protein per lane. The E. coli-produced Cry1A.105 reference standard was analyzed at 96 ng of purity corrected full-length protein. All samples were heated in a thermo-block at 99 °C for 3 min and applied to a pre-cast tris-glycine 4→20% polyacrylamide gradient 10-well mini- gel (Invitrogen). Electrophoresis was performed at a constant voltage of 125 V for 10 min followed by a constant voltage of 170 V for 70 min.

The gel was stained using the SilverXpress® Silver Staining Kit Protocol (Invitrogen,and and regime. Carlsbad, CA). The gel was fixed for 10 minutes AGin 200 ml of fixing solution (90 ml ultra pure water, 100 ml methanol, and 20 ml acetic acid). This was followed by 10 minutes in 100 ml sensitizing solution (105 ml ultra pure water, 100 ml methanol, and 5 ml sensitizer) and repeated once. The excessBayer of the sensitizingproperty solution waspublishing removed using protectioncontents two 5 minutes washes in 200 ml ultra pureof water. The gel was stained for 15 minutes in its 100 ml staining solution (5 ml Stainer A, 5 ml Stainerparties. B,data and 90 ml ultratherefore pure water). and/oror The stain was removed using two 5 minutes washes of 200 ml ultra pure water. affiliates. Developing occurred in 100 mlproperty of solution intellectual (5 mlthird developer and 95 mlmay ultra pure water) its as for 3 - 15 minutes, and wasthe stopped of by additioandn of 5 ml stopping solution for 10 minutes. The gel was washed threeis times for 10 minutesregulatory each with 200 ml ofowner. ultra pure water. a document Analysis of the gel was performedany such using a Bio-Rad GS-800 densitometerits with the reproductiondocument supplied Quantity One software (versionowner 4.4.0, Hercules,this CA).of Molecular weight under of markers (Bio-Rad Broad-Range)rightsthe were used to estimatethis the apparent molecular weight of documentand/or rights the plant-produced Cry1A.105to of protein. fall For theuse purity of evaluation, all visible bands within each lane were quantified. Stained bands correspondingthe to immunoreactive bands This may distribution,and identified by anti-Cry1A.105rights antibody and ownermigrating from ~56 to ~130 kDa were included in the subjectpurity calculation for the protein of interest as they represent various lengths of thebe insecticidal protein from thethe ~56 violate kDa tryptic core to the ~130 kDa full- copy of length protein (Bietlot et al., 1989). Theand purity and estimated full-length molecular weight mayof the plant-produceddocumentpublication, Cry1A.105 protein were reported as the average of the six It exploitation values obtained by densitometric analysis. this any

permission 1.10. Determination of Functionalprohibited Activity by Insect Bioassay the be In order to assesscommercial the functional activity of the plant-produced Cry1A.105 protein and to compareFurthermore, its anyactivity to the E. coli-produced Cry1A.105 reference standard, aliquots of the plant-producedConsequently, withoutCry1A.105 protein and E. coli-produced Cry1A.105 reference standard protein were used to estimate the effective protein concentration necessary to inhibit the growth of the target insect by 50% (EC50 value) and the rate of weight loss as test protein concentration in the diet increased (slope parameter). The functional activity of the plant- produced Cry1A.105 was considered equivalent to that of the E. coli-produced protein activity if there was less than a 4-fold difference in mean EC50 values. Logistic regression was used to model concentration-response curves for growth inhibition and for EC50 determinations.

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1.10.1. Insects

Helicoverpa zea (CEW) eggs were obtained from Benzon Research Inc. Insect eggs were incubated at a temperatures ranging from 10o C to 27o C, to achieve the desired hatch time.

1.10.2. Bioassays

CEW were used to measure activity of the plant- and E. coli-produced Cry1A.105 protein samples in diet-incorporation bioassay. The bioassay was replicated three times on separate days with separate batches of insects. The plant- and E. coli-produced proteins regime.and were run in parallel during each bioassay. Each bioassay andreplicate for the E. coli- AG produced and plant-produced Cry1A.105 proteins consisted of a series of five protein levels yielding a dose series ranging from 0.00048 – 0.039 µg Cry1A.105 protein/mL diet with a 3-fold separation factor between Bayerdose levels. property This doseprotection seriespublishing was chosen to adequately characterize the dose-effect ofrelationship for the proteins fromcontents both sources. its Each dose level, including the control, had an equivalentparties. datavolume of buffertherefore added to the and/or dosing solution. The Cry1A.105 protein dosing solutions were preparedor by diluting the affiliates. protein with purified water andproperty incorporating intellectual thethird solution into an agar-basedmay insect diet. its Diet mixture was then dispensed in 1 asmL aliquots into a 128 well tray (#BIO-BA-128, the of and CD International, Pitman,is NJ). Insect larvaeregulatory were placed on theseowner. diets using a fine a document paintbrush, with a target numberany such of 16 insects per treatment. Theits infested wells were reproductiondocument covered by a ventilated adhesive coverowner (#BIO-CV-16, this CD International,of Pitman, NJ) and under of the insects were allowed torights feedthe for a period of 6-7 daysthis in an environmental chamber documentand/or rights programmed at 27° C, toambient of relativefall humidityuse ofand a lighting regime of 14h:10h, light:dark. The combined weight of the surviving insectsthe at each dose level for each source ofThis protein was recordedmay at the distribution, end andof the 6-7 day incubation period. rights owner subject the violate 1.10.3. Statisticalbe copy analysis of and The followingmay three-parameterdocumentpublication, logistic model, with an extra parameter for the change in It exploitation variation with thisthe expected weight (equation below), was used to model the dose- response curves forany each protein source and each replicate under the PROC NLMIXED permission procedure in SAS: prohibited the commercial be Furthermore,any Consequently,without

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

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

and and regime. where Wt is the average CEW larvae weight andAG DietDose is the Cry1A.105 protein diet dose level. The residual variation was assumed to be proportional to the expected mean weight. The parameters that are includedBayer in the modelproperty are W0 whichpublishing represents the protectioncontents expected weight at DietDose = 0.0, ECof50 which represents the concentration needed to its inhibit the growth of the target insect by 50%, B whichparties. reflectsdata the rate of thetherefore weight loss as DietDose increases, s which represents the proportion of theand/or standardor deviation to the affiliates. expected weight, and e which denotesproperty the intellectualresidualthird (error). may its as the of and 1.11. Glycosylation Analysisis regulatory owner. a document any such its reproductiondocument Glycosylation analysis was used toowner determine whetherthis the plant-producedof Cry1A.105 under of protein was post-translationallyrightsthe modified with covalentlythis bound carbohydrate moieties. documentand/or rights Aliquots of the plant-producedto of Cry1A.105fall useprotein, of the E. coli-produced Cry1A.105 reference standard, and the positive controls, transferrinthe (Amersham Biosciences) and horseradishThis peroxidase (Sigma),may were distribution, eachand mixed with 5× LB. These samples were rights owner heated at 95 °Csubject for 4 min, cooled, and loaded on a tris-glycine 4→20% polyacrylamide gradient 10-well mini-gel. Each sample wasthe loadedviolate at 48 and 96 ng (purity corrected for be copy the full length protein) per lane. Precisionof Plus Dual Color pre-stained protein molecular and weight maymarkers (Bio-Rad)documentpublication, were loaded to verify electrotransfer of the proteins to the It exploitation membrane, andthis the CandyCane™ Glycoprotein Molecular Weight Standards (Molecular Probes, Eugene, OR)any were loaded as positive/negative controls and markers for permission molecular weight. Electrophoresisprohibited was perfor med at a constant voltage of 125 V for 10 min followed by a constant voltage of 170 V for 70 min. Electrotransfer to a 0.2 µm the be PVDF membranecommercial was performed for 90 min at a constant voltage of 25 V. Furthermore,any CarbohydrateConsequently, detectionwithout was performed directly on the PVDF membrane using the Pro- Q® Emerald 488 Glycoprotein Gel and Blot Stain Kit (Molecular Probes). The manufacturer’s protocol was followed. All steps were performed at room temperature. The PVDF membrane was fixed in 25 ml of a solution containing 50% methanol and 5% glacial acetic acid for 1 hour, and then the solution was changed and the membrane was incubated overnight. Two 15 minute washes (50 ml each) of 3% (v/v) glacial acetic acid (wash solution), were followed by a 20 minute oxidation in 25 ml of the kit supplied oxidizing solution. After oxidation, three 15 minute washes (50 ml each) prepared the

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membrane for staining. The blot was incubated in 25 ml of Pro-Q Emerald Staining Solution that was prepared using the kit reagents. After 1 hour of staining in the dark, two 30 minute, 50 ml wash cycles were followed by two 45 minute, 50 ml wash cycles. The final wash cycles included two 25 ml, 1 minute deionized water washes followed by three 25 ml, 5 minute methanol washes (B&J Brand, Muskegon, MI). The blot was then scanned using the BioRad Molecular Imager FX using the Alexa 488 illumination setting (Qunatity One software; version 4.6, build 036) in order to visualize the fluorescing glycosylated proteins.

2.0. Materials and Methods used to Characterize Cry2Ab2 Protein

2.1. Plant-Produced Cry2Ab2 Protein regime.and and AG The plant-produced Cry2Ab2 protein was isolated from ground corn grain of MON 89034. The identity of the grain sample containing MON 89034 was confirmed by event specific PCR. The isolated plant-producedBayer Cry2Ab2property protein protection waspublishing stored in a –80 °C freezer in a buffer solution containingof 50 mM CAPS, 2 mM DTT, pHcontents 11, at a total its protein concentration of 0.25 mg/ml. parties.data therefore and/oror affiliates. 2.2. E. coli-Produced Cry2Ab2property Protein intellectual third may its as the of and The E. coli-produced Cry2Ab2is protein was regulatoryused as a reference owner.standard in select a document analyses. These analyses anyincluded such apparent molecular weight determinationits by SDS- reproductiondocument PAGE, western blot analysis, glycosylationowner analysis,this BioRad ofprotein assay, and the functional activity assay. The E. coli-producedunder Cry2Ab2of protein reference standard has rightsthe this previously beendocument characterizedand/or and is referred to as “Cry2Ab2.820”rights on the Certificate of to of fall use of Analysis. the This may distribution,and rights owner 2.3. Assay Controlssubject the violate be copy Protein molecular weight standards wereof used to calibrate SDS polyacrylamide gels and and verify proteinmay transferdocument topublication, PVDF membranes. The E. coli-produced Cry2Ab2 reference It exploitation standard proteinthis was used to estimate the total protein concentration in the BioRad protein assays and anyalso used as the positive control in western blot analysis. Beta- permission lactoglobulin protein and PTH-aminoprohibited acid standards were used to verify the performance of the amino acid sequencer. A peptide mixture and an analytical BSA standard were the be used to calibratecommercial the MALDI-TOF mass spectrometer for tryptic mass analysis and molecularFurthermore, weightany determination, respectively. Transferrin and E. coli-produced Cry2Ab2 proteinConsequently, were usedwithout as the positive and negative control, respectively, in glycosylation analysis.

2.4. Protein Purification

The plant-produced Cry2Ab2 protein was purified from the grain of MON 89034 prior to initiation of this characterization. Although the purification procedure was not performed under a GLP study or plan, procedures were documented on worksheets and, where

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applicable, SOPs were followed. The Cry2Ab2 protein was purified from an extract of ground grain of MON 89034 using a combination of ammonium sulfate fractionation, anion exchange chromatography, and immunoaffinity chromatography.

The isolation of Cry2Ab2 protein from ground corn grain was performed in two 10 kg batches. Prior to extraction of Cry2Ab2 protein, each batch was extracted with 1X PBS buffer (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) to remove contaminant proteins. The PBS extraction procedure consisted of soaking ground grain in PBS for 2 hrs in a 4 °C cold room at approximately 1:10 sample weight to buffer volume ratio. The slurry was clarified by filtration using an Ertel Alsop filter press (Kingston, NY), and the PBS washed solid particle (cake) was retained. Subsequently, the Cry2Ab2 protein in the cake was extracted with extraction buffer containingregime.and 50 mM CAPS, 1 mM EDTA, 2.5 mM DTT, 1 mM PMSF, 2 mM benzamidine-HCl,and 0.5 mM AG CHAPS, 1% (w/v) PVPP, pH 10.8 at approximately 1:10 sample weight to buffer volume ratio for 2-3 hrs. During extraction, lipids were removed from the extract by adding CelPure P65 diatomaceous earth (AdvancedBayer Mineralsproperty Corp, protection Goleta,publishing CA) to the homogenate at ~7.5% (w/v) and allowedof to mix for ~10 minutes. The slurrycontents was clarified its by filtration using the filter press and the resultant extractparties. datafrom both batchestherefore was pooled and/or for a total volume of ~230 L. The pooled extract was concentratedor using a 30,000 affiliates. NMWC Hollow Fiber Cartridgeproperty (Amersham intellectual thirdBiosciences, Piscataway,may NJ) to a final its volume of ~35 L. To remove plant genomicas DNA, polyethyleneimine, 10% (w/v), was the of and added to the concentratedis extract to a final concentrationregulatory of 0.05% (w/v),owner. the extract was a document clarified by centrifugation toany remove such precipitated DNA, and the supernatantits was retained. reproductiondocument The Cry2Ab2 protein in the supernatantowner was precipitatedthis by of0% – 35% ammonium under of sulfate saturation. The 35%rights ammoniumthe sulfate pelletthis was recovered by centrifugation documentand/or rights and the pellet was dissolvedto ofin 20 fallL of resuspensionuse of buffer containing 50 mM CAPS, 1 mM EDTA, 2.5 mM DTT, 1 mM PMSF, 2 mM benzamidine-HCl,the pH 10.8 by mixing in 4 °C coldThis room. The suspensionmay wasdistribution, clarifiedand by centrifugation and 21 L supernatant rights owner was retained, subjectconcentrated, and buffer exchanged by diafiltration against Buffer the violate containing 50be mMcopy CAPS, 1 mM EDTA, 2.5 mM DTT, 1 mM PMSF, 2 mM benzamidine-HCl, pH 10.8 to removeof any residual ammonium sulfate salt. The and concentratedmay sampledocument of publication,13 L was loaded onto an anion exchange column in two batches, It exploitation Run 1 (6L) andthis Run 2 (7L). The elution parameters were identical for both runs and thus only the Run1 columnany parameters are described below. permission prohibited A portion of concentratedthe samplebe was loaded onto a 4.5 L (20 cm x 14.4 cm column) Q Sepharose Fast Flowcommercial anion exchange resin column, which was equilibrated with Buffer. TheFurthermore, bound Cry2Ab2any protein was eluted with step gradients as follows: 0-20% buffer B (BufferConsequently, containingwithout 1M NaCl) in 10 column volumes (CV), and then the gradient was held at 20% buffer B for 4 CV, and then the gradient was increased to 65% buffer B over 10 CV and finally the gradient was stepped up to 100% buffer B and held at 100% buffer B in 2 CV. Fractions, each ~ 4 L, containing Cry2Ab2 protein were identified by Cry2A QuickStix™ (Portland, ME) for further analysis. Based on western blot analysis and SDS-PAGE analysis, fraction 7 from Run 2 was selected for affinity purification.

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Subsequently, fraction 7 was concentrated to 400 mL using a 30,000 NMWC Hollow Fiber Cartridge. Approximately one half of the aforementioned sample was buffer exchanged into EPPS buffer (50 mM EPPS, 2 mM DTT, pH 7.2) using centrifuge concentrators (30 kDa MWCO), resulting in a final volume of 200 mL. This sample, in two separate batches, was applied to an affinity column (1.0 cm × 2.7 cm) containing Protein G agarose conjugated with Cry2Aa-specific mAb and equilibrated with EPPS buffer (50 mM EPPS, 2 mM DTT, pH 7.2). The sample was re-circulated through the column for 2 hrs at 100 mL/hr. The column was then washed with 17-20 CV of EPPS buffer (50 mM EPPS, 2 mM DTT, pH 7.2), followed by 5-7 CV of EPPS buffer (50 mM EPPS, 2 mM DTT, pH 8.5). The bound Cry2Ab2 protein was eluted with 100 mM Triethylamine, pH 11.4, in five 1.5 mL fractions. Based on SDS-PAGE analysis, fractions containing Cry2Ab2 protein from batch one and two were concentratedregime.and and buffer exchanged into a storage buffer containing 50 mM CAPS,and 2 mM DTT, pH 11.0, AG using centrifuge concentrators (30 kDa MWCO). Concentrated samples were then pooled, resulting in a final volume of 2 ml. Bayer property protectionpublishing The affinity chromatography procedure ofwas repeated beginning with 100 contentsmL of fraction its 7 from Run2. As previously described, the sampleparties. datawas concentratedtherefore and buffer exchanged into EPPS buffer (50 mM EPPS, 2 mM DTT, pH 7.05)and/or toor a final volume of 50 affiliates. mL using centrifuge concentratpropertyors (30 kDa intellectual MWCO).third This sample wasmay re-circulated for 2 its hr (60 mL/hr) through an affinity columnas (1.0 cm × 2.75 cm) prepared with Cry2Aa- the of and specific mAb equilibratedis with EPPS buffer (50regulatory mM EPPS, 2 mM DTT,owner. pH 7.05). The a document column was then washed withany 15such CV of EPPS buffer (50 mM EPPS,its 2 mM DTT, pH reproductiondocument 7.05) followed by 5 CV of EPPS bufferowner (50 mM EPPS,this 2 mM DTT,of pH 8.5). The bound Cry2Ab2 protein was eluted with 100under mM Trieofthylamine, pH 11.4, in five 1.5 mL rightsthe this fractions. documentand/or rights to of fall use of the Based onThis SDS-PAGE analysis,may distribution,Cry2Ab2and enriched fractions were individually rights owner concentrated andsubject buffer exchanged into a storage buffer containing 50 mM CAPS, 2 mM DTT, pH 11, using a centrifuge concentratthe orviolate (30 kDa MWCO). The concentrated be copy samples were pooled into a final volumeof of 2.4 mL. The aforementioned sample and the and sample mayfrom the firstdocument runpublication, (2 ml) were pooled resulting in a final volume of 4.4 ml. This It exploitation sample was a clearthis colorless solution and was assigned lot # Cry2Ab.820_040705. any permission 2.5. Determination of the Totalprohibited Protein Concentration

the be The total proteincommercial concentration of the purified plant-produced Cry2Ab2 protein was estimatedFurthermore, usingany a BioRad protein assay. The E. coli-produced Cry2Ab2 reference standardConsequently, proteinwithout (concentrations ranging from 0.05 to 0.5 mg/mL) was used to prepare a standard curve. The plant-produced Cry2Ab2 total protein concentration was estimated by comparison of absorbance values obtained for the sample to the values of the standard curve. Data were collected using a Bio-Tek Instruments, Inc. Powerwave Xi microplate scanning spectrophotometer (Winooski, VT) employing KC4 software version 3.3 revision 10. Readings were taken at a wavelength of 595 nm.

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2.6. Western Blot Analysis

Aliquots of the stock solutions of the plant-produced Cry2Ab2 and reference standard were diluted to a final purity-corrected protein concentration of 2 ng/µl in dilution buffer (50 mM CAPS, 2 mM DTT, pH 11) and 5× sample loading buffer [5x concentrated Laemmli buffer (312 mM Tris-HCl, 20% (v/v) 2-mercaptoethanol, 10% (w/v) sodium dodecyl sulfate, 0.025% (w/v) bromophenol blue, 50% (v/v) glycerol, pH 6.8)]. Samples were then heated to ~100 °C for 5 min and applied to a pre-cast tris-glycine 4→20% polyacrylamide gradient 10-well gel. The plant-produced Cry2Ab2 protein was loaded in duplicate at three different loadings of 20, 30, and 40 ng per lane. The E. coli-produced Cry2Ab2 reference standard was loaded at 20 ng per lane. Electrophoresis was performed at a constant voltage of 140 V for 20 min followed by a constant voltageand of and regime. 200 V for 43 min. Pre-stained molecular weight AGmarkers included during electrophoresis (BioRad Precision Plus Dual Color, Hercules, CA) were used to verify electrotransfer of protein to the membrane and to estimate the molecular weight of the immunoreactive bands. Samples were electrotransferred toBayer a 0.45 micronproperty PVDF membranepublishing (Invitrogen, protectioncontents Carlsbad, CA) for 60 min at a constant currentof of 300 mA. its parties.data therefore and/oror The membrane was blocked for one hour with 5% (w/v) NFDM in PBST. The affiliates. membrane was probed with propertya 1:3000 intellectualdilutionthird of goat anti-Cry2Ab2may antibody (lot its as 7227632) in 2% (w/v) NFDMthe ofin PBST forand one hour. Excess antibody was removed using three 10 min washesis with PBST. The regulatorymembrane was probedowner. with peroxidase- a document conjugated rabbit anti-goat anyIgG (Sigma,such St. Louis, MO) at a dilutionits of 1:10,000 in 2% reproductiondocument (w/v) NFDM in PBST for one hour.owner Exce ss peroxidase-conjugatethis of was removed using under of three 10 min washes withrights PBST.the All procedures, includingthis blocking, and all other documentand/or rights incubations were performedto atof room fall temper ature.use Immunoreactiveof bands were visualized using the ECL detection system (Amersham Biosciences,the Piscataway, NJ) and exposed This may distribution,and (30 sec, 1 min, 2 min,rights 3 min, and 7 min)owner to Hyperfilm ECL high performance chemiluminescencesubject film (Amersham Biosciences, Piscataway, NJ). Films were developed usingbe a Konica SRX-101A automatedthe violate film processor. copy of and 2.7. Analysismay of thedocument N-terminalpublication, Sequence It exploitation

this any An aliquot of the plant-produced Cry2Ab2 protein was diluted with 5× sample loading permission buffer to a final purity correctedprohibited protein concentration of 80 ng/µl. Molecular weight markers (BioRad Precisionthe Plus Dual Color, Hercules, CA) were used to confirm the commercial be transfer of protein to the PVDF membrane. The plant-produced Cry2Ab2 protein was electrophoresedFurthermore,any in eight lanes at 2 µg per lane. The Cry2Ab2 containing samples were heatedConsequently, to ~99 °withoutC for 5 min prior to electrophoresis on a pre-cast tris-glycine 4→20% SDS polyacrylamide gel at 140V for 20 min followed by 200 V for 43 min. The gel was then electroblotted to a 0.2 micron PVDF membrane for 60 min at a constant current of 300 mA in a solution containing 10 mM CAPS, 10% (v/v) methanol, pH 11. Protein bands were stained by briefly soaking the membrane with Coomassie Blue R-250 stain (BioRad) and visualized by destaining with a Coomassie R-250 destain solution (BioRad).

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2.8. MALDI-TOF MS Tryptic Mass MapBayer Analysis property protectionpublishing of contents its MALDI-TOF mass spectrometry was used to confirmparties. the dataidentity of the plant-producedtherefore and/or Cry2Ab2 protein. A protein can be typically identified when 40% ofor the mass fragments affiliates. are identified from the analyzedproperty protein (Jiménezintellectualthird et al., 1998). may its as the of and 2.8.1. SDS-PAGE Separationis of Proteins regulatory owner. a document any such its reproductiondocument An aliquot of the plant-produced Cry2Ab2owner protein wasthis diluted ofwith 5× sample loading under of buffer to a final purity correctedrightsthe protein concentrationthis of 80 ng/µl and 2 µg was documentand/or rights electrophoresed in each toof fiveof lanes.fall Broad useRange ofmolecular weight markers (BioRad, Hercules, CA) were used to estimate molecular weights.the Samples were heated to ~99 °C This may distribution,and for 5 min prior to electrophoresisrights on a pre-castowner tris-glycine 4→20% SDS polyacrylamide gel at 140 V forsubject 20 min followed by a constant voltage of 200 V for 46 min. Proteins the were fixed bybe placing the gel in a solution of violate40% (v/v) methanol and 7% (v/v) glacial copy of acetic acid for 30 min, stained 2 hr withand Brilliant Blue G-Colloidal stain (Sigma, St. may documentpublication, Louis,It MO), destained 30 secexploitation with a solu tion containing 10% (v/v) acetic acid and 25% (v/v) methanol,this and any followed by 25% (v/v) methanol for 1 hr. Two protein bands, band-1 and band-2, migrating at 61 kDa and 50 kDa, respectively, were identified for tryptic permission mass map analysis. prohibited the commercial be 2.8.2. In-gel Protein Digestion and Sample Preparation Furthermore,any In-gelConsequently, protein digestionwithout The plant-produced full length Cry2Ab2 protein, band-1 migrating at ~61 kDa, and band- 2, a proteolytic fragment of the full length Cry2Ab2 protein migrating at ~50 kDa, were excised, destained, reduced, alkylated, and subjected to an in-gel trypsin (Promega, Madison, WI) digestion (Williams et al., 1997). Each gel band was individually destained for 30 min by incubation in 100 µL of 40% (v/v) methanol and 10% (v/v) glacial acetic acid in its own microfuge tube. Following destaining, the gel bands were incubated in 100 µL of 100 mM ammonium bicarbonate buffer for 30 min at room

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 210 of 323

temperature. Proteins were reduced in 100 µl of 10 mM dithiothreitol solution for 2 hrs at 37 °C. Proteins were then alkylated by the addition of 100 µl of buffer containing 200 mM iodoacetic acid. The alkylation reaction was allowed to proceed at room temperature for 20 min in the dark. The gel bands were incubated in 100 µl of 100 mM ammonium bicarbonate for 30 min at room temperature at which time 100 µl of acetonitrile was added and the incubation was continued for an additional 30 minutes. The ammonium bicarbonate/acetonitrile incubations were repeated two additional times to remove the reducing and alkylating reagents, and salts from the gel. The gel bands were dried in a SpeedVac concentrator (Savant, Holbrook, NY), rehydrated with 40 µl 25 mM ammonium bicarbonate containing 33 µg/ml trypsin, and the protein contained in the gel band was digested for 16 hours at 37 °C. Digested peptides were extracted for one hour at room temperature with 50 µl 70% (v/v) acetonitrile containing 0.1%and (v/v) and regime. TFA per gel band. Supernatants for each extractionAG were combined into a single tube and dried in a SpeedVac concentrator. This process of extracting the peptides was repeated two more times. The final dried materials were reconstituted in 10 µl of 0.1% (v/v) TFA. Bayer property publishing protectioncontents Sample Preparation of its parties.data therefore A portion (5 µl) of the digested samples was desalted (Bagshawand/or et al., 2000) using ® or Millipore (Bedford, MA) ZipTip C18affiliates. pipette tips. Prior to desalting, the tips were property intellectualthird may wetted with methanol and equilibratedits with 0.1% (v/v) TFA. Each sample was applied to ® as a ZipTip C18 and eluted thewith of5 µL of Washand 1 [0.1% (v/v) TFA], followed by 5 µl of is regulatory owner. Wash 2 [20% (v/v) acetonitrile containing 0.1%a (v/v) TFA],document followed by 5 µl of Wash 3 any such its [50% (v/v) acetonitrile containing 0.1% (v/v) TFA],reproduction and finallydocument withof 5 µl of Wash 4 [90% (v/v) acetonitrile containing 0.1% (v/v)owner TFA]. this under of rightsthe this documentand/or rights 2.8.3. MALDI-TOF Instrumentationto of fall and Massuse Analysisof the This may distribution,and Mass spectral analysesrights were performed as follows:owner mass calibration of the instrument was performed subjectusing an external peptide mixture from a Sequazyme™ Peptide Mass be the violate Standards kit (Appliedcopy Biosystems). Samplesof (0.3 µl) from each of the desalting steps, as well as a sample of the solution taken priorand to desalting, were co-crystallized with 0.75 µl may documentpublication, α-cyano-4-hydroxyIt cinnamicexploitation acid (Waters, Milford, MA) on the analysis plate. All samples were analyzedthis any in the 500 to 5000 dalton range using 100 shots at a laser intensity setting of 2318-2460 (a unit-less MALDI-TOF instrument specific value). Protonated permission (MH+) peptide masses were observedprohibited monoisot opically in reflector mode (Aebersold, the 1993; Billeci andcommercial Stults, 1993). be GPMAW32 software (Applied Biosystems, version 4.23) was used to generate a theoretical trypsin digest of the expected Cry2Ab2 protein sequenceFurthermore, deducedany from the nucleotide sequence. Masses were calculated for each theoreticalConsequently, peptidewithout and compared to the raw mass data. Experimental masses (MH+) were assigned to peaks in the 500 to 1000 Da range if there were two or more isotopically resolved peaks, and in the 1000 to 5000 Da range if there were three or more isotopically resolved peaks in the spectra. Peaks were not assessed if the peak heights were less than approximately twice the baseline noise, or when a mass could not be assigned due to overlap with a stronger signal of ±2 Daltons from the mass analyzed. Known autocatalytic fragments from trypsin digestion were identified in the raw data. The identity of the Cry2Ab2 protein is confirmed if ≥ 40 % of the protein sequence was

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 211 of 323

identified by matching experimental masses for the tryptic peptide fragments to the expected masses for the fragments.

2.9. Determination of Purity and Molecular Weight

2.9.1. SDS-PAGE

Aliquots of the E. coli- and plant-produced Cry2Ab2 proteins were each diluted with sample dilution buffer and 5× sample loading buffer to a final protein concentration of 0.2 µg/µl. Molecular weight markers (BioRad Broad-Range, Hercules, CA) that were used to estimate the apparent molecular weight of the test substance, were diluted to a final total protein concentration of 0.9 µg/µl. The plant-produced Cry2Ab2 proteinand was and regime. analyzed in duplicate at 1, 2, and 3 µg total proteinAG loads per lane. The E. coli-produced Cry2Ab2 reference standard was analyzed at 1 µg total protein. All samples were heated at ~102 °C for 5 min and applied to a pre-cast tris-glycine 4→20% polyacrylamide Bayer property protectionpublishing gradient 10-well mini-gel (Invitrogen, Carlsbof ad, CA). Electrophoresis wascontents performed at a constant voltage of 150 V for 82 min. Proteins were fixed by placingits the gel in a parties.data therefore solution of 40% (v/v) methanol and 7% (v/v) glacial acetic acidand/or for 30or min, stained 16 hr with Brilliant Blue G-Colloidal stainaffiliates. (Sigma, St. Louis, MO), destained 30 sec with a property intellectualthird may solution containing 10% (v/v) aceticits acid and 25% (v/v) methanol, and finally destained as with 25% (v/v) methanol forthe 6 hr.of and is regulatory owner. a document any such its Analysis of the gel was performed using a BioRadreproduction GS-800document of densitometer with the supplied Quantity One software (versionowner 4.4.0, Hercules,this CA). Values for the markers under of supplied by the manufacturerrights werethe used to estimate thethis apparent molecular weight of documentand/or rights each observed band. Allto visibleof bandsfall withinuse each oflane were quantified using Quantity One software. For the plant-produced Cry2Ab2 protein,the purity was estimated as the This may distribution,and percent optical densityrights of the ~61 kDa bandowner relative to all bands detected in the lane. Apparent molecularsubject weight and purity were reported as an average of all six loadings be the violate containing the plant-producedcopy Cry2Ab2of protein. and may documentpublication, 2.9.2.It MALDI-TOF MS Trypticexploitation Mass Analysis this any Determination of the intact mass was attempted for the plant-produced Cry2Ab2 protein permission using MALDI-TOF mass spectrometryprohibited analysis. Prior to analysis, the plant-produced Cry2Ab2 protein and BSAthe reference protein (NIST, Gaithersberg, MD) were desalted commercial be using drop dialysis (Görisch, 1988). A portion of each protein sample (0.3 µl) was spottedFurthermore, on anany analysis plate, mixed with 0.75 µl sinapinic acid solution and air-dried. EachConsequently, sample waswithout analyzed in triplicate. Mass spectral analysis was performed using an Applied Biosystems Voyager DE-Pro Biospectrometry Workstation MALDI-TOF instrument with the supplied Data Explorer software (version 4.0.0.0, Foster City, CA). Mass calibration of the instrument was performed using desalted BSA reference protein.

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2.10. Determination of Functional Activity by Insect Bioassay

The purpose of this analysis was to compare the biological activity of the plant- the E. coli-produced Cry2Ab2 proteins by determining EC50 values in a CEW (Helicoverpa zea) diet-incorporation insect bioassay. The EC50 value is defined as the concentration of Cry2Ab2 protein in the diet that results in 50% larval growth inhibition. In order to assess the functional activity of the plant-produced Cry2Ab2 protein and to compare its activity to the E. coli-produced Cry2Ab2 reference standard, aliquots of the plant- and E. coli-produced Cry2Ab2 proteins were used to estimate the effective protein concentration necessary to inhibit the growth of the target insect by 50%. The plant- and E. coli-produced Cry2Ab2 proteins were be determined to have comparable functional activity if the difference in mean EC values between plant- and E. coli-produced 50 regime.and proteins is less than or equal to three fold. and AG

2.10.1. Insects Helicoverpa zea (CEW) eggs were obtainedBayer from Benzonproperty Research protection Inc.publishing Insect eggs were incubated at temperatures ranging from of10 o C to 27o C, to achieve the desiredcontents hatch time. its parties.data therefore 2.10.2. Bioassays and/oror affiliates. CEW larvae were used to measureproperty activity intellectual of thirdthe plant- and E. coli-producedmay Cry2Ab2 its protein samples in a laboratory diet-incorporas ation bioassay. The bioassay was replicated the of and three times on separate isdays with separate batchesregulatory of insects. The owner.plant- and E. coli- a document produced proteins were runany in parallel such during each bioassay. Each itsbioassay replicate for reproductiondocument the E. coli-produced and plant-producedowner Cry2Ab2 proteithis ns consistedof of a series of seven dilutions and a buffer control yieldingunder a dose seriesof with a 2-fold separation factor rightsthe this documentand/or rights ranging from 0.016 – 1.0to µgof Cry2Ab2 fall protein/mLuse ofdiet. The dose-response curves for each protein included a buffer control. Each buffer controlthe contained an amount of their respectiveThis buffer equivalent tomay the distribution,amountand of protein in the highest dose level. The rights owner Cry2Ab2 proteinsubject dosing solutions were prepared by diluting the protein with purified water and incorporating the dilution into thean agar-basedviolate insect diet. This dose series in be copy diet was chosen to adequately characteriof ze the dose-effect relationship on CEW weight and gain formay the proteinsdocument frompublication, both sources. The diet mixture was then dispensed in 1 mL It exploitation aliquots into a 128this well tray (#BIO-BA-128, CD International, Pitman, NJ). Insect larvae were placed on theseany diets using a fine paintbrush, with a target number of 16 insects per permission treatment. The infested wells wereprohibited covered by a ventilated adhesive cover (#BIO-CV-16, CD International, Pitman, NJ) and the insects were allowed to feed for a period of seven the be days in an environmentalcommercial chamber programmed at 27° C, ambient relative humidity and a lightingFurthermore, regimeany of 14 h light:10 h dark. The combined weight of the surviving insects at eachConsequently, dose levelwithout for each source of protein was recorded at the end of the 7-day incubation period.

2.10.3. Statistical analysis Data were entered into an Excel spreadsheet and transferred to the Statistics Technology Center for analysis. Dose response modeling and EC50 determinations were performed using a 3-parameter logistic regression model (equation 1) under the PROC NLIN procedure in SAS.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 213 of 323

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

where Wt is the average CEW larvae weight and DietDose is the Cry1Ab protein diet dose level. Three parameters that are included in the model; W0 represents the weight at DietDose = 0.0, EC50 represents effective concentration to reduce the growth of the target insect by 50%, and B reflects the rate of the weight loss as DietDose increases,regime.and and e denotes the residual (error). and AG

2.11. Glycosylation Analysis Bayer property protectionpublishing Glycosylation analysis was used to deteof rmine whether the plant-producedcontents Cry2Ab2 its protein was post-translationally modified with covalentlyparties. databound carbohydratetherefore moieties. Aliquots of the plant-produced Cry2Ab2 protein, the E. coli-producedand/oror Cry2Ab2 reference affiliates. standard (in this instance, a propertynegative intellectualcontrol),third and the positive maycontrol transferrin its (Amersham Biosciences, Piscataway, NJ)as were each diluted in dilution buffer and in 5× the of and sample loading buffer isto a final purity coregulatoryrrected (total proteinowner. for transferrin) a document concentration of 50 ng/µl. anyThese such samples were heated to ~100 °C itsfor 5 min, and loaded reproductiondocument along with Precision Plus Dual Colorowner pre-st ained thisprotein molecuof lar weight markers under of (BioRad , Hercules, CA) onrights a pre-castthe tris-glycine 4→this20% polyacrylamide gradient 10- documentand/or rights well mini-gel. All threeto samples of werefall loadeduse at a singleof loading of 0.5 and 1 µg protein per lane. Electrophoresis was performed at a constantthe voltage of 140 V for 20 min This may distribution,and followed by a constant rightsvoltage of 200 V for 47owner min. After electrophoresis, proteins were electrotransferedsubject to a 0.45 micron PVDF membrane for one hour at a constant current of 300 mA. be the violate copy of and may documentpublication, CarbohydrateIt detection was exploitationperformed directly on the PVDF membrane containing the 0.5 and 1.0 µgthis sample any loads using the ECL detection system (Amersham Biosciences, Piscataway, NJ). After the electrotransfer of the proteins, the PVDF membrane was permission incubated in PBS for 10 min, andprohibited transferred to a solution of 100 mM acetate buffer, pH 5.5, containing the oxidationthe reagent, 10 mM sodium metaperiodate. The membrane was commercial be incubated in the dark for 20 minutes. The oxidation solution was removed from the membraneFurthermore, byany two brief rinses followed by three sequential 10 min washes in PBS. The membraneConsequently, was withouttransferred to a solution of 100 mM acetate buffer, pH 5.5, containing 25 nM biotin hydrazide and incubated for 60 minutes. Biotin hydrazide solution was removed by washing in PBS as previously described. The membrane was blocked with 5% blocking agent in PBS for 60 minutes. The blocking solution was removed by washing in PBS as previously described. The membrane was incubated with streptavidin-HRP conjugate (diluted 1:6000) in acetate buffer for 30 min to detect carbohydrate moieties bound to biotin. Excess streptavidin-HRP was removed by washing in PBS as previously described. Bands were visualized using the ECL detection

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 214 of 323

system (Amersham Biosciences, Piscataway, NJ). Films were exposed (1 min, 3 min, and 6 min) to Hyperfilm ECL high performance chemiluminescence film (Amersham Biosciences, Piscataway, NJ). Films were developed using a Konica SRX-101A automated film processor.

References

Bagshaw, R. D., Callahan, J. W., and Mahuran, D. J. 2000. Desalting of in-gel-digested protein sample with mini-C18 columns for matrix-assisted laser desorption ionization time of flight peptide mass fingerprinting. Anal Biochem 284:432-435.

and Jiménez, C. R., L. Huang, Y. Qui, and A.L. Burlingame. 1998.and In-gelregime. digestion of proteins for MALDI-MS fingerprint mapping.AG Current Protocols in Protein Science, Supplement 14, pp. 16.4.1-16.4.5.

Bayer property protectionpublishing Williams, K., M. LoPresti, and K Stone.of 1997. Internal protein sequencingcontents of SDS- PAGE-separated proteins: Optimization of an in-gel digest protocol.its Tech in Protein parties.data therefore Chem 8:79-98. and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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APPENDIX C: Materials and Methods for the Safety Assessment of the Cry1A.105 and Cry2Ab2 Proteins

1. Assessment of Digestibility of the Cry1A.105 Protein in Simulated Gastric Fluid

1.1. Cry1A.105 Protein

The test substance was the Cry1A.105 protein. The Cry1A.105 protein was isolated from a fermentation batch of E. coli containing pMON96851 expression plasmid. This protein has been characterized and has a total protein concentration of 1.2 mg/ml and a purity of 92 %. Functional activity was confirmed using an insect bioassay with the larvaeand of a and regime. susceptible pest, corn earworm (CEW). The ECAG50 value was 5.8 ng/ml of diet. Prior to its application to the test system, the test substance was stored in a –80 ºC freezer in a test substance storage buffer containing 25 mM CAPS, 1 mM benzamidine-HCl, 0.1 mM EDTA, and 0.2 mM DTT, pH ~10.3. Bayer property protectionpublishing of contents its parties.data therefore 1.2. Test System and/oror affiliates. property intellectualthird may The test system for this study wasits simulated gastric fluid (SGF) that contains the proteolytic enzyme pepsin.the The SGF wasas prepared using a highly purified form of pepsin of and owner. (Catalog number P-6887,is Sigma Company, St. regulatoryLouis, MO). The SGF formulation, time a documentits course, and experimental parametersany such used in this study followed the conditions used in reproductiondocument of the ILSI multi-laboratory evaluationowner test (Thom as et al.,this 2004). The SGF was formulated under of so that ten units of pepsin rightsactivitythe per microgram of totalthis protein from the test substance documentand/or rights would be present in theto digeofstion fall reactions. use The ofamount of pepsin powder used to prepare SGF was calculated from the specific activity reportedthe on the product label. One This may distribution,and unit of activity is definedrights as a change in A280owner nm of 0.001 per minute at 37 °C, measured as trichloroaceticsubject acid (TCA) soluble products using hemoglobin as the substrate. The be the violate stock SGF solutioncopy was prepared by addingof pepsin powder (26.6 mg) to 33.2 ml of an acidic sodium chloride solution (2 mg/ml NaCland , 10 mM HCl, pH 1.3). After the activity may documentpublication, of pepsinIt in SGF was confirmed,exploitation the stock SGF solution was diluted to provide approximately this1500 any units pepsin activity/ml of solution.

permission 1.3. Sample Preparation prohibited the commercial be Digestion of the Cry1A.105protein in SGF was evaluated over time by analyzing specimensFurthermore, fromany all incubation time points. The target digestion temperature was 37 ± 2 ºC. Consequently,without

The Cry1A.105 protein was diluted to 0.6 mg total protein /ml by mixing 200 µl of the protein at 1.2 mg/ml and 200 µl of storage buffer (25 mM CAPS, 1 mM benzamidine- HCl, 0.1 mM EDTA, and 0.2 mM DTT, pH 10.3) and then heated at 44.5 ºC for 10 minutes. Digestion samples were prepared by adding 200 µl of the diluted test substance to a tube containing 800 µl of SGF. The tube contents were vortex mixed and immediately placed in a 37.2 ºC water bath and subsequently vortex mixed every 30 - 60

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sec throughout the digestion experiment. Samples (100 µl) were removed at targeted times of 0.5, 2, 5, 10, 20, 30, and 60 min (specimens T1 to T7, respectively) and placed in a tube containing quenching mixture. Quenching mixture contained 35 µl of carbonate buffer [700 mM Na2CO3, pH 11.0], and 35 µl of 5× Laemmli buffer (LB) [312.5 mM Tris-HCl, 25% (v/v) 2-mercaptoethanol, 10% (w/v) sodium dodecyl sulfate, 0.025% (w/v) Bromophenol Blue, and 50% (v/v) glycerol, pH 6.8].

The zero incubation time point (T0) was prepared in a separate tube. SGF (80 µl) was quenched by adding 35 µl of carbonate buffer and 35 µl of 5× LB prior to the addition of 20 µl of the diluted test substance.

All quenched samples were heated to 75-100 ºC for 5-10 min, frozen on dry andice, and and regime. stored in a -80 ºC freezer until analyzed. AG

1.4. Experimental Controls Bayer property publishing protectioncontents Experimental controls were prepared to ofdetermine the stability of the test substance in the its test system buffer lacking pepsin [10 mM HCl,parties. 2 mg/mldata NaCl, pH therefore1.3]. These and/oror experimental controls were identified with the letter "P". The zero incubation time point affiliates. (P0) was prepared in a separateproperty tube. intellectualTest thirdsystem buffer (80 µl) maywas quenched by its as addition of 35 µl of carbonatethe bufferof and 35and µl of 5× LB prior to the addition of 20 µl of the diluted test substance.is The 60 min incubationregulatory time point (P7) was preparedowner. by adding a document 20 µl of the diluted test substanceany such to te st system buffer lacking pepsinits (80 µl). The tube reproductiondocument was vortex mixed and immediatelyowner placed in a 37.1this ºC water ofbath. After 60 min of under of incubation, the sample wasrights quenchedthe by addition of 35this µl of carbonate buffer and 35 µl documentand/or rights of 5× LB. to of fall use of the This may distribution,and Additional experimentalrights controls were preparedowner to evaluate the stability of the pepsin in the test systemsubject (SGF) lacking the test substance and to determine if non-specific interaction occursbe between the test systemthe components violate and the antibodies during western copy of blot analysis of the specimens. These experimentaland controls contained an aliquot of the test systemmay incubated document withpublication, test substance storage buffer instead of the test substance and It exploitation were identified with the letter "N". The zero incubation time point (N0) was in a separate this any tube. Test system (80 µl) was quenched by addition of 35 µl of carbonate buffer and 35 permission µl of 5× LB prior to addition ofprohibited 20 µl of the storage buffer (25 mM CAPS, pH 10.3, 1 mM benzamidine-HCl, the0.1 mM EDTA, and 0.2 mM DTT). The 60 min incubation time commercial be point (N7) was prepared by adding 20 µl storage buffer to 80 µl of test system. The tube wasFurthermore, vortex anymixed and placed in a 37.1 ºC water bath. After 60 min of incubation, the sampleConsequently, was quenchedwithout by addition of 35 µl of carbonate buffer and 35 µl of 5× SB.

All experimental controls were heated at 75-100 ºC for 5-10 min, frozen on dry ice, and stored in a -80 ºC freezer until analyzed.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 217 of 323

1.5. SGF Activity Assay

Activity of the SGF was assessed using a pepsin activity assay. The digestibility of the Cry1A.105 protein in SGF was assessed using stained polyacrylamide gels and western blot analysis. The limit of detection (LOD) of the Cry1A.105 protein for these methods was determined concurrently.

The SGF activity assay was used to confirm the suitability of the test system before its use with the test substance. Acceptable activity was defined as a pepsin activity per mg of pepsin powder (0.03 mg of powder per ml of diluted SGF) equal to the activity of pepsin per mg of pepsin powder as determined by the manufacturer (±1000 units/mg). One unit of pepsin activity in this assay is defined as the amount of pepsin that will regime.and produce a change in the absorbance at 280 nm of 0.001 per minand at pH 1.2-2.0 at 37 ± 2 AG ºC. The assay is used to estimate the amount of soluble peptides present in a TCA solution after pepsin digestion of denatured hemoglobin. Undigested hemoglobin was precipitated with TCA, and the amount ofBayer soluble peptidesproperty was protection estimatedpublishing by measuring the absorbance at 280 nm. The amountof of soluble peptide is directly proportionalcontents to the its amount of protease activity. parties.data therefore and/oror affiliates. Briefly, the SGF was diluted propertyto 0.03 mg intellectual of solidthird material (pepsin) permay ml of SGF [the its dilution factor (DF) was 26.7]. Acidifiedas hemoglobin [2% (w/v), 5 ml] was added to the of and each of three replicatesis of the test sample regulatoryand blank samples andowner. pre-warmed at a document 37 ± 2 °C for 5-10 minutes anyprior suchto starting the reactions. Diluted SGFits (1 ml) was added reproductiondocument to each replicate of test samples andowner both test andthis blank samplesof were incubated at under of 37.0 °C for an additional rights10 min.the The reaction was thisstopped by addition of 10 ml of documentand/or rights chilled 5% (v/v) TCA toto the testof andfall blank samples.use of Diluted SGF (1 ml) was then added to the blank samples. Samples were mixed and thenthe incubated another 5-10 min at This may distribution,and 37.4 °C. Precipitated rightsprotein was removed byowner filtering the test and the blank samples using 0.8 µm syringesubject filters. Samples of the clarified test and blank samples were read at 280 nm in a beBeckman DU-650 Spectrophotometer.the violate The activity of pepsin was calculated copy of using the following equation: and may documentpublication, It exploitation TestMean − BlankMean this any 280nmA 280nmA × DF , ×× 1min10001.0 ml permission prohibited the where 0.001 is thecommercial change in be the absorbance at 280 nm per min at pH 1.2-2.0 and 37 ± 2 ºC produced by one unit of pepsin activity; 10 min is the reaction time, 1 ml is the amountFurthermore, of SGFany added to the reaction; and DF is the dilution factor for the SGF. Consequently,without

1.6. SDS-PAGE

Samples containing 1× LB from the SGF in vitro digestion of the Cry1A.105 protein were separated by SDS-PAGE using pre-cast tricine 10-20% polyacrylamide gradient mini-gels and tricine running buffer (Invitrogen, Carlsbad, CA). The protein loaded per lane was based on the pre-digestion total protein concentration of the Cry1A.105 protein. All experimental controls were loaded at the same volume as those containing Cry1A.105

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protein so that all other components would be comparable. All samples were heated at 100.3 °C for 5 min prior to loading on the gels. Protein markers were used to estimate the relative molecular weight. Electrophoresis was performed at a constant voltage of 125 V for 85 minutes. After electrophoresis, proteins were either visualized by staining the gel with colloidal Brilliant Blue G, or the gel was subjected to electrotransfer of proteins to nitrocellulose membrane for western blot analysis.

1.7. Colloidal Brilliant Blue G Staining

The colloidal Brilliant Blue G staining method was selected because it is an effective method for detecting nanogram quantities of protein on a gel (Neuhoff et al., 1988). Mark12 molecular weight markers (Invitrogen, Carlsbad, CA) were used toregime. estimateand the relative molecular weight of visualized proteins and peptides.and Based on pre-digestion AG concentrations, approximately 0.7 µg of total protein was loaded per lane. After separation of proteins, the gels were fixed in a solution containing 7% (v/v) acetic acid and 40% (v/v) methanol for 30 min and Bayerstained for propertyapproximately 20publishing h in 1× Brilliant protectioncontents Blue G-colloidal stain solution containingof 20% (v/v) methanol. The gels were briefly its destained for 30 s in a 10% (v/v) acetic acid, 25% (v/v)parties. methanoldata solution andtherefore completely and/oror destained for ~5 h in a 25% (v/v) methanol solution. Images were captured using a Bio- affiliates. Rad GS-800 densitometer. Theproperty results ofintellectual the inthird vitro digestibility of Cry1A.105may protein its as were determined by visual theexamin of ation of theand stained gels. is regulatory owner. a document The approximate molecularany weights such of the full-size protein and itsproteolytic fragment reproductiondocument observed on the colloidal Brilliant Blueowner G stained gelsthis were visuallyof determined relative under of to the positions of the molecularrightsthe weight markers. this documentand/or rights to of fall use of The LOD of the Cry1A.105 protein using the colloidalthe Brilliant Blue G staining This may distribution,and procedure was determined.rights Various dilutionsowner of the zero time point (T0) digestion specimen were loadedsubject onto a separate gel that was run concurrently with the gel used to assess digestibility.be Aliquots of the T0 digestionthe violate sample representing approximately 700, copy of 350, 100, 50, 20, 10, 5, and 2.5 ng total protandein were used for the stained LOD gel. may documentpublication, It exploitation 1.8. Western Blot Analysis this any

permission Specimens from the SGF in vitroprohibited digestions were also analyzed using western blotting. Based on pre-digestion theconcentr be ations, approximately 20 ng of total protein were loaded per lane. Followingcommercial electrophoresis, pre-stained molecular weight markers (Precision PlusFurthermore, Proteinany Standards, Bio-Rad, Hercules CA) were used to verify electrotransfer of proteinsConsequently, to the withoutmembrane. Proteins were electrotransferred to nitrocellulose membranes (0.45 µm pore size, Invitrogen) for 90 min at a constant voltage of 25 V.

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washes with PBST. The membrane was incubated with HRP–conjugated goat anti-rabbit IgG (Sigma) at a dilution of 1:10,000 in PBST containing 1% (w/v) NFDM for 1 h and again washed (three 10 min washes) with PBST. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences) and exposed (2, 5, and 10 minutes) to Hyperfilm ECL high performance chemiluminescence film (Amersham Biosciences). Films were developed using a Konica SRX101A automated film processor (Tokyo, Japan).

The approximate molecular weights of the full-size protein observed on the western blots were visually determined relative to the positions of the molecular weight markers.

The LOD for the Cry1A.105 protein using the western blot analysis procedure was regime.and determined. Various dilutions of the zero time point (T0) anddigestion specimen were AG loaded onto a separate gel that was run concurrently with the digestion western blot gel and subjected to the same western blot procedure as described above. Aliquots of the T0 digestion sample representing approximatelyBayer 7, 3.5, 2,property 1, 0.5, 0.2,protection 0.1,publishing and 0.05 ng total protein were used for the western blot LODof analysis. contents its parties.data therefore and/oror affiliates. 2.0. Digestibility of the Cry2Ab2property Protein intellectual in Simulatedthird Gastric Fluidmay its as the of and 2.1. Cry2Ab2 Protein is regulatory owner. a document any such its reproductiondocument The test substance is the E. coli-ownerproduced Cry2Ab2this protein. of The E. coli-produced Cry2Ab2 protein is referred to as Cry2Ab2.820under of in the Certificate of Analysis. The rightsthe this protein was isolateddocumentand/or from a fermentation batch of E. colirights containing the pMON70520 to of fall use of expression plasmid. This protein has been characterizedthe and has a total protein concentrationThis of 0.5 mg/ml, andmay a purity distribution, ofand 87%. Activity was confirmed using an insect rights owner bioassay with thesubject larvae of a susceptible pest, corn earworm (Helicoverpa zea, CEW). The EC value was 0.25 µg/ml of diet. Thethe testviolate substance was stored prior to use in a – 50 be copy 80 ºC freezer in buffer containing 50 mMof CAPS, 2 mM DTT, pH 11. and may documentpublication, It exploitation 2.2. Test Systemthis any permission The test system for this studyprohibited was simulate d gastric fluid (SGF) that contains the proteolytic enzyme pepsin. The SGF was prepared using a highly purified form of pepsin the be (Catalog numbercommercial P-6887, Sigma Company, St. Louis, MO). The SGF was formulated so thatFurthermore, ten unitsany of pepsin activity per microgram of total protein from the test substance wouldConsequently, be presentwithout in the digestion reactions. The amount of pepsin powder used to prepare SGF was calculated from the specific activity reported on the product label. One unit of activity is defined as a change in A280 nm of 0.001 per minute at 37 °C, measured as trichloroacetic acid (TCA) soluble products using hemoglobin as the substrate. The stock SGF solution was prepared by adding pepsin powder (26.6 mg) to 33.2 ml of an acidic sodium chloride solution (2 mg/ml NaCl, 10 mM HCl, pH ~1.3). After the activity of pepsin in SGF was confirmed, the stock SGF solution was diluted to provide approximately 1944 units pepsin activity/ml of solution.

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2.3. Sample Preparation

Digestion of the test substance in SGF was evaluated over time by analyzing specimens from all incubation time points. The target digestion temperature was 37 ± 2 ºC.

Digestion samples were prepared by adding 252 µl of the test substance to a tube containing 648 µl of SGF. The tube contents were vortex mixed and immediately placed in a 37.2 ºC water bath. The tube with the digestion mixture was regularly vortexed (every 30-60 sec) throughout the digestion experiment to prevent test substance precipitation. Samples (100 µl) were removed at targeted times of 0.5, 2, 5, 10, 20, 30, regime.and and 60 min (specimens T1 to T7, respectively) and placed in a andtube containing quenching AG mixture. Quenching mixture contained 35 µl of carbonate buffer [700 mM Na2CO3, pH 11], and 35 µl of 5× Laemmli buffer (LB) [312.5 mM Tris-HCl, 25% (v/v) 2- mercaptoethanol, 10% (w/v) sodium dodecylBayer sulfate, property 0.025% (w/v)protection Bromophenolpublishing Blue, and 50% (v/v) glycerol, pH 6.8]. of contents its parties.data therefore and/or The zero incubation time point (T0) was prepared in a separate tube.or SGF (72 µl) was affiliates. quenched by adding 35 µl of carbonateproperty bufferintellectual andthird 35 µl of 5× LB priormay to adding 28 µl of its test substance. as the of and is regulatory owner. a document All quenched samples wereany heated such at 75-100 ºC for 5-10 min, frozenits on dry ice, and reproductiondocument stored in a -80 ºC freezer until analyzed.owner this of under of rightsthe this documentand/or rights 2.4. Experimental Controlsto of fall use of the ExperimentalThis controls were preparedmay distribution, to determineand the stability of the test substance in the rights owner test system buffersubject lacking pepsin [10 mM HCl, 2 mg/ml NaCl, pH 1.3]. These the violate experimentalbe controls copy were identified with the letter "P". The zero incubation time point (P0) was prepared in a separate tube.of Test system buffer (72 µl) was quenched by and additionmay of 35 µl ofdocument carbonatepublication, buffer and 35 µl of 5× LB prior to addition of 28 µl of the It exploitation test substance. The 60 min incubation time point (P7) was prepared by adding 28 µl test this any substance to test system buffer lacking pepsin (72 µl). The tube was vortex mixed and permission immediately placed in a 37.4 ºCprohibited water bath. After 60 min of incubation, the sample was quenched by addition ofthe 35 µbel of carbonate buffer and 35 µl of 5× LB. commercial AdditionalFurthermore, experimentalany controls were prepared to evaluate the stability of the pepsin in the Consequently,test systemwithout (SGF) lacking the test substance and to determine if non-specific interaction occurs between the test system components and the antibodies during western blot analysis of the specimens. These experimental controls contained an aliquot of the test system incubated with test substance storage buffer instead of the test substance and were identified with the letter "N". The zero incubation time point (N0) was in a separate tube. Test system (72 µl) was quenched by addition of 35 µl of carbonate buffer and 35 µl of 5× LB prior to addition of 28 µl of storage buffer (50 mM CAPS, 2 mM DTT, pH 11). The 60 min incubation time point (N7) was prepared by adding 28 µl storage buffer

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 221 of 323

to 72 µl of test system. The tube was vortex mixed and placed in a 37.4 ºC water bath. After 60 min of incubation, the sample was quenched by addition of 35 µl of carbonate buffer and 35 µl of 5× LB.

All experimental controls were heated at 75-100 ºC for 5-10 min, frozen on dry ice, and stored in a -80 ºC freezer until analyzed.

2.5. SGF Activity Assay

Activity of the SGF was assessed using a pepsin activity assay. The digestibility of the Cry2Ab2 protein in SGF was assessed using stained polyacrylamide gels and western blot analysis. The limit of detection (LOD) of the Cry2Ab2 protein for regime.these methodsand was determined concurrently. and AG

The SGF activity assay was used to confirm the suitability of the test system before its use with the test substance. Acceptable activBayerity was propertydefined asprotection a pepsinpublishing activity per mg of pepsin powder (0.03 mg of powder perof ml of SGF) equal to the activitycontents of pepsin per its mg of pepsin powder as determined by the manufacturerparties. (±1000data units/mg).therefore One unit of and/or pepsin activity in this assay is defined as the amount of pepsin that willor produce a change affiliates. in the absorbance at 280 nm ofproperty 0.001 per minintellectual atthird pH 1.2-2.0 at 37 ± 2 ºC.may its as the of and The assay is used to estimateis the amount of solubleregulatory peptides present owner.in a TCA solution a document after pepsin digestion ofany denatured such hemoglobin. Undigestedits hemoglobin was reproductiondocument precipitated with TCA, and the amountowner of soluble peptidesthis was ofestimated by measuring under of the absorbance at 280 nm.rights Thethe amount of soluble peptidethis is directly proportional to the documentand/or rights amount of protease activity.to of fall use of the The SGFThis was diluted to 0.03 maymg of distribution, solidand mate rial (pepsin) per ml of SGF [the dilution rights owner factor (DF) wassubject 26.7]. Acidified hemoglobin [2% (w/v), 5 ml] was added to each of the violate three replicatesbe ofcopy the test sample and blank samples and pre-warmed at 37 ± 2 °C for 5- 10 minutes prior to starting the reactions.of Diluted SGF (1 ml) was added to each and replicatemay of test samplesdocumentpublication, and both test and blank samples were incubated at 37.0 °C for an It exploitation additional 10 min. The reaction was stopped by addition of 10 ml of 5% (v/v) chilled this any TCA to the test and blank samples. Diluted SGF (1 ml) was then added to the blank permission samples. Samples were mixedprohibited and then incubated another 5-10 min at 37.4 °C. Precipitated protein wasthe removed be by filtering the test and the blank samples using 0.8 µm syringe filters. Samplescommercial of the clarified test and blank samples were read at 280 nm in a BeckmanFurthermore, DU-650any Spectrophotometer. The activity of pepsin was calculated using the followingConsequently, equation:without

TestMean − BlankMean 280nmA 280nmA × DF , ×× 1min10001.0 ml where 0.001 is the change in the absorbance at 280 nm per min at pH 1.2-2.0 and 37 ± 2 ºC produced by one unit of pepsin activity; 10 min is the reaction time; 1 ml is the amount of SGF added to the reaction; and DF is the dilution factor for the SGF.

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2.6. SDS-PAGE

Samples containing 1× LB from the SGF in vitro digestion of the Cry2Ab2 protein were separated by SDS-PAGE using pre-cast tricine10-20% polyacryamide gradient mini-gels and tricine running buffer (Invitrogen, Carlsbad, CA). The protein loading per lane was based on pre-digestion total protein concentration of the Cry2Ab2 protein. All experimental controls were loaded at the same volume as those containing Cry2Ab2 protein so that all other components would be comparable. All samples were heated at 95.3 °C for 5 min prior to loading on the gels. Mark12 molecular weight markers (Invitrogen, Carlsbad, CA) were loaded to estimate the relative molecular weight of proteins and peptides visualized by staining. Electrophoresis was performed at a constantand and regime. voltage of 125 V for 90 min. After electrophorAGesis, proteins were either visualized by staining the gel with colloidal Brilliant Blue G, or the gel was subjected to electrotransfer of proteins to polyvinylidene difluoride (PVDF) membrane for western blot analysis. Bayer property publishing protectioncontents 2.7. Colloidal Brilliant Blue G Stainingof its parties.data therefore and/oror The colloidal Brilliant Blue G staining method was selected because it is an effective affiliates. method for detecting nanogramproperty quantities intellectual of thirdprotein on a gel (Neuhoffmay et al., 1988). its as Based on pre-digestion concenthe trations,of approximatelyand 0.8 µg of total protein was loaded per lane. After separationis of proteins, the gelsregulatory were fixed in a solutionowner. containing 7% a document (v/v) acetic acid and 40% (v/v)any methanolsuch for 30 min and stained forits 15 h in 1× Brilliant reproductiondocument of Blue G-colloidal stain solution containingowner 20% (v/v)this methanol. The gels were briefly under of destained for 30 s in 10% (v/v)rights aceticthe acid, 25% (v/v) methanolthis and completely destained documentand/or rights for 23 h in a 25% (v/v)to methanol of solution.fall Imagesuse ofwere captured using a Bio-Rad GS- 800 densitometer. The results of the in vitro digestibilitythe of Cry2Ab2 protein were This may distribution,and determined by visual examinrights ation of the stainedowner gels. subject The LOD of bethe Cry2Ab2 protein using thethe collo violateidal Brilliant Blue G staining procedure copy of was determined. Various dilutions of the andzero time point (T0) digestion specimen were loaded mayonto a separatedocumentpublication, gel that was run concurrently with the gel used to assess It exploitation digestibility. Aliquots of the T0 digestion sample representing approximately 1, 0.5, 0.2, this any 0.1, 0.05, 0.02, 0.01, 0.005, and 0.0025 µg total protein were used for the stained LOD permission gel. prohibited the commercial be 2.8. Western Blot Analysis Furthermore,any SpecimensConsequently, fromwithout the SGF in vitro digestions were also analyzed using western blotting. Based on pre-digestion concentrations, approximately 20 ng of total protein were loaded per lane. Following electrophoresis, pre-stained molecular weight markers (Precision Plus Protein Standards, Bio-Rad, Hercules CA) were used to verify electrotransfer of proteins to the membrane and estimate size of proteins and peptides. Proteins were electrotransferred to PVDF membranes (0.45 µm pore size, Invitrogen) for 90 min at a constant current of 25 V.

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Membranes were blocked overnight in a 4 ºC refrigerator with 5% (w/v) non-fat dry milk (NFDM) in phosphate buffered saline containing Tween-20 (PBST) buffer. All subsequent incubations (described below) were performed at room temperature. Membranes were incubated with goat anti-Cry2Ab2 antibody diluted 1:3,000 in PBST containing 1% (w/v) NFDM for 70 min. Excess serum was removed by three 5 min washes with PBST. The membrane was incubated with HRP–conjugated anti-goat IgG (Sigma) at a dilution of 1:10,000 in PBST containing 1% (w/v) NFDM for 1 h and again washed (three 5 min washes) with PBST. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Piscataway, NJ) and exposed (1, 2, 5, and 10 min) to Hyperfilm ECL high performance chemiluminescence film (Amersham Biosciences). Films were developed using a Konica SRX101A automated film processor (Tokyo, Japan). regime.and and AG The LOD for the Cry2Ab2 protein using the western blot analysis procedure was determined. Various dilutions of the zero time point (T0) digestion specimen were loaded onto a separate gel that was run concurBayerrently property with the digesprotectiontionpublishing western blot gel and subjected to the same western blot proceof dure as described above. Aliquotscontents of the T0 its digestion sample representing approximately 10, 5, parties.2, 1, 0.5,data 0.2, 0.1, andtherefore 0.05 ng total protein were used for the western blot LOD analysis. and/oror affiliates. property intellectualthird may its as the of and 3. Materials and Methodsis Used to Establish regulatoryEquivalence of the Cry1A.105owner. Proteins a document Produced in E. coli andany in MON such 89034 its reproductiondocument owner this of under of 3.1. Plant-Produced Cry1A.105rightsthe Protein this documentand/or rights to of fall use of The plant-produced Cry1A.105 protein was purified thefrom grain of MON 89034. The identity Thisof the grain containingmay MON distribution, 89034and was confirmed by event-specific PCR. The rights owner purified plant-producedsubject Cry1A.105 protein was stored in a 4 ºC refrigerator in a buffer solution containing 50 mM CAPS, 1 mM thePMSF, violate 2 mM benzamidine-HCl, 1 mM EDTA, be copy 0.8 M NaCl, and 30% (v/v) ethylene glycol,of pH 10.0. and may documentpublication, It exploitation 3.2. E. coli-producedthis Cry1A.105 Reference Standard any permission E. coli-produced Cry1A.105 proteinprohibited was used as a reference standard for determination of protein concentration and immunoblot analysis using N-terminal peptide antibody. the be This protein wascommercial also used as a reference standard to evaluate equivalence between plant- andFurthermore, E. coli-producedany Cry1A.105 proteins for the molecular weight and functional activity assay,Consequently, as a referencewithout and a negative control in glycosylation analysis, and as a reference and a positive control in immunoblot analysis using anti-Cry1A.105 antibody. The E. coli-produced Cry1A.105 reference standard was previously characterized. The Cry1A.105 protein was stored in a -80 °C freezer in a buffer solution (25 mM CAPS, 1 mM benzamidine-HCl, 0.1 mM EDTA, and 0.2 mM DTT, pH ~10.3) at a total protein concentration of 1.2 mg/ml by amino acid analysis.

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3.3. Assay Controls

Protein molecular weight standards (Bio-Rad, Hercules, CA) were used to calibrate SDS- PAGE gels and verify protein transfer to PVDF membranes. The E. coli-produced Cry1A.105 reference standard was used to create a standard curve for the quantitative western blot. The Cry1A.105 trypsin-resistant core was used as a negative control for the western blot analysis using N-terminal peptide antibody. A peptide mixture (Sequazyme Peptide Mass Standards kit, Applied Biosystems) was used to calibrate the MALDI-TOF mass spectrometer for tryptic mass analysis. Transferrin (Amersham Biosciences, Piscataway, NJ) and horseradish peroxidase (Sigma, St. Louis, MO) were used as positive controls for glycosylation analysis. CandyCane™ glycoprotein molecular regime.and weight standards were used as molecular weight markers andand positive and negative AG controls for glycosylation analysis.

3.4. Protein Purification Bayer property protectionpublishing of contents its The Cry1A.105 protein was purified at 4°C from parties.an extractdata of ground thereforegrain using a combination of ammonium sulfate fractionation, anion exchangeand/or chromatography,or and affiliates. immunoaffinity chromatography.property intellectualthird may its the as of and owner. The ground grain (10 kg)is was mixed in PBS extractionregulatory buffer [1× PBS: 1 mM KH2PO4, a document 10 mM Na2HPO4, 137 mMany NaCl,such and 2.7 mM KCl, pH 7.4]its for 2.5 hours at reproductiondocument approximately a 1:10 sample weightowner to buffer volumethis ratio. The ofslurry was filtered using an Ertel Alsop filter press (Kingston, NY)under and the ofPBS washed filter cakes were retained. rightsthe this The filter cakesdocument wereand/or resuspended in ~100 L of CAPS extractionrights buffer [50 mM CAPS, 1 to of fall use of mM EDTA, 10 mM DTT, 1 mM PMSF, 2 mM benzamidine-the HCl, and 1% PVPP (w/v), pH 10.8]This and stirred for 2 hours.may distribution, To andremo ve lipids from the extract, CelPure P65 rights owner Diatomaceous Earthsubject (Advanced Minerals Corp, Goleta, CA) was added to the slurry at 7.5% (w/v) and allowed to mix for ~10 minutesthe violate. The extract was clarified by filtration be copy using the Ertel Alsop filter press andof concentrated using a Hollow Fiber Cartridge (Amersham Biosciences,document Piscataway, NJ).and The contaminant DNA was removed by a may publication,exploitation combinationIt of benzonase treatment and precipitation with polyethyleneimine. this any Benzonase was added to a final concentration of ~7 U/ml in the presence of 5 mM MgCl2 permission and allowed to mix overnight. prohibitedA polyethyle neimine solution [10% (w/v)] was added to the concentrated extract to a final concentration of 0.05% (w/v) and the extract was the be clarified by centrifugationcommercial to remove the remaining contaminant DNA. An ammonium sulfateFurthermore, pelletany (0% – 35% saturation) was prepared by the addition of ammonium sulfate salt Consequently,to the clarifiedwithout extract and was allowed to dissolve overnight. After centrifugation, the ammonium sulfate pellet was dissolved over the weekend in ~15 L of resuspension buffer [50 mM CAPS, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, and 2 mM benzamidine-HCl, pH 10.8]. The resuspended sample was clarified by centrifugation, diafiltrated against fresh buffer (same as the resuspension buffer) to remove any residual ammonium sulfate and concentrated to final volume of ~8 L and CHAPS was added to a final concentration of 0.5 mM.

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The concentrated sample was loaded onto a 2.1 L (6.7 cm x 20 cm) Q Sepharose Fast Flow anion exchange resin column, which was equilibrated with AEX buffer A [50 mM CAPS, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, 2 mM benzamidine-HCl, and 0.5 mM CHAPS, pH 10.8]. The bound Cry1A.105 protein was eluted with a linear salt gradient that increased from 0 M to 0.20 M sodium chloride over ~12 L and then maintained a 0.20 M sodium chloride concentration for ~4 L. Next, the salt gradient increased to 0.65 M sodium chloride over ~21 L, and finally stepped up to 1.0 M sodium chloride instantly. Fractions containing the Cry1A.105 protein (based on stained SDS-PAGE gel and western blot analysis of all fractions) were pooled to a final volume of ~10 L. Salt was removed from these pooled fractions by diafiltration with fresh buffer [50 mM EPPS, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, and 2 mM benzamidine-HCl, pH 7.5].

regime.and The buffer exchanged sample was loaded onto a 2.1 L (13.7 cmand x 14.0 cm) Q Sepharose AG XL anion exchange resin column, which was equilibrated with AEX buffer A [50 mM EPPS, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, 2 mM benzamidine-HCl, and 0.5 mM CHAPS, pH 7.5]. The bound Cry1A.105Bayer protein wasproperty eluted wprotectionith a publishinglinear salt gradient that increased from 0 M to 0.25 M sodiumof chloride over ~ 4 L and then contentsheld at 0.25 M its sodium chloride for ~ 10 L. Next, the salt gradient increasedparties.data to 0.65 M sodiumtherefore chloride over ~21 L and finally increased to 1.0 M sodium chloride overand/or ~ 4or L and held constant affiliates. for ~8L. The fractions containingproperty Cry1A. intellectual105 proteinthird (based on stainedmay SDS-PAGE gel its and western blot analysis of all fractions)as were pooled to a final volume of ~10 L. These the of and fractions were diafiltratedis into fresh buffer [50regulatory mM EPPS, 1 mM EDTA,owner. 10 mM DTT, a document 1mM PMSF, and 2 mM benzamidine-HCl,any such pH 7.5] to remove saltits and concentrated to reproductiondocument ~2.0 L using a hollow fiber cartridge.owner this of under of rightsthe this Prior to the affinitydocument purificationand/or step, the sample was diafiltratedrights into fresh buffer [50 mM to of fall use of EPPS, 1mM EDTA, 1 mM PMSF, 2 mM benzamidine-HCl,the and 150 mM NaCl, pH 7.6] and concentratedThis down to ~1L.may Thedistribution, soluandtion containing Cry1A.105 protein was re- rights owner circulated over subjectthe 9.3 ml (1.75 cm x 2.6 cm) protein A agarose column (Sigma) conjugated with monoclonal anti-Cry1Ac theantibody violate (Strategic Biosolutions, Newark DE). be copy Bound Cry1A.105 protein was eluted usingof 50 mM CAPS, 1 mM EDTA, 1 mM PMSF, 2 and mM benzamidine-HCl,may document publication,0.8 M NaCl, and 30% (v/v) ethylene glycol, pH 10.0. After It exploitation analysis of fractionsthis by lateral flow strips, stained SDS-PAGE gel and western blot, fraction 25 was determinedany to contain the majority of the full-length Cry1A.105. permission prohibited 3.5. Protein Concentration the be commercial TheFurthermore, concentrationany of the full-length (~130 kDa) plant-produced Cry1A.105 protein was estimatedConsequently, usingwithout quantitative immunoblot analysis. The E. coli-produced Cry1A.105 protein (amounts ranging from 1 to 6 ng purity corrected for the full-length protein, which was 82%) was used to create a standard curve. Aliquots of the plant-produced Cry1A.105 protein and reference standard were diluted in deionized water and 5x Laemmli buffer (5x LB), heated at 95.5 °C for 5 min, and applied to a pre-cast tris- glycine 4→20% polyacrylamide gradient 12-well gel. Three different amounts of the plant-produced protein were loaded in duplicate. Electrophoresis was performed at a constant voltage of 125 V for 15 min followed by a constant voltage of 170 V for 75 min.

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Pre-stained molecular weight markers (Bio-Rad Precision Plus Dual Color, Hercules, CA) were used to verify electrotransfer of protein to the membrane and estimate the size of the immunoreactive bands observed. Electrotransfer to a 0.45 µm PVDF membrane (Invitrogen, Carlsbad, CA) was performed for 90 min at a constant voltage of 25 V.

The membrane was blocked for 1 hour with 5% (w/v) NFDM in 1× PBST. The membrane was probed with a 1:1000 dilution of rabbit anti-Cry1A.105 antibody in 1% (w/v) NFDM in PBST for 60 min. Excess antibody was removed using four 5 min washes with PBST. Finally, the membrane was probed with horseradish peroxidase- conjugated goat anti-rabbit IgG (Sigma, St. Louis, MO) at a dilution of 1:7500 in 1% (w/v) NFDM in PBST for 60 minutes. Excess HRP-conjugate was removed using three 5 min washes with PBST. All incubations were performed at roomregime. temperature.and Immunoreactive bands were visualized using the ECL detectionand system (Amersham AG Biosciences, Piscataway, NJ) and exposed (5 min, 10 min, 20 min) to Hyperfilm ECL high performance chemiluminescence film (Amersham Biosciences). Films were developed using a Konica SRX-101A automatedBayer film propertyprocessor protection(Tokyo,publishing Japan). of contents its The immunoreactive band of the plant-producedparties. Cry1A.105data protein thereforein each lane and/or migrating at the same level as the full-length reference standard proteinor was quantitated affiliates. relative to the standard curve. propertyQuantitation intellectual wasthird performed using the volumemay tool and the its linear regression method in the Quantityas One software (version 4.4.0) after scanning on the of and the Bio-Rad GS-800 densitometer.is The concenregulatorytration of the plant-producedowner. Cry1A.105 a document was determined by dividingany the amountsuch of protein in each lane by itsthe volume of protein reproductiondocument loaded in that lane [Microsoft Excelowner 2002 (10.6730.6718)this SP3].of All load quantities of under of plant-produced Cry1A.105rights referthe to the amount of the full-lengththis protein. documentand/or rights to of fall use of 3.6. Western Blot Analysis the This may distribution,and rights owner Immunoblot analysissubject was performed to confirm the identity of the Cry1A.105 protein, the intactness of its N-terminus, and comparethe immunoreactivity violate of the plant- and E. coli- be copy produced proteins. of and may documentpublication, It exploitation 3.6.1. Westernthis Blot Analysis Using anti-Cry1A.105 Antibody any permission The western blot described in sectionprohibited 5.2 was also used to establish the identity of the plant-produced protein and to compare the immunoreactivity of the plant-produced and the be E. coli-producedcommercial Cry1A.105 proteins. Equivalence was demonstrated for bands representingFurthermore,any full-length (~130 kDa) proteins that were identified by the anti-Cry1A.105 antibodyConsequently, and showedwithout similar electrophoretic mobility.

3.6.2. Western Blot Analysis Using the N-terminal Peptide Antibody

A western blot analysis using the N-terminal peptide antibody was performed to confirm the intactness of the N-terminus of the plant-produced Cry1A.105 protein. The N- terminal peptide antibodies were produced against a synthetic peptide consisting of the first 14 amino acids of the Cry1A.105 protein N-terminus. The trypsin resistant core

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was used as a negative control because its N-terminus was removed by trypsin. The plant-produced Cry1A.105 protein, the E. coli-produced Cry1A.105 reference standard (corrected for the purity of the full-length protein), and the Cry1A.105 trypsin-resistant core (~56 kDa) were each loaded on gels at 20 ng and 40 ng per lane. Each protein was mixed with 5× LB, heated at 96.0 °C for 5 min, and applied to a pre-cast tris-glycine 4→20% polyacrylamide gradient 10-well gel. Electrophoresis was performed at a constant voltage of 150 V for 5 min followed by a constant voltage of 200 V for 60 min. Pre-stained molecular weight markers (Bio-Rad Precision Plus Dual Color,) were used to verify electrotransfer of protein to the membrane and estimate the size of the immunoreactive bands. Electrotransfer to a 0.45 µm PVDF membrane (Invitrogen) was performed for 90 min at a constant voltage of 25 V. and and regime. The membrane was blocked overnight with 5%AG (w/v) NFDM in 1× PBST. The membrane was probed with a 1:33.33 dilution of rabbit N-terminal peptide antibody (lot 42005MG) in 1% (w/v) NFDM in PBST for 60 min. Excess antibody was removed using three 5 min washes with PBST. Finally, thBayere membrane property was probed withpublishing HRP-conjugated protectioncontents anti-rabbit IgG (Sigma) at a dilution ofof 1:7500 in 1% (w/v) NFDM in PBST for its 60 minutes. Excess HRP-conjugate was removed usingparties. threedata 5 min washestherefore with PBST. and/oror The blocking step was performed at 4 °C. All other incubations were performed at room affiliates. temperature. Immunoreactiveproperty bands were intellectual visualizedthird using the ECLmay detection system its as (Amersham Biosciences) andthe exposedof (1 min,and 2 min, 5 min, 10 min) to Hyperfilm ECL high performance chemiluminescenceis film (Amershamregulatory Biosciences).owner. Films were a document developed using a Konica SRX-101Aany such automated film processor. its reproductiondocument owner this of under of 3.7. MALDI-TOF Trypticrights Massthe Analysis this documentand/or rights to of fall use of MALDI-TOF mass spectrometry was used to confirmthe the identity of the plant-produced This may distribution,and Cry1A.105 protein. rights owner subject 3.7.1. Concentrationbe of Protein the violate copy of and The plant-producedmay document proteinpublication, (4.5 ml) was diluted to 9.0 ml with Cry1A.105 concentration It exploitation buffer [25 mM CAPS, 0.02% (w/v) sodium dodecyl sulfate] followed by concentration this any down to 64 µl using Amicon Ultrafree CL concentrators (Millipore Corporation, Bedford, permission MA). Sixty microliters of 5prohibited× LB was used to wash the concentrator walls and membranes. The membranesthe be were heated with 5× LB for 5 minutes at 95.1°C. Sixteen microliters of thiscommercial 5× LB was then used as the loading buffer for the Cry1A.105 sample forFurthermore, SDS-PAGEany separation. Consequently,without 3.7.2. SDS-PAGE Separation

The concentrated plant–produced Cry1A.105 protein was subjected to electrophoresis on an SDS-polyacrylamide gel. The protein sample was loaded in two lanes. Broad Range molecular weight markers (Bio-Rad) were used to estimate molecular weights. Plant- produced Cry1A.105 protein and molecular weight markers were heated at 95.3 °C for 5 min and then applied to a pre-cast tris-glycine 4→20% polyacrylamide gradient 10-well

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mini-gel. Electrophoresis was performed at constant voltage (125 V for 10 min followed by 170 V for 70 min). Proteins were stained with Bio-Rad Coomassie 1 × stain for 2 hours, and destained by washing with Coomassie R-250 1× destain solution (Bio-Rad) for 2 hours with one change of the destain solution.

3.7.3. In-gel Protein Digestion

The bands representing full-length plant-produced Cry1A.105 protein (~130 kDa) were excised from two gel lanes, destained, reduced, alkylated, and subjected to an in-gel trypsin digestion (Williams et al., 1997). Briefly, each gel band was individually destained for 30 min by incubation in 100 µL of 40% (v/v) methanol and 10% (v/v) glacial acetic acid in a microfuge tube. This was repeated two additional times. regime.and Following destaining, the gel bands were incubated in 100 µandL of 100 mM ammonium AG bicarbonate buffer for 30 min at room temperature. The protein was reduced in 100 µL of 10 mM dithiothreitol solution for 2 hours at 37 °C. The protein was alkylated by the addition of 100 µL of 200 mM iodoaceticBayer acid. Theproperty alkylation protection reactionpublishing was allowed to proceed at room temperature for 20 min ofin the dark. The gel bands were incubatedcontents in 100 its µL of 100 mM ammonium bicarbonate buffer for 30parties. min dataat room temperaturetherefore at which and/oror time 100 µL of acetonitrile was added and the incubation was continued for an additional affiliates. 30 minutes. The ammoniumproperty bicarbonate/aceton intellectualthirditrile incubations weremay repeated two its additional times to remove the reducingas and alkylating reagents and other salts from the the of and gel. The gel bands wereis dried in a SpeedVac concentratorregulatory , rehydrated owner.with 40 µL 25 mM a document ammonium bicarbonate containingany such 33 µg/ml trypsin, and the proteinits contained in the gel reproductiondocument of band was digested for 16 hours at 37.1owner °C. Digestedthis peptides were extracted for 60-80 under of min at room temperature withrights 50the µ L 70% (v/v) acetonitrilethis containing 0.1% (v/v) TFA documentand/or rights per gel band. Both extractionto of s upernatantsfall wereuse combined of into a single tube and dried in a SpeedVac concentrator. This process of extracting thethe peptides was repeated two more This may distribution,and times. The final dried materialsrights were reconstitutedowner in 5 µL of 0.1% (v/v) TFA. subject 3.7.4. Samplebe Preparation the violate copy of and may documentpublication, An aliquotIt (4 µl) of the digestedexploitation sample was desalted (Bagshaw et al., 2000) using Millipore (Bedford, MA) ZipTip C18 pipette tips. Prior to desalting, the tips were wetted this any with methanol and equilibrated with 0.1% (v/v) TFA. The sample was applied to a permission ZipTip and eluted with 4 µL ofprohibited Wash 1 [0.1% (v/v) TFA], followed by 4 µL of Wash 2 [20% (v/v) acetonitrile thecontaining 0.1% (v/v) TFA], followed by 4 µL of Wash 3 [50% commercial be (v/v) acetonitrile containing 0.1% (v/v) TFA], and finally with 4 µL of Wash 4 [90% (v/v)Furthermore, acetonitrileany containing 0.1% (v/v) TFA]. Consequently,without 3.7.5. MALDI-TOF Instrumentation and Mass Analysis

Mass spectral analyses were performed as follows. Mass calibration of the instrument was performed using an external peptide mixture from a Sequazyme Peptide Mass Standards kit (Applied Biosystems). Samples (0.5 µL) from each of the desalting steps, as well as a sample of solution taken prior to desalting, were co-crystallized with 0.8 µL α-cyano-4-hydroxy cinnamic acid on the analysis plate. The sample was analyzed in the

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500 to 5000 Dalton range using 100 shots at a laser intensity setting of 2781 (a unit-less MALDI-TOF instrument specific value). Protonated (MH+) peptide masses were observed monoisotopically in reflector mode (Aebersold, 1993; Billeci and Stults, 1993). GPMAW32 software (Applied Biosystems, version 4.23) was used to generate a theoretical trypsin digest of the expected Cry1A.105 (plant) protein sequence based upon the nucleotide sequence. Masses were calculated for each theoretical peptide and compared to the raw mass data. Experimental masses (MH+) were assigned to peaks in the 500 to 1000 Da range if there were two or more isotopically resolved peaks, and in the 1000 to 5000 Da range if there were three or more isotopically resolved peaks in the spectra. Peaks were not assessed if the peak heights were less than approximately twice the baseline noise, or when a mass could not be assigned due to overlap with a stronger signal ±2 Daltons from the mass analyzed. Known autocatalytic fragmentsregime. fromand trypsin digestion were identified in the raw data. The tryptic mass mapand coverage was considered AG acceptable if ≥ 40 % of the protein sequence was identified by matching experimental masses observed for the tryptic peptide fragments to the expected masses for the fragments. Bayer property publishing protectioncontents of its 3.8. Molecular Weight and Purity Estimation by SDS-PAGEparties.data therefore and/oror

affiliates. Aliquots of the E. coli-producedproperty reference intellectual standardthird and plant-producedmay Cry1A.105 its as proteins were mixed with the5× LBof to a finaland protein concentration of 10 ng/µl and 2.4 is regulatory owner. ng/µl, respectively. The plant-produced proteia n was analyzeddocument in duplicate at 48, 72, and 96 ng of total protein per lane.any Thesuch E. coli-produced Cry1A.105 referenceits standard was reproductiondocument of analyzed at 96 ng of purity correctedowner full-lengt h protein.this All samples were heated in a under of thermo-block at 99 °C forrights 3the min and applied to thisa pre-cast tris-glycine 4→20% documentand/or rights polyacrylamide gradientto 10-well of mini-gelfall (Invitrogen).use of Electrophoresis was performed at a constant voltage of 125 V for 10 min followed by athe constant voltage of 170 V for 70 This may distribution,and min. rights owner subject the The gel wasbe stained using the SilverXpress® violateSilver Staining Kit Protocol (Invitrogen, copy of Carlsbad, CA). The gel was fixed for 10 minutand es in 200 ml of fixing solution (90 ml ultra may documentpublication, pure water,It 100 ml methanol,exploitation and 20 ml acetic acid). This was followed by 10 minutes in 100 ml sensitizingthis any solution (105 ml ultra pure water, 100 ml methanol, and 5 ml sensitizer) and repeated once. The excess of the sensitizing solution was removed using permission two 5 minutes washes in 200 mlprohibited ultra pure water. The gel was stained for 15 minutes in 100 ml staining solutionthe (5 ml Stainer A, 5 ml Stainer B, and 90 ml ultra pure water). commercial be The stain was removed using two 5 minutes washes of 200 ml ultra pure water. DevelopingFurthermore, anyoccurred in 100 ml of solution (5 ml developer and 95 ml ultra pure water) for 3Consequently, - 15 minutes,without and was stopped by addition of 5 ml stopping solution for 10 minutes. The gel was washed three times for 10 minutes each with 200 ml of ultra pure water. Analysis of the gel was performed using a Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA). Broad-Range molecular weight markers (Bio-Rad) were used to estimate the apparent molecular weight of the plant-produced Cry1A.105 protein. For the purity evaluation, all visible bands within each lane were quantified. Stained bands corresponding to immunoreactive bands identified by anti-Cry1A.105 antibody and migrating from ~56 to ~130 kDa were

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 230 of 323

included in the purity calculation for the protein of interest as they represent various lengths of the insecticidal protein from the ~56 kDa tryptic core to the ~130 kDa full- length protein (Bietlot et al., 1989). The purity and estimated full-length molecular weight of the plant-produced Cry1A.105 protein were reported as the average of the six values obtained by densitometric analysis.

3.9. Glycosylation Analysis

Glycosylation analysis was used to determine whether the plant-produced Cry1A.105 protein was post-translationally modified with covalently bound carbohydrate moieties. Aliquots of the plant-produced Cry1A.105 protein, the E. coli-produced Cry1A.105 reference standard, and the positive controls, transferrin (Amersham Biosciences) and regime.and horseradish peroxidase (Sigma), were each mixed with 5× LB.and These samples were AG heated at 95 °C for 4 min, cooled, and loaded on a tris-glycine 4→20% polyacrylamide gradient 10-well mini-gel. Each sample was loaded at 48 and 96 ng (purity corrected for the full length protein) per lane. PrecisionBayer Plus Dual propertyColor pre-st protectionainedpublishing protein molecular weight markers (Bio-Rad) were loadedof to verify electrotransfer of the contentsproteins to the its membrane, and the CandyCane™ Glycoprotein Molecularparties.data Weight Standardstherefore (Molecular and/or Probes, Eugene, OR) were loaded as positive/negative controlsor and markers for affiliates. molecular weight. Electrophoresisproperty was perforintellectualmedthird at a constant voltagemay of 125 V for 10 its min followed by a constant voltage ofas 170 V for 70 min. Electrotransfer to a 0.2 µm the of and PVDF membrane was performedis for 90 min at aregulatory constant voltage of 25owner. V. a document any such its reproductiondocument Carbohydrate detection was performedowner direct ly on thethis PVDF membraneof using the Pro- under of Q® Emerald 488 Glycoproteinrightsthe Gel and Blot Stainthis Kit (Molecular Probes). The documentand/or rights manufacturer’s protocolto was offollowed. fall All stepsuse wereof performed at room temperature. The PVDF membrane was fixed in 25 ml of a solutionthe containing 50% methanol and 5% glacial aceticThis acid for 1 hour, mayand thendistribution, theand solution was changed and the membrane was rights owner incubated overnight.subject Two 15 minute washes (50 ml each) of 3% (v/v) glacial acetic acid (wash solution), were followed by a 20 theminut violatee oxidation in 25 ml of the kit supplied be copy oxidizing solution. After oxidation, threeof 15 minute washes (50 ml each) prepared the and membranemay for staining.documentpublication, The blot was incubated in 25 ml of Pro-Q Emerald Staining It exploitation Solution that wasthis prepared using the kit reagents. After 1 hour of staining in the dark, two 30 minute, 50 ml washany cycles were followed by two 45 minute, 50 ml wash cycles. The permission final wash cycles included two 25prohibited ml, 1 mi nute deionized water washes followed by three 25 ml, 5 minute methanol washes (B&J Brand, Muskegon, MI). The blot was then the be scanned using thecommercial BioRad Molecular Imager FX using the Alexa 488 illumination setting (QunatityFurthermore, Oneany software; version 4.6, build 036) in order to visualize the fluorescing glycosylatedConsequently, proteins.without

3.10. Functional Activity Assay

In order to assess the functional activity of the plant-produced Cry1A.105 protein and to compare its activity to the E. coli-produced Cry1A.105 reference standard, aliquots of the plant-produced Cry1A.105 protein and E. coli-produced Cry1A.105 reference standard protein were analyzed in diet-incorporation insect bioassay.

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The total protein concentration of the E. coli-produced Cry1A.105 protein aliquots was 1.2 mg/mL, with a purity of 92%, and a purity corrected concentration of 1.1 mg Cry1A.105/mL. The Cry1A.105 protein concentration of the plant-produced Cry1A.105 protein aliquots was 3 µg/mL. The control substances used in the bioassays were buffers of the same composition used for storing the E. coli-produced and plant-produced proteins. The E. coli-produced reference standard was suspended in 25 mM CAPS, pH ~10.3, 1 mM benzamidine-HCl, 0.1 mM EDTA, 0.2 mM DTT buffer solution. The composition of the plant storage buffer was 50 mM CAPS, 1.0 mM PMSF, 2.0 mM benzamidine-HCl, 1 mM EDTA, 0.8 M NaCl, 30% (v/v) ethylene glycol, pH 10.0. The plant-produced and E. coli-produced Cry1A.105 proteins were stored at 4° C and -80° C, respectively, and the buffers for both proteins were stored at 4° C. and and regime. AG 3.10.1. Insects

CEW were obtained from Benzon ResearchBayer In c. property Insect eggs werepublishing incubated at a protectioncontents temperatures ranging from 10o C to 27o C,of to achieve the desired hatch time. its parties.data therefore and/oror 3.10.2. Bioassays affiliates. property intellectualthird may its as CEW were used to measurethe activity of of the plant-and and E. coli-produced Cry1A.105 protein samples. The bioassay wasis replicated three timregulatoryes on separate days withowner. separate batches a document of insects. The plant- andany E. colisuch-produced proteins were run inits parallel during each reproductiondocument bioassay. Each bioassay replicateowner for the E. thiscoli -producedof and plant-produced under of Cry1A.105 proteins consistedrights ofthe a series of five proteinthis levels yielding a dose series documentand/or rights ranging from 0.00048 –to 0.039 of µg fallCry1A.105 use protein/mL of diet with a 3-fold separation factor between dose levels. This dose series was chosenthe to adequately characterize the This may distribution,and dose-effect relationshiprights for the proteins fromowner both sources. Each dose level, including the control, hadsubject an equivale nt volume of buffer added to the dosing solution. The Cry1A.105 proteinbe dosing solutions werethe prep violateared by diluting the protein with purified copy of water and incorporating the dilution into anand aga r-based insect diet. Diet mixture was then dispensedmay in 1 mL aliquotsdocumentpublication, into a 128 well tray (#BIO-BA-128, CD International, Pitman, It exploitation NJ). Insect larvae were placed on these diets using a fine paintbrush, with a target this any number of 16 insects per treatment. The infested wells were covered by a ventilated permission adhesive cover (#BIO-CV-16,prohibited CD International, Pitman, NJ) and the insects were allowed to feed for a periodthe of 6-7 days in an environmental chamber programmed at 27° commercial be C, ambient relative humidity and a lighting regime of 14h:10h, light:dark. The combined weightFurthermore, of theany surviving insects at each dose level for each source of protein was recorded at theConsequently, end of thewithout 6-7 day incubation period.

3.10.3. Dose-response Modeling and Results

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

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

2 ⎧ ⎫ ⎪ ⎪ ⎪ ⋅Ws 0 ⎪ ()eVar = ⎨ B ⎬ ⎪ ⎛ DietDose ⎞ ⎪ 1 + ⎜ ⎟ ⎩⎪ ⎝ EC50 ⎠ ⎭⎪ and and regime. AG where Wt is the average CEW larvae weight and DietDose is the Cry1A.105 protein diet dose level. The residual variation was assumeBayerd to beproperty proportional to publishingthe expected mean protectioncontents weight. The parameters that are includedof in the model are W0 which represents the its expected weight at DietDose = 0.0, EC50 which representsparties.data the concentrationtherefore needed to inhibit the growth of the target insect by 50%, B which reflectsand/or the rateor of the weight loss affiliates. as DietDose increases, s whichproperty represents intellectualthe proportionthird of the standardmay deviation to the its expected weight, and e which denotes theas residual (error). the of and is regulatory owner. a document any such its reproductiondocument 4. Materials and Methods Used toowner Estab lish Equivalencethis ofof the Cry2Ab2 Proteins under of Produced in E. coli andrights in MONthe 89034 this documentand/or rights to of fall use of 4.1. Cry2Ab2 Protein the This may distribution,and rights owner The plant-producedsubject Cry2Ab2 protein was isolated from ground grain of MON 89034. The identity of the grain sample containingthe MON violate 89034 was confirmed by event specific be copy PCR. The isolated plant-produced Cry2Ab2of protein was stored in a –80 °C freezer in a and buffer maysolution containingdocumentpublication, 50 mM CAPS, 2 mM DTT, pH 11 at a total protein It exploitation concentration ofthis 0.25 mg/mL. any permission 4.2. Reference Standard prohibited

the be The E. coli-producedcommercial Cry2Ab2 protein was used as a reference standard in select analyses.Furthermore, Theseany analyses included apparent molecular weight determination by SDS- PAGE,Consequently, westernwithout blot analysis, glycosylation analysis, BioRad protein assay, and the functional activity assay.

4.3. Assay Controls

Protein molecular weight standards were used to calibrate SDS polyacrylamide gels and verify protein transfer to PVDF membranes. The E. coli-produced Cry2Ab2 reference standard protein was used to estimate the total protein concentration in the BioRad

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protein assays and also used as the positive control in western blot analysis. Beta- lactoglobulin protein and PTH-amino acid standards were used to verify the performance of the amino acid sequencer. A peptide mixture and an analytical BSA standard were used to calibrate the MALDI-TOF mass spectrometer for tryptic mass analysis and molecular weight determination, respectively. Transferrin and E. coli-produced Cry2Ab2 protein were used as the positive and negative control, respectively, in glycosylation analysis.

4.4. Protein Purification

The Cry2Ab2 protein was purified from an extract of ground grain of MON 89034 using a combination of ammonium sulfate fractionation, anion exchange chromatography, and regime.and immunoaffinity chromatography. and AG

The isolation of Cry2Ab2 protein from ground corn grain was performed in two 10 kg batches. Prior to extraction of Cry2Ab2 protBayerein, eachproperty batch was extractedpublishing with 1X PBS protectioncontents buffer (1 mM KH2PO4, 10 mM Na2HPOof 4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) to its remove contaminant proteins. The PBS extraction procedureparties.data consis ted of soakingtherefore ground and/or grain in PBS for 2 hrs in a 4 °C cold room at approximately 1:10 sampleor weight to buffer affiliates. volume ratio. The slurry wasproperty clarified intellectualby filtrationthird using an Ertel mayAlsop filter press its (Kingston, NY), and the PBS washed assolid particle (cake) was retained. Subsequently, the of and the Cry2Ab2 protein in isthe cake was extracted regulatorywith extraction buffer owner.containing 50 mM a document CAPS, 1 mM EDTA, 2.5 anymM DTT,such 1 mM PMSF, 2 mM benzamidine-HCl,its 0.5 mM reproductiondocument CHAPS, 1% (w/v) PVPP, pH 10.8 atowner approxima tely 1:10this sample ofweight to buffer volume under of ratio for 2-3 hrs. Duringrights extraction,the lipids were removedthis from the extract by adding documentand/or rights CelPure P65 diatomaceousto ofearth fall (Advanced use Mineralsof Corp, Goleta, CA) to the homogenate at ~7.5% (w/v) and allowed to mix for ~10the minutes. The slurry was clarified by filtrationThis using the filter pressmay and distribution, theand resultant extract from both batches was pooled rights owner for a total volumesubject of ~230 L. The pooled extract was concentrated using a 30,000 NMWC Hollow Fiber Cartridge (Amershamthe violateBiosciences, Piscataway, NJ) to a final be copy volume of ~35 L. To remove plant genomicof DNA, polyethyleneimine, 10% (w/v), was and added tomay the concentrateddocumentpublication, extract to a final concentration of 0.05% (w/v), the extract was It exploitation clarified by centrifugatiothis n to remove precipitated DNA, and the supernatant was retained. The Cry2Ab2 proteinany in the supernatant was precipitated by 0% – 35% ammonium permission sulfate saturation. The 35% ammoniumprohibited sulfate pellet was recovered by centrifugation and the pellet was dissolved in 20 L of resuspension buffer containing 50 mM CAPS, 1 the be mM EDTA, 2.5 commercialmM DTT, 1 mM PMSF, 2 mM benzamidine-HCl, pH 10.8 by mixing in 4Furthermore, °C cold room.any The suspension was clarified by centrifugation and 21 L supernatant wasConsequently, retained, withoutconcentrated, and buffer exchanged by diafiltration against Buffer containing 50 mM CAPS, 1 mM EDTA, 2.5 mM DTT, 1 mM PMSF, 2 mM benzamidine-HCl, pH 10.8 to remove any residual ammonium sulfate salt. The concentrated sample of 13 L was loaded onto an anion exchange column in two batches, Run 1 (6L) and Run 2 (7L). The elution parameters were identical for both runs and thus only the Run1 column parameters are described below.

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A portion of concentrated sample was loaded onto a 4.5 L (20 cm x 14.4 cm column) Q Sepharose Fast Flow anion exchange resin column, which was equilibrated with Buffer. The bound Cry2Ab2 protein was eluted with step gradients as follows: 0-20% buffer B (Buffer containing 1M NaCl) in 10 column volumes (CV), and then the gradient was held at 20% buffer B for 4 CV, and then the gradient was increased to 65% buffer B over 10 CV and finally the gradient was stepped up to 100% buffer B and held at 100% buffer B in 2 CV. Fractions, each ~ 4 L, containing Cry2Ab2 protein were identified by Cry2A QuickStix™ (Portland, ME) for further analysis. Based on western blot analysis and SDS-PAGE analysis, fraction 7 from Run 2 was selected for affinity purification.

Subsequently, fraction 7 was concentrated to 400 mL using a 30,000 NMWC Hollow Fiber Cartridge. Approximately one half of the aforementioned sampleregime. wasand buffer exchanged into EPPS buffer (50 mM EPPS, 2 mM DTT, andpH 7.2) using centrifuge AG concentrators (30 kDa MWCO), resulting in a final volume of 200 mL. This sample, in two separate batches, was applied to an affinity column (1.0 cm × 2.7 cm) containing Protein G agarose conjugated with Cry2Aa-specificBayer property mAb and protection equilibratedpublishing with EPPS buffer (50 mM EPPS, 2 mM DTT, pHof 7.2). The sample was re-circulatedcontents through the its column for 2 hrs at 100 mL/hr. The column was thenparties. washeddata with 17-20therefore CV of EPPS and/or buffer (50 mM EPPS, 2 mM DTT, pH 7.2), followed by 5-7 CV of EPPSor buffer (50 mM affiliates. EPPS, 2 mM DTT, pH 8.5). property The bound intellectual Cry2Ab2third protein was elutedmay with 100 mM its Triethylamine, pH 11.4, in five 1.5 asmL fractions. Based on SDS-PAGE analysis, the of and fractions containing Cry2Ab2is protein from batchregulatory one and two wereowner. concentrated and a document buffer exchanged into a storageany suchbuffer containing 50 mM CAPS, 2its mM DTT, pH 11.0, reproductiondocument using centrifuge concentrators (30owner kDa MWCO). this Concentrated of samples were then under of pooled, resulting in a final rightsvolumethe of 2 mL. this documentand/or rights to of fall use of The affinity chromatography procedure was repeated beginningthe with 100 mL of fraction 7 from ThisRun2. As previouslymay described, distribution,and the sample was concentrated and buffer rights owner exchanged into EPPSsubject buffer (50 mM EPPS, 2 mM DTT, pH 7.05) to a final volume of 50 mL using centrifuge concentrators (30 kDathe MWCO). violate This sample was re-circulated for 2 be copy hr (60 mL/hr) through an affinity columnof (1.0 cm × 2.75 cm) prepared with Cry2Aa- and specificmay mAb equilibrateddocumentpublication, with EPPS buffer (50 mM EPPS, 2 mM DTT, pH 7.05). The It exploitation column was thenthis washed with 15 CV of EPPS buffer (50 mM EPPS, 2 mM DTT, pH 7.05) followed by 5any CV of EPPS buffer (50 mM EPPS, 2 mM DTT, pH 8.5). The bound permission Cry2Ab2 protein was eluted withprohibited 100 mM Triethylamine, pH 11.4, in five 1.5 mL fractions. the be commercial BasedFurthermore, on anySDS-PAGE analysis, Cry2Ab2 enriched fractions were individually concentratedConsequently, andwithout buffer exchanged into a storage buffer containing 50 mM CAPS, 2 mM DTT, pH 11, using a centrifuge concentrator (30 kDa MWCO). The concentrated samples were pooled into a final volume of 2.4 mL. The aforementioned sample and the sample from the first run (2 mL) were pooled resulting in a final volume of 4.4 mL.

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4.5. Total Protein Concentration by BioRad Assay

The total protein concentration of the purified plant-produced Cry2Ab2 protein was estimated using a BioRad protein assay. The E. coli-produced Cry2Ab2 reference standard protein (concentrations ranging from 0.05 to 0.5 mg/mL) was used to prepare a standard curve. The plant-produced Cry2Ab2 total protein concentration was estimated by comparison of absorbance values obtained for the sample to the values of the standard curve. Data were collected using a Bio-Tek Instruments, Inc. Powerwave Xi microplate scanning spectrophotometer (Winooski, VT) employing KC4 software version 3.3 revision 10. Readings were taken at a wavelength of 595 nm.

4.6. Western Blot Analysis regime.and and AG Equivalence would be demonstrated if the full-length plant-produced Cry2Ab2 protein was identified by the anti-Cry2Ab2 antibody and exhibited similar electrophoretic mobility compared to the E. coli-producedBayer Cry2Ab2 propertyreference stprotectionandard.publishing Aliquots of the stock solutions of the plant-produced Cry2Ab2of and reference standard werecontents diluted to a its final purity-corrected protein concentration of 2 ng/µparties.L in dilutiondata buffer (50therefore mM CAPS, and/oror 2 mM DTT, pH 11) and 5× sample loading buffer [5x concentrated Laemmli buffer affiliates. (312 mM Tris-HCl, 20% (v/v)property 2-mercaptoet intellectualhanol,third 10% (w/v) sodiummay dodecyl sulfate, its 0.025% (w/v) bromophenol blue, 50%as (v/v) glycerol, pH 6.8)]. Samples were then the of and heated to ~100 °C foris 5 min and appliedregulatory to a pre-cast tris-glycineowner. 4→20% a document polyacrylamide gradient 10-wellany suchgel. The plant-produced Cry2Ab2its protein was loaded in reproductiondocument duplicate at three different loadingsowner of 20, 30, and 40this ng per lane.of The E. coli-produced under of Cry2Ab2 reference standardrights wasthe loaded at 20 ng thisper lane. Electrophoresis was documentand/or rights performed at a constantto volta ofge offall 140 V foruse 20 minof followed by a constant voltage of 200 V for 43 min. Pre-stained molecular weight markersthe included during electrophoresis This may distribution,and (BioRad Precision Plusrights Dual Color, Hercules,owner CA) were used to verify electrotransfer of protein to the membranesubject and to estimate the molecular weight of the immunoreactive bands. Samplesbe were electrotransferred tothe a 0.45violate micron PVDF membrane (Invitrogen, copy of Carlsbad, CA) for 60 min at a constant currentand of 300 mA. may documentpublication, It exploitation The membrane was blocked for one hour with 5% (w/v) NFDM in PBST. The this any membrane was probed with a 1:3000 dilution of goat anti-Cry2Ab2 antibody (lot permission 7227632) in 2% (w/v) NFDM prohibitedin PBST for one hour. Excess antibody was removed using three 10 min washesthe bewith PBST. The membrane was probed with peroxidase- conjugated rabbitcommercial anti-goat IgG (Sigma, St. Louis, MO) at a dilution of 1:10,000 in 2% (w/v)Furthermore, NFDMany in PBST for one hour. Excess peroxidase-conjugate was removed using threeConsequently, 10 min washeswithout with PBST. All procedures, including blocking, and all other incubations were performed at room temperature. Immunoreactive bands were visualized using the ECL detection system (Amersham Biosciences, Piscataway, NJ) and exposed (30 sec, 1 min, 2 min, 3 min, and 7 min) to Hyperfilm ECL high performance chemiluminescence film (Amersham Biosciences, Piscataway, NJ). Films were developed using a Konica SRX-101A automated film processor.

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4.7. MALDI-TOF Tryptic Mass Analysis

MALDI-TOF mass spectrometry was used to confirm the identity of the plant-produced Cry2Ab2 protein. A protein can be typically identified when 40% of the mass fragments are identified from the analyzed protein (Jiménez et al., 1998).

4.7.1. SDS-PAGE Separation of Proteins

An aliquot of the plant-produced Cry2Ab2 protein was diluted with 5× sample loading buffer to a final purity corrected protein concentration of 80 ng/µL and 2 µg was electrophoresed in each of five lanes. Broad Range molecular weight markers (BioRad, Hercules, CA) were used to estimate molecular weights. Samples were heated toand ~99 °C and regime. for 5 min prior to electrophoresis on a pre-cast tris-glycineAG 4→20% SDS polyacrylamide gel at 140 V for 20 min followed by a constant voltage of 200 V for 46 min. Proteins were fixed by placing the gel in a solution of 40% (v/v) methanol and 7% (v/v) glacial acetic acid for 30 min, stained 2 hr withBayer Brilliant propertyBlue G-Colloidal protectionpublishing stain (Sigma, St. of contents Louis, MO), destained 30 sec with a solution containing 10% (v/v) acetic acid and 25% its parties.data therefore (v/v) methanol, and followed by 25% (v/v) methanol for 1 hr. Twand/oro proteinor bands, band-1 and band-2, migrating at 61 kDa and 50 kDa, respectively, were identified for tryptic affiliates. may mass map analysis. property intellectualthird its as the of and is regulatory owner. 4.7.2. In-gel Protein Digestion a document any such its reproductiondocument of The plant-produced full length Cry2Ab2owner protein, band-1this migrating at ~61 kDa, and band- under of 2, a proteolytic fragment ofrights the the full length Cry2Ab2 proteinthis migrating at ~50 kDa, were documentand/or rights excised, destained, reduced,to ofalkylated, fall and usesubjected of to an in-gel trypsin (Promega, Madison, WI) digestion (Williams et al., 1997). theEach gel band was individually This may distribution,and destained for 30 min byrights incubation in 100 µownerL of 40% (v/v) methanol and 10% (v/v) glacial acetic acidsubject in its own microfuge tube. Following destaining, the gel bands were the incubated inbe 100 µL of 100 mM ammoniumviolate bicarbonat e buffer for 30 min at room copy of temperature. Proteins were reduced in 100and µL of 10 mM dithiothreitol solution for 2 hrs may documentpublication, at 37 It° C. Proteins were thenexploitation alkylated by the addition of 100 µL of buffer containing 200 mM iodoaceticthis any acid. The alkylation reaction was allowed to proceed at room temperature for 20 min in the dark. The gel bands were incubated in 100 µL of 100 mM permission ammonium bicarbonate for 30prohibited min at room temperature at which time 100 µL of acetonitrile was added theand the incubation was continued for an additional 30 minutes. commercial be The ammonium bicarbonate/acetonitrile incubations were repeated two additional times toFurthermore, remove theany reducing and alkylating reagents, and salts from the gel. The gel bands wereConsequently, dried in awithout SpeedVac concentrator (Savant, Holbrook, NY), rehydrated with 40 µL 25 mM ammonium bicarbonate containing 33 µg/mL trypsin, and the protein contained in the gel band was digested for 16 hours at 37 °C. Digested peptides were extracted for one hour at room temperature with 50 µL 70% (v/v) acetonitrile containing 0.1% (v/v) TFA per gel band. Supernatants for each extraction were combined into a single tube and dried in a SpeedVac concentrator. This process of extracting the peptides was repeated two more times. The final dried materials were reconstituted in 10 µL of 0.1% (v/v) TFA.

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4.7.3. Sample Preparation

A portion (5 µl) of the digested samples was desalted (Bagshaw et al., 2000) using Millipore (Bedford, MA) ZipTip®C18 pipette tips. Prior to desalting, the tips were wetted with methanol and equilibrated with 0.1% (v/v) TFA. Each sample was applied to a ZipTip®C18 and eluted with 5 µL of Wash 1 [0.1% (v/v) TFA], followed by 5 µL of Wash 2 [20% (v/v) acetonitrile containing 0.1% (v/v) TFA], followed by 5 µL of Wash 3 [50% (v/v) acetonitrile containing 0.1% (v/v) TFA], and finally with 5 µL of Wash 4 [90% (v/v) acetonitrile containing 0.1% (v/v) TFA].

4.7.4. MALDI-TOF Instrumentation and Mass Analysis and and regime. AG Mass spectral analyses were performed as follows: mass calibration of the instrument was performed using an external peptide mixture from a Sequazyme™ Peptide Mass Standards kit (Applied Biosystems). SamplesBayer (0.3 µL)property from eachprotection of publishingthe desalting steps, of contents as well as a sample of the solution taken prior to desalting, were co-crystallized with 0.75 its parties.data therefore µL α-cyano-4-hydroxy cinnamic acid (Waters, Milford, MA) and/oron theor analysis plate. All samples were analyzed in the 500 to 5000affiliates. dalto n range using 100 shots at a laser intensity property intellectualthird may setting of 2318-2460 (a unit-less itsMALDI-TOF instrument specific value). Protonated as (MH+) peptide masses werethe observedof monoisotand opically in reflector mode (Aebersold, is regulatory owner. 1993; Billeci and Stults, 1993). GPMAW32 softa ware (Applieddocument Biosystems, version 4.23) was used to generate a theoanyretical such trypsin digest of the expectedits Cry2Ab2 protein reproductiondocument of sequence deduced from the nucleotideowner sequence. this Masses were calculated for each under of theoretical peptide and comprightsaredthe to the raw mass data.this Experimental masses (MH+) documentand/or rights were assigned to peaks into the of500 tofall 1000 Da rausenge ifof there were two or more isotopically resolved peaks, and in the 1000 to 5000 Da range if therethe were three or more isotopically This may distribution,and resolved peaks in the spectra.rights Peaks were notowner assessed if the peak heights were less than approximately twicesubject the baseline noise, or when a mass could not be assigned due to the overlap withbe a stronger signal of ±2 daltonsviolate from the mass analyzed. Known copy of autocatalytic fragments from trypsin digestionand were identified in the raw data. The may documentpublication, identityIt of the Cry2Ab2 proteinexploitation is confirmed if ≥ 40 % of the protein sequence was identified by matchingthis any experimental masses for the tryptic peptide fragments to the expected masses for the fragments. permission prohibited 4.8. Analysis of the N-terminalthe Sequence commercial be

AnFurthermore, aliquot ofany the plant-produced Cry2Ab2 protein was diluted with 5× sample loading bufferConsequently, to a finalwithout purity corrected protein concentration of 80 ng/µL. Molecular weight markers (BioRad Precision Plus Dual Color, Hercules, CA) were used to confirm the transfer of protein to the PVDF membrane. The plant-produced Cry2Ab2 protein was electrophoresed in eight lanes at 2 µg per lane. The Cry2Ab2 containing samples were heated to ~99 °C for 5 min prior to electrophoresis on a pre-cast tris-glycine 4→20% SDS polyacrylamide gel at 140V for 20 min followed by 200 V for 43 min. The gel was then electroblotted to a 0.2 micron PVDF membrane for 60 min at a constant current of 300 mA in a solution containing 10 mM CAPS, 10% (v/v) methanol, pH 11. Protein

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bands were stained by briefly soaking the membrane with Coomassie Blue R-250 stain (BioRad) and visualized by destaining with a Coomassie R-250 destaining solution (BioRad).

The protein bands with molecular weights of approximately 61.3, 49.6 and 27 kDa, (also referred to as band-1, band-2, and band-3, respectively) were excised from the membrane. N-terminal sequence analysis was performed using automated Edman degradation chemistry (Hunkapillar et al., 1983). An Applied Biosystems 494 Procise Sequencing System with 140C Microgradient system and 785 Programmable Absorbance Detector and Procise™ Control Software (version 2.1) was used. Chromatographic data were collected using Atlas99 software (version 2003R1.1, LabSystems, Altrincham, Cheshire, England). A PTH-amino acid standard mixture (Applied Biosystems,regime.and Foster City, CA) was used to calibrate the instrument for each analysis.and This mixture served to AG verify system suitability criteria such as percent peak resolution and relative amino acid chromatographic retention times. A control protein (10 picomole β-lactoglobulin, Applied Biosystems) was analyzed beforeBayer and afterproperty the analysis protection ofpublishing the three protein bands to verify that the sequencer metof performance criteria for repetitivecontents yield and its sequence identity. parties.data therefore and/or or affiliates. 4.9. Molecular Weight Estimationproperty by SDS-PAGEintellectualthird may its as the of and Aliquots of the test substanceis and reference regulatorystandard protein were owner.each diluted with a document sample dilution buffer and any5× samplesuch loading buffer to a final proteinits concentration of reproductiondocument 0.2 µg/µL. Molecular weight markersowner (BioRad Broad-Range,this Hercules,of CA) that were under of used to estimate the apparentrights molecularthe weight of thethis test substance, were diluted to a documentand/or rights final total protein concentrationto of of 0.9fall µg/µL.use The plant-producedof Cry2Ab2 protein was analyzed in duplicate at 1, 2, and 3 µg total protein loadsthe per lane. The E. coli-produced This may distribution,and Cry2Ab2 reference standardrights was analyzed at 1owner µg total protein. All samples were heated at ~102 °C forsubject 5 min and applied to a pre-cast tris-glycine 4→20% polyacrylamide be the violate gradient 10-well mini-gelcopy (Invitrogen, Carlsbof ad, CA). Electrophoresis was performed at a constant voltage of 150 V for 82 min. and Pr oteins were fixed by placing the gel in a may documentpublication, solutionIt of 40% (v/v) methanolexploitation and 7% (v/v) glacial acetic acid for 30 min, stained 16 hr with Brilliant Bluethis anyG-Colloidal stain (Sigma, St. Louis, MO), destained 30 sec with a solution containing 10% (v/v) acetic acid and 25% (v/v) methanol, and finally destained permission with 25% (v/v) methanol for 6 hr.prohibited the commercial be Analysis of the gel was performed using a BioRad GS-800 densitometer with the suppliedFurthermore, Quantityany One software (version 4.4.0, Hercules, CA). Values for the markers suppliedConsequently, by thewithout manufacturer were used to estimate the apparent molecular weight of each observed band. All visible bands within each lane were quantified using Quantity One software. For the plant-produced Cry2Ab2 protein, purity was estimated as the percent optical density of the ~61 kDa band relative to all bands detected in the lane. Apparent molecular weight and purity were reported as an average of all six loadings containing the plant-produced Cry2Ab2 protein.

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4.10. Molecular Weight Determination using MALDI-TOF MS

Determination of the intact mass was attempted for the plant-produced Cry2Ab2 protein using MALDI-TOF mass spectrometry analysis. This analysis was not specified in the characterization plan and is therefore a plan deviation. However, there is no impact on the protein characterization.

Prior to analysis, the plant-produced Cry2Ab2 protein and BSA reference protein (NIST, Gaithersberg, MD) were desalted using drop dialysis (Görisch, 1988). A portion of each protein sample (0.3 µL) was spotted on an analysis plate, mixed with 0.75 µL sinapinic acid solution and air-dried. Each sample was analyzed in triplicate. Mass spectral analysis was performed using an Applied Biosystems Voyager DE-Pro Biospectrometryregime.and Workstation MALDI-TOF instrument with the supplied Data Eandxplorer software (version AG 4.0.0.0, Foster City, CA). Mass calibration of the instrument was performed using desalted BSA reference protein. Bayer property protectionpublishing 4.11. Functional Activity Assay of contents its parties.data therefore and/or The purpose of this analysis was to compare the biological activityor between plant- affiliates. produced Cry2Ab2 protein andproperty the E. coliintellectual-producedthird Cry2Ab2 proteinmay by determining its EC50 values in a CEW diet-incorporationas insect bioassay. The EC50 value is defined as the of and the concentration of Cry2Ab2is protein in the dietregulatory that results in 50% growthowner. inhibition. In a document order to assess the functionalany activitysuch of the plant-produced Cry2Ab2its protein and to reproductiondocument compare its activity to the E. coli-producedowner Cry2Ab2this reference ofstandard, aliquots of the under of plant-produced Cry2Ab2 rightsproteinthe and E. coli-producedthis Cry2Ab2 reference standard documentand/or rights protein were used to estimateto of the effectivefall proteinuse concentrationof necessary to inhibit the growth of the target insect by 50%. the This may distribution,and rights owner The total proteinsubject concentration of the E. coli-produced Cry2Ab2 protein aliquots was 0.50 mg/mL, with a purity of 87%, andthe a purviolateity corrected concentration of 0.4 mg be copy Cry2Ab2/mL. The total protein concentraof tion of the plant-produced Cry2Ab2 protein and aliquotsmay was 0.25 mg/mLdocumentpublication, with a purity of 33%, and a purity corrected concentration of It exploitation 0.1 mg Cry2Ab2/mL.this Both proteins were suspended in 50 mM CAPS, 2 mM DTT, pH 11.0, buffer. The anycontrol substance used in the bioassays was buffer of the same permission composition used to store the prohibitedE. coli-produced and plant-produced proteins, 50 mM CAPS, 2 mM DTT, pH 11, buffer. The plant-produced and E. coli-produced Cry2Ab2 the be proteins were storedcommercial in a -80 ° C freezer and the buffer was stored in a 4° C refrigerator. Furthermore,any 4.11.1.Consequently, Insectswithout

CEW (Helicoverpa zea) eggs were obtained from Benzon Research Inc. Insect eggs were incubated at temperatures ranging from 10o C to 27o C, to achieve the desired hatch time.

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4.11.2. Bioassays

CEW were used to measure activity of the plant- and E. coli-produced Cry2Ab2 protein samples in accordance with the Monsanto SOP BR-ME-0044-03. The bioassay was replicated three times on separate days with separate batches of insects. The plant- and E. coli-produced proteins were run in parallel during each bioassay. Each bioassay replicate for the E. coli-produced and plant-produced Cry2Ab2 proteins consisted of a series of seven dilutions and a buffer control yielding a dose series with a 2-fold separation factor ranging from 0.016 – 1.0 µg Cry2Ab2 protein/mL diet. The dose- response curves for each protein included a buffer control. Each buffer control contained an amount of their respective buffer equivalent to the amount of protein in the highest dose level. The Cry2Ab2 protein dosing solutions were prepared by dilutingregime. theand protein with purified water and incorporating the dilution into an agar-basedand insect diet. This AG dose series in diet was chosen to adequately characterize the dose-effect relationship on CEW weight gain for the proteins from both sources. The diet mixture was then dispensed in 1 mL aliquots into a 128 well Bayertray (#BIO-BA-128, property CDprotection International,publishing Pitman, NJ). Insect larvae were placed on theseof diets using a fine paintbrush,contents with a target its number of 16 insects per treatment. The infested parties.wells datawere covered bytherefore a ventilated and/or adhesive cover (#BIO-CV-16, CD International, Pitman, NJ) andor the insects were affiliates. allowed to feed for a period ofproperty seven days intellectual in anthird environmental chambermay programmed at its 27° C, ambient relative humidity andas a lighting regime of 14 h light:10 h dark. The the of and combined weight of theis surviving insects at eachregulatory dose level for eachowner. source of protein a document was recorded at the end of thanye 7-day such incubation period. its reproductiondocument owner this of under of 4.11.3. Dose Response Modelingrightsthe this documentand/or rights to of fall use of Data were entered into an Excel spreadsheet and transferredthe to the Statistics Technology Center forThis analysis. Dose responsemay distribution,modelingand and EC determinations were performed rights owner 50 using a 3-parametersubject logistic regression model (equation 1) under the PROC NLIN the violate procedure in beSAS. copy of and Equationmay 1: documentpublication,W It exploitation0 Wt = B + e this any ⎛ DietDose ⎞ 1 + ⎜ ⎟ ⎝ permissionEC50 ⎠ prohibited the commercial be Furthermore,any whereConsequently, Wt is thewithout average CEW larvae weight and DietDose is the Cry1Ab protein diet dose level. Three parameters that are included in the model; W0 represents the weight at DietDose = 0.0, EC50 represents effective concentration to reduce the growth of the target insect by 50%, and B reflects the rate of the weight loss as DietDose increases, and e denotes the residual (error).

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4.12. Glycosylation Analysis

Glycosylation analysis was used to determine whether the plant-produced Cry2Ab2 protein was post-translationally modified with covalently bound carbohydrate moieties. Aliquots of the plant-produced Cry2Ab2 protein, the E. coli-produced Cry2Ab2 reference standard (in this instance, a negative control), and the positive control transferrin (Amersham Biosciences, Piscataway, NJ) were each diluted in dilution buffer and in 5× sample loading buffer to a final purity corrected (total protein for transferrin) concentration of 50 ng/µL. These samples were heated to ~100 °C for 5 min, and loaded along with Precision Plus Dual Color pre-stained protein molecular weight markers (BioRad , Hercules, CA) on a pre-cast tris-glycine 4→20% polyacrylamide gradient 10- well mini-gel. All three samples were loaded at a single loading of 0.5 and 1 µgand protein and regime. per lane. Electrophoresis was performed at a AGconstant voltage of 140 V for 20 min followed by a constant voltage of 200 V for 47 min. After electrophoresis, proteins were electrotransfered to a 0.45 micron PVDF membrane for one hour at a constant current of 300 mA. Bayer property protectionpublishing of contents

its parties.data therefore Carbohydrate detection was performed directly on the PVDFand/or membraneor containing the 0.5 and 1.0 µg sample loads using theaffiliates. ECL detec tion system (Amersham Biosciences, property intellectualthird may Piscataway, NJ). After the electrotransfeits r of the proteins, the PVDF membrane was incubated in PBS for 10 min,the and transferredas to a solution of 100 mM acetate buffer, pH of and owner. 5.5, containing the oxidationis reagent, 10 mM sodiumregulatory metaperiodate. The membrane was a documentits incubated in the dark for any20 minutes. such The oxidation solution was removed from the reproductiondocument of membrane by two brief rinses followedowner by three sequentialthis 10 min washes in PBS. The under of membrane was transferredrights to a solutionthe of 100 mM acetatethis buffer, pH 5.5, containing 25 documentand/or rights nM biotin hydrazide andto incubatedof fall for 60 useminutes. of Biotin hydrazide solution was removed by washing in PBS as previously described. the The membrane was blocked with This may distribution,and 5% blocking agent in rightsPBS for 60 minutes.owner The blocking solution was removed by washing in PBSsubject as previously described. The membrane was incubated with the streptavidin-HRPbe conjugate (diluted 1:6000) violatein acetate buffer for 30 min to detect copy of carbohydrate moieties bound to biotin. and Exce ss streptavidin-HRP was removed by may documentpublication, washingIt in PBS as previouslyexploitation described. Bands were visualized using the ECL detection system (Amershamthis any Biosciences, Piscataway, NJ). Films were exposed (1 min, 3 min, and 6 min) to Hyperfilm ECL high performance chemiluminescence film (Amersham permission Biosciences, Piscataway, NJ). prohibited Films were developed using a Konica SRX-101A automated film processor.the commercial be

Furthermore,any ReferencesConsequently, without

Aebersold, R. (1993). Mass spectrometry of proteins and peptides in biotechnology. Curr. Opin. Biotechnol. 4:412-419.

Billeci, T. M. and Stults, J. T. (1993). Tryptic mapping of recombinant proteins by matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 65:1709- 1716.

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Bietlot, H., P.R. Carey, C. Choma, H. Kaplan, T. Lessard, and M Pozsgay. 1989. Facile preparation and characterization of the toxin from Bacillus thuringiensis var. kurstaki. Biochem. J. 260: 87-91.

Jiménez, C. R., L. Huang, Y. Qui, and A.L. Burlingame. 1998. In-gel digestion of proteins for MALDI-MS fingerprint mapping. Current Protocols in Protein Science, regime.and Supplement 14, pp. 16.4.1-16.4.5. and AG

Thomas, K, G.A. Bannon, S. Hefle, C. Herouet, M. Holsapple, G. Ladics, S. MacIntosh, and L. Privalle. 2005. In silico methodsBayer for evaluatingproperty human protection allergenicitypublishing to novel proteins: international bioinformaticsof work shop meeting report, 23-24 contentsFebruary 2005. its Toxicol. Sci. 88: 307-310. parties.data therefore and/oror affiliates. Williams, K., M. LoPresti, andproperty K Stone. intellectual 1997.third Internal protein sequencingmay of SDS- its PAGE-separated proteins: Optimizationas of an in-gel digest protocol. Tech in Protein the of and Chem 8:79-98. is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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APPENDIX D: Summary of The Tryptic Masses of the Cry1A.105 And Cry2Ab2 Proteins Identified Using MALDI-TOF MS

and and regime. AG

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Table D.1. Summary of the Tryptic Masses Identified for the Full-length Plant-produced Cry1A.105 Protein Using MALDI- TOF Mass Spectrometry Only experimental masses that matched expected masses are listed in the table.

regime.and Observed Mass (Da) and 4 Expected 1 AG AA No Desalting ∆ 2 Fragment Sequence(s) Mass (Da) Position Desalting3 Wash 1 Wash 2 Wash 3 Wash 4 515.15 515.22 515.25 515.27 515.27 515.34 0.19Bayer property 525-528 protectionARIRpublishing of contents 529.16 529.27 0.11 695-698 QPER its parties.data therefore 579.13 579.21 579.25 579.28 579.33 0.20 749-752 and/orYQLRor 5 589.31 affiliates. 0.19 225-228 DWIR 589.12 589.18 589.23 589.26 589.25 property5 intellectualthird may 589.28its 0.16 1023-1027 VCPGR as 606.01 the 605.31of 0.70and 666-670 ELSEK is regulatory owner. 611.25 611.29 611.32 611.29 611.36 0.11a 936-940document VHSIR any such its reproductiondocument 621.17 621.29 621.31 621.32 621.37owner 0.20 this 1028-1032 of GYILR 649.16 649.94 649.29 649.32 649.99 649.37 under 0.21 of 254-258 TYPIR rightsthe this 688.26 688.30 document and/or 688.37 0.11 94-99rights NQAISR to of fall use of 727.14 727.25 727.28 727.31 727.35 0.21 229-233the YNQFR 731.15 731.24 731.29 731.33This 731.36may distribution, and 0.21 424-429 QGFSHR rights owner 764.18 764.27 764.33 764.35 subject764.35 764.39 0.21 88-93 IEEFAR 781.28 781.32 be 781.34 781.38 the 0.10 violate 193-198 YNDLTR copy of and may documentpublication, It exploitation this any 1 A difference of less than one dalton between the observed (first column where this mass is documented) and expected masses was necessary for consideration permission as a match. prohibited 2 AA position refers to amino acid position within the predictedthe Cry1A.105 sequence. commercial be 3 Sample, 0.5 µL, was analyzed prior to Zip Tip desalting 4 The ZipTip was washed with 0.1%Furthermore, (v/v) trifluoroaceticany acid containing acetonitrile at varying concentrations of acetonitrile: Wash 1 – 0% (v/v), Wash 2 – 20% (v/v), Wash 3 – 50% (v/v), and WashConsequently, 4 - 90% (v/v)without acetonitrile.

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Table D.1 (cont.). Summary of the Tryptic Masses Identified for the Full-length Plant-produced Cry1A.105 Protein Using MALDI-TOF Mass Spectrometry

and and regime. AG Observed Mass (Da) AA2 Fragment Desalting3 Bayer propertyPosition Sequence(s)publishing No Expected protectioncontents 4 Wash 1 Wash 2 Wash 3 Wash 4 of 1 Desalting Mass (Da) ∆ its parties.data therefore 784.26 784.37 0.11 853-859 and/or TQDGHARor 804.22 804.33 804.39 804.43 804.41 804.46 affiliates. 0.24 259-265 TVSQLTR property intellectualthird may 816.17 816.29 816.33 816.36 816.40its 0.23 218-224 VWGPDSR as 854.94 855.00 the 854.41of 0.53and 1114-1120 SYTDGRR is regulatory owner. a document 907.21 907.40 907.43 907.43 any 907.46 such 0.25 174-181 DVSVFGQRits reproductiondocument 925.24 925.37 925.42 925.46 925.45 925.47owner 0.23 this 529-536 ofYASTTNLR 940.26 940.45 940.47 940.48 940.51 under 0.25 of 361-368 TLSSTLYR rightsthe this documentand/or rights 976.26 976.44 976.48 976.47to of 976.50 fall 0.24use of 430-437 LSHVSMFR 1007.31 1007.44 1007.49 1007.52 1007.55 0.24 537-545the IYVTVAGER This may distribution,and 1066.17 1065.97 1066.38 1066.03rights 1066.43 0.26owner 1121-1128 ENPCEFNR 1074.28 1074.50 subject 1074.52 1074.55 0.27 282-292 GSAQGIEGSIR be the violate copy 1078.51 1078.55 of 0.04 682-690 NLLQDSNFK 1089.30 1089.52 1089.54 1089.55 1089.57 0.27and 491-501 GPGFTGGDILR may documentpublication, It exploitation this any 1 A difference of less than one dalton between the observed (first column where this mass is documented) and expected masses was necessary for consideration permission as a match. prohibited 2 AA position refers to amino acid position within the predictedthe Cry1A.105 sequence. commercial be 3 Sample, 0.5 µL, was analyzed prior to Zip Tip desalting 4 The ZipTip was washed with 0.1%Furthermore, (v/v) trifluoroaceticany acid containing acetonitrile at varying concentrations of acetonitrile: Wash 1 – 0% (v/v), Wash 2 – 20% (v/v), Wash 3 – 50% (v/v), and WashConsequently, 4 - 90% (v/v)without acetonitrile.

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Table D.1 (cont.) Summary of the Tryptic Masses Identified for the Full-length Plant-produced Cry1A.105 Protein Using MALDI-TOF Mass Spectrometry

regime.and Observed Mass (Da) Expected AA2 andFragment AG No Desalting4 Mass (Da) Position Sequence(s) Desalting3 Wash 1 Wash 2 Wash 3 Wash 4 ∆1 1144.29 1144.53 1144.55 1144.54 1144.57 0.28Bayer property 450-458 protectionAPMFSWIHRpublishing of contents 1203.40 1203.64 1203.67 1203.66 1203.68 0.28 350-360 IVAQLGQGVYR its parties.data therefore 1237.30 1237.55 1237.58 1237.57 1237.60 0.30 182-192 and/orWGFDAATINSRor 1253.36 1253.61 1253.64 1253.63 1253.65affiliates. 0.29 438-449 SGFSNSSVSIIR property intellectualthird may 1258.36 1258.61 1258.63 1258.64 1258.65its 0.29 199-209 LIGNYTDHAVR as 1269.40 1269.65 the 1269.69of 0.29and 479-490 AHTLQSGTTVVR is regulatory owner. 1398.34 1398.63 1398.65 1398.65 1398.67 0.33a 116-127document EWEADPTNPALR any such its reproductiondocument 1424.32 1424.53 1424.62 1424.65owner 0.33 this 994-1005 of GHVDVEEQNNQR 5 1576.81 under 0.00 of 682-694this NLLQDSNFKDINR 1576.81 rightsthe 5 documentand/or 1576.87 0.06 623-637rights AVNALFTSTNQLGLK to of fall use of 1598.81 1598.71 0.10 1120-1131RENPCEFNRGYRthe 1800.47 1800.84 1800.84This 1800.84 1800.87may distribution, and 0.40 753-767 GYIEDSQDLEIYSIR rights owner 1900.48 1900.88 1900.89 subject1900.89 1900.91 0.43 266-281 EIYTNPVLENFDGSFR 1902.52 1902.92be 1902.90 1902.96 the0.44 violate 100-115 LEGLSNLYQIYAESFR copy of and may documentpublication, It exploitation 1 A difference of less than one dalton between thethis observed any (first column where this mass is documented) and expected masses was necessary for consideration as a match. 2 permission AA position refers to amino acid position within the predicted Cry1A.105prohibited sequence. 3 Sample, 0.5 µL, was analyzed prior to Zip Tip desalting the commercial be 4 The ZipTip was washed with 0.1% (v/v) trifluoroacetic acid containing acetonitrile at varying concentrations of acetonitrile: Wash 1 – 0% (v/v), Wash 2 – 20% (v/v), Wash 3 – 50% (v/v), and WashFurthermore, 4 - 90%any (v/v) acetonitrile. 5 Two expected fragments having nearlyConsequently, identicalwithout masses were matched to one observed mass.

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Table D.1 (cont.) Summary of the Tryptic Masses Identified for the Full-length Plant-produced Cry1A.105 Protein Using MALDI-TOF Mass Spectrometry

regime.and Observed Mass (Da) and AG No Desalting4 Expected AA2 Fragment Desalting3 Wash 1 Wash 2 Wash 3 Wash 4 Mass (Da) ∆Bayer1 propertyPosition Sequence(s)publishing protectioncontents 1955.58 1955.98 1955.97 1956.01 of 0.43 1006-1022 SVLVVPEWEAEVSQEVR its parties.data therefore 2097.69 2098.14 2098.12 2098.15 0.46 860-878 and/or LGNLEFLEEKPLVGEALARor 2107.63 2108.07 2108.05 2108.09affiliates. 0.46 602-619 FELIPVTATLEAEYNLER property intellectualthird may 2133.62 2134.08 2133.11its 0.51 503-522 TSGGPFAYTIVNINGQLPQR as 2148.59 2149.03 2149.06 2149.02 the 2149.05of 0.46and 404-423 SGTVDSLDEIPPQNNNVPPR is regulatory owner. 2196.64 2197.11 2197.06 2197.11 0.47a 293-311document SPHLMDILNSITIYTDAHR any such its reproductiondocument 2211.11 2211.05 2211.13owner 0.02 this 430-449 of LSHVSMFRSGFSNSSVSIIR 2277.105 0.02 753-771 GYIEDSQDLEIYSIRYNAK 2277.12 under of this and/orrights 2277.15the 5 0.03 403-423 KSGTVDSLDEIPPQNNNVPPR document to of fall of rights 2615.78 2616.33 2616.37 2616.36 0.58use 941-964 EAYLPELSVIPGVNAAIFEELEGR the This may distribution,and RPFNIGINNQQLSVLDGTEFAYGTSS 3729.00 rights owner 3728.03 subject 3728.87 0.84 369-402 NLPSAVYR be the violate copy of and may documentpublication, It exploitation 1 A difference of less than one dalton between thethis observed any (first column where this mass is documented) and expected masses was necessary for consideration as a match. 2 permission AA position refers to amino acid position within the predicted Cry1A.105prohibited sequence. 3 Sample, 0.5 µL, was analyzed prior to Zip Tip desalting the commercial be 4 The ZipTip was washed with 0.1% (v/v) trifluoroacetic acid containing acetonitrile at varying concentrations of acetonitrile: Wash 1 – 0% (v/v), Wash 2 – 20% (v/v), Wash 3 – 50% (v/v), and WashFurthermore, 4 - 90%any (v/v) acetonitrile. 5 Two expected fragments having nearlyConsequently, identicalwithout masses were matched to one observed mass.

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Table D.2. Summary of the Tryptic Masses Identified Using MALDI-TOF Mass Spectrometry for the 61 kDa Plant-Produced Cry2Ab2 Protein

1 Observed Mass (Da) 3 and 5 Expected 2 AA and regime. No Desalting ∆ Mass (Da) AG Position Fragment Sequence(s) Desalting4 Wash 1 Wash 2 Wash 3 Wash 4 506.58 506.25 0.33 100-103 ETEK Bayer 552.44 552.45 552.45 552.31 0.13 property 538-541 protectionYTLRpublishing of contents 553.35 553.40 553.27 0.08 218-221 NYTRits parties.data therefore 560.38 560.44 560.45 560.32 0.06 237-241 and/orGLNTRor 646.39 646.48 646.47 646.32 affiliates. 0.07 439-443 NEDLR property intellectualthird may 659.44 659.44 659.38its 6 0.06 563-568 VTINGR as the 659.41of 6 0.03and 69-75 VGSLVGK is regulatory owner. a document 677.47 677.53 677.54 677.54 677.54 any677.37 such 0.10 104-108 FLNQRits reproductiondocument 709.46 709.52 709.54 709.54 709.54 709.36owner 0.10 this 410-416 ofSGAFTAR 724.57 724.39 under0.18 of 514-519 TFISEK rightsthe this 730.54 730.63 730.63document 730.63and/or 730.45 0.09 77-82rights ILSELR to of fall use of 903.61 903.72 903.72 903.72 903.49 0.12 109-116the LNTDTLAR 993.62 993.70 993.73 993.73This 993.73 993.48may distribution, and 0.14 520-528 FGNQGDSLR rights owner 1022.73 1022.72 subject 1022.45 0.28 380-388 SWLDSGSDR be the violate copy of and may documentpublication, It exploitation 1 Only experimental masses that matched to an expected mass are listed in the table. this any 2 A difference of less than one dalton between the observed (first column where this mass is documented) and expected mass was necessary for consideration as permission a match (Except for mass fragment 144-164). prohibited 3 AA position refers to amino acid position within the predictedthe Cry2Ab2be sequence as depicted in Figure 9.VI. 4 Sample, 0.3 µL, was analyzed prioto desalting. commercial 5 The ZipTip was washed with 0.1%Furthermore, (v/v) trifluoroaceticany acid containing acetonitrile at varying concentrations [0, 20, 50, and 90% (v/v) acetonitrile]. 6 Two expected fragments having nearlyConsequently, identicalwithout masses were matched to one observed mass.

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Table D.2 (cont.) Summary of the Tryptic Masses Identified Using MALDI-TOF Mass Spectrometry for the 61 kDa Plant- Produced Cry2Ab2 Protein

1 Observed Mass (Da) 3 and 5 Expected 2 AA andFragment regime. No Desalting ∆ Mass (Da) AG Position Sequence(s) Desalting4 Wash 1 Wash 2 Wash 3 Wash 4 1033.71 1033.83 1033.82 1033.82 1033.56 0.15 553-562 VISSIGNSTIR Bayer 1053.79 1053.91 1053.91 1053.91 1053.64 0.15 property 429-438 protectionNISGVPLVVRpublishing of contents 1060.21 1060.80 1060.79 1060.79 1060.52 0.31 242-249 LHDMLEFRits parties.data therefore 1076.69 1076.80 1076.80 1076.80 1076.53 0.16 466-474 and/orAYMVSVHNRor 1080.67 1080.76 1080.79 1080.79 1080.51affiliates. 0.16 529-537 FEQNNTTAR property intellectualthird may 1163.92 1164.60its 0.68 100-108 ETEKFLNQR as 1184.78 1184.92 1184.91 1184.90 the 1184.60of 0.18and 453-465 NIASPSGTPGGAR is regulatory owner. 1197.82 1197.94 1197.96 1197.95 1197.95 1197.65 0.17a 444-452document RPLHYNEIR any such its reproductiondocument 1216.79 1216.92 1216.92 1216.91 1216.61owner 0.18 this 134-143 ofQVDNFLNPNR 6 1492.91 1493.13 1493.06 1493.06 1492.69 under0.22 of 417-428this GNSNYFPDYFIR rightsthe 6 document and/or 1492.75 0.16 211-221rights TYRDYLKNYTR to of fall use of 1680.15 1680.31 1680.94 0.79 429-443the NISGVPLVVRNEDLR 1904.24 1904.45 1904.46 1904.43This 1904.41 1903.95may distribution, and 0.29 117-133 VNAELTGLQANVEEFNR rights owner subject be the violate copy of and may documentpublication, It exploitation 1 Only experimental masses that matched to an expected mass are listed in the table. this any 2 A difference of less than one dalton between the observed (first column where this mass is documented) and expected mass was necessary for consideration as permission a match (Except for mass fragment 144-164). prohibited 3 AA position refers to amino acid position within the predictedthe Cry2Ab2be sequence as depicted in Figure 9.VI. 4 Sample, 0.3 µL, was analyzed prior to desalting. commercial 5 The ZipTip was washed with 0.1%Furthermore, (v/v) trifluoroaceticany acid containing acetonitrile at varying concentrations [0, 20, 50, and 90% (v/v) acetonitrile]. 6 Two expected fragments having nearlyConsequently, identicalwithout masses were matched to one observed mass.

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Table D.2 (cont.) Summary of the Tryptic Masses Identified Using MALDI-TOF Mass Spectrometry for the 61 kDa Plant- Produced Cry2Ab2 Protein

1 Observed Mass (Da) 3 and 5 Expected 2 AA andFragment regime. No Desalting ∆ Mass (Da) AG Position Sequence(s) Desalting4 Wash 1 Wash 2 Wash 3 Wash 4 1919.28 1919.47 1919.47 1919.01 0.27 83-99 NLIFPSGSTNLMQDILR 2311.25 2311.68 2311.06 0.19Bayer property 569-590 protectionVYTATNVNTTTNNDGVNDNGARpublishing of contents 2334.77 2334.78 2333.23 1.54 144-164 NAVPLSITSSVNTMQQLFLNRits parties.data therefore 2339.51 2339.75 2339.69 2339.15 0.36 389-409 and/orEGVATVTNWQTESFETTLGLRor 2451.70 2451.98 2451.97 2451.34affiliates. 0.36 320-343 LSNTFPNIVGLPGSTTHALLAAR 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 1 Only experimental masses that matched to an expectedthis any mass are listed in the table. 2 A difference of less than one dalton between the observed (firstpermission column where this mass is documented) and expected mass was necessary for consideration as a match (Except for mass fragment 144-164). prohibited 3 the AA position refers to amino acid position within thecommercial predicted Cry2Ab2 be sequence as depicted in Figure 9.VI. 4 Sample, 0.3 µL, was analyzed prior to desalting. 5 The ZipTip was washed withFurthermore, 0.1% (v/v) anytrifluoroacetic acid containing acetonitrile at varying concentrations [0, 20, 50, and 90% (v/v) acetonitrile], as described in the methods section (SectionConsequently, 5.4.3).without

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Table D.3. Summary of the Tryptic Masses Identified Using MALDI-TOF Mass Spectrometry for the 50 kDa Fragment of the Plant-Produced Cry2Ab2 Protein

1 Observed Mass (Da) 3 and 5 Expected 2 AA andFragment regime. No Desalting ∆ Mass (Da) AG Position Sequence(s) Desalting4 Wash 1 Wash 2 Wash 3 Wash 4 552.44 552.45 552.31 0.13 394-397 YTLR 553.37 553.41 553.27 0.10Bayer property74-77 protectionNYTRpublishing of contents 560.40 560.45 560.45 560.46 560.32 0.08 93-97 GLNTRits parties.data therefore 646.47 646.48 646.32 0.15 295-299 and/orNEDLRor 659.40 659.45 660.20 659.38 affiliates.0.02 419-424 VTINGR property intellectualthird may 709.49 709.54 709.54 709.55 709.57 709.36its 0.13 266-272 SGAFTAR as 993.66 993.73 993.74 993.77 the 993.48of 0.18and 376-384 FGNQGDSLR is regulatory owner. 1022.73 1022.45 0.28a 236-244document SWLDSGSDR any such its reproductiondocument 1033.74 1033.84 1033.85 1033.56owner 0.18 this 409-418 ofVSSIGNSTIR 1053.83 1053.92 1053.93 1053.97 1053.64 under 0.19 of 285-294 NISGVPLVVR rightsthe this 1060.25 1060.80 1060.81document and/or 1060.52 0.27 98-105rights LHDMLEFR to of fall use of 1072.30 1072.54 0.24 70-77the DYLKNYTR 1076.73 1076.81 1076.82This 1076.84 1076.53may distribution, and 0.20 322-330 AYMVSVHNR rights owner 1080.71 1080.79 1080.79 1080.80 subject 1080.51 0.20 385-393 FEQNNTTAR be the violate copy of and may documentpublication, It exploitation

this any 1 Only experimental masses that matched to an expected mass are listed in the table. 2 permission A difference of less than one dalton between the observed (first columnprohibited where this mass is documented) and expected mass was necessary for consideration as a match. the be 3 AA position refers to amino acid position within thecommercial predicted Cry2Ab2 sequence as depicted in Figure 10.VI. 4 Sample, 0.3 µL, was analyzed priorFurthermore, to desalting.any 5 The ZipTip was washed with 0.1%Consequently, (v/v) trifluoroaceticwithout acid containing acetonitrile at varying concentrations [0, 20, 50, and 90% (v/v) acetonitrile].

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Table D.3 (cont.) Summary of the Tryptic Masses Identified Using MALDI-TOF Mass Spectrometry for the 50 kDa Fragment of the Plant-Produced Cry2Ab2 Protein.

1 Observed Mass (Da) 3 and 5 Expected 2 AA andFragment regime. No Desalting ∆ Mass (Da) AG Position Sequence(s) Desalting4 Wash 1 Wash 2 Wash 3 Wash 4 1184.81 1184.91 1184.93 1184.60 0.21 309-321 NIASPSGTPGGAR Bayer property publishing 1197.87 1197.97 1197.98 1198.02 1197.65 0.22 300-308 protectionRPLHYNEIRcontents 6 of 1492.96 1493.09 1493.10 1493.12 1492.69 0.27 273-284 GNSNYFPDYFIRits 6 parties.data therefore 1492.75 0.21 67-77 and/or TYRDYLKNYTRor 1680.22 1680.31 1680.35 1680.94affiliates. 0.72 285-299 NISGVPLVVRNEDLR property intellectualthird may 1844.28 1843.96its 0.32 50-66 DVILNADEWGISAATLR as the of and 1875.16 is 1874.79 0.37 regulatory78-92 DYSNYCINTYQSAFKowner. a document 2311.67 any 2311.06 such 0.61 425-446 VYTATNVNTTTNNDGVNDNGARits reproductiondocument 2339.79 2339.15owner 0.64 this245-265 ofEGVATVTNWQTESFETTLGLR under of 2451.81 2452.02 2452.03 rights2451.34the 0.47 176-199this LSNTFPNIVGLPGSTTTHALLAAR documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and 1 may documentpublication, Only experimental masses that matchedIt to an expected mass are exploitationlisted in the table. 2 A difference of less than one dalton between the observed (first column where this mass is documented) and expected mass was necessary for consideration as this any a match. 3 permission AA position refers to amino acid position within the predicted Cry2Ab2prohibited sequence as depicted in Figure 10.VI. 4 Sample, 0.3 µL, was analyzed prior to desalting. the be 5 The ZipTip was washed with 0.1% (v/v) trifluoroaceticcommercial acid containing acetonitrile at varying concentrations [0, 20, 50, and 90% (v/v) acetonitrile] 6 Two expected fragments havingFurthermore, nearly identicalany masses were matched to one observed mass. Consequently,without

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APPENDIX E: Materials and Methods used for the Estimation of Cry1Ab.105 and Cry2Ab2 Protein Levels in Tissues of MON 89034

1. Test, control, and reference substances

1.1. Test substance

The test substance was MON 89034 grown in 2005 U.S. field trials at five sites.

1.2. Control substance and and regime. The negative control substance was a conveAGntional corn with a similar genetic background to the test plants grown in 2005 U.S. field trials at five sites. Bayer property publishing protectioncontents 1.3. Characterization of test and controlof substances its parties.data therefore and/oror The identities of the test and control substances were confirmed by verifying the chain- affiliates. of-custody documentation priorproperty to analysis. intellectual Tothird further confirm the identitiesmay of the test its and control substances, event-specificas polym erase chain reaction (PCR) analyses were the of and conducted on seed and grainis samples. The identitiesregulatory of grain samples owner.harvested from the a document field were verified by PCRany and suchthe verification of identity was referencedits back to the reproductiondocument seed used for planting. owner this of under of this rightsthe documentand/or of rights 1.4. Reference substancesto of fall use the This may distribution,and Four E. coli-produced rightsprotein standards wereowner used and the certificates of analysis were subject archived with the study data. A Cry1A.105 protein standard (lot 20-100086) was used as be the violate the reference substancecopy for the analysisof of Cry1A.105 protein levels. The purity- corrected protein concentration of the purandified standard was 1.0 mg/ml by amino acid may documentpublication, compositionIt analysis. The exploitationpurity was 80% as determined by sodium dodecyl sulfate polyacrylamidethis gel anyelectrophoresis (SDS-PAGE) and densitometric analysis. permission A Cry2Ab2 protein standard (lotprohibited 20-100071) was used as the reference substance for the the analysis of Cry2Ab2commercial protein be le vels. The purity-corrected protein concentration of the purified standard was 0.4 mg/ml by amino acid composition analysis. The purity was 87%Furthermore, as determinedany by SDS-PAGE and densitometric analysis. without Consequently, 2.0. Generation of plant samples

2.1. Summary of field design

Plants were grown at five field sites in the U.S. during the 2005 field season: Jefferson County, IA; Warren County, IL; Clinton County, IL; York County, NE; and Fayette

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 254 of 323

County, OH. These field sites were located within the major corn-growing regions of the U.S. and provided a variety of environmental conditions. At each site, three replicated plots of MON 89034, as well as the conventional control, were planted using a randomized complete block field design. Overseason leaf (OSL 1-4), overseason root (OSR 1-4), over season whole plant (OSWP 1-4), pollen, silk, forage, stover, forage-root, senescent root, and grain tissues were collected from each replicated plot at all field sites. The over season samples (leaf, root, and whole plant) were collected four times at different growth stages: (1) V2 – V4 stage, (2) V6 – V8 stage, (3) V10 – V12 stage, and (4) pre-VT stage. The identification of corn growth and development stages was based on the descriptions in “How a Corn Plant Develops” (Ritchie et al., 1997). Throughout the field production, sample identity was maintained by using unique sample identifiers and proper chain-of-custody documentation. All tissue samples, except grain, were regime.and stored and shipped on dry ice to the Monsanto processingand facility in Creve Coeur, AG Missouri. Grain samples were stored and shipped at ambient temperature.

2.2. Description of the collected tissues Bayer property protectionpublishing of contents its Overseason Leaf parties.data therefore The youngest immature whorl leaf (2 - 4 inches) samples wereand/or collector ed from 15 plants affiliates. from each of the test and controlproperty plots. Ovintellectualerseasonthird leaf (OSL) samplesmay were collected at its the four growth stages defined in Sectionas 2.1. The leaves corresponding to each growth the of and stage were pooled from eisach plot during collection.regulatory owner. a documentits any such reproduction this documentof Overseason Whole Plant owner under of Two whole plants were collectedrightsthe from each of the test thisand control plots. A whole plant documentand/or rights sample consists of shootto tissueof (above-grfall ounduse portionof of the plant including leaves, tassels, ears, etc.), minus the roots. The two whole plantthe samples were pooled from each This may distribution,and plot during collection. rights OSWP samples were ownercollected at the four growth stages defined in Section 2.1. subject be the violate copy of Overseason Root document and may publication,exploitation The OSRIt samples were the below ground root mass that had been cut from the this corresponding OSWPany sample. The two root samples were pooled from each plot during collection. OSR tissue samplespermission were collected were collected at the four growth stages prohibited defined in Section 2.1. the commercial be

PollenFurthermore, any ApproximatelyConsequently, without5 g of pollen 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

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 255 of 323

(the R1 plant growth stage). The silk tissue samples were pooled from each plot during collection.

Forage Root Forage root is defined as the root ball removed from the plant sampled for forage. Forage root tissue samples were collected at the early dent growth stage (R4 - R6) from each test and control plot. Collection 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. regime.and Two senescent root samples were collected from each testand and control plot and AG 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 Bayerduring collection.property protectionpublishing of contents its Grain parties.data therefore All hand-pollinated ears in each test and control plot were collectedand/oror at the R6 growth affiliates. stage (physiological maturity).property The ears intellectualwere thirdshucked and dried to moisturemay content of its 10-15%. All cobs were shelled prior toas shipping. the of and is regulatory owner. a document 2.3. Tissue processing andany pr oteinsuch extraction methods its reproductiondocument owner this of under of 2.3.1. Processing rightsthe this documentand/or rights to of fall use of All tissue samples produced at the field sites were shippedthe to Monsanto for processing. During theThis processing step, drymay ice distribution,was combinand ed with the samples (except pollen) and rights owner then vertical cutterssubject or mixers were used to thoroughly grind and mix the tissues. All processed tissue samples were stored in a –80the °Cviolate freezer during the study. be copy of and 2.3.2. Extractionmay documentpublication, It exploitation this The Cry1A.105 andany Cry2Ab2 proteins were extracted from corn tissues as using standard permission operating procedures. All processedprohibited tissu es were kept on dry ice during extract preparation. All tissues were extracted using a Harbil mixer. Insoluble material was the be removed from thecommercial extracts by using a Serum Filter System (Fisher Scientific, Pittsburgh, PA),Furthermore, or by centrifugation.any The extracts were aliquoted and stored in a -80°C freezer until ELISAConsequently, analyses.without

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 256 of 323

2.4. ELISA reagents and methods

2.4.1. Cry1A.105 antibodies

Goat polyclonal antibodies (lot 7509175) specific for the Cry1A.105 protein were purified using Protein-G Agarose affinity chromatography. The concentration of the purified IgG was determined to be 0.93 mg/ml by spectrophotometric methods. The purified antibody was stored in a phosphate buffered saline (1X PBS) buffer (pH 7.4) containing 0.001 M KH2PO4, 0.01 M Na2HPO4 · 7H2O, 0.137 M NaCl, and 0.0027 M KCl.

and The purified Cry1A.105 antibodies were coupled with biotinand (Sig ma, St.regime. Louis, MO) according to the manufacturer’s instructions andAG assigned lot 7509180. The detection reagent was NeutrAvidin (Pierce, Rockford, IL) conjugated to horseradish peroxidase (HRP). Bayer property protectionpublishing of contents 2.4.2. Cry2Ab2 antibodies its parties.data therefore Mouse monoclonal antibody (lot G-800601) specific for theand/or Cry2Ab2or protein was affiliates. purified using Protein-A Agaroseproperty affinity intellectual chromatography.third The concentrationmay of the its purified IgG was determined to beas 1.0 mg/ml by spectrophotometric methods. the of and Production of the Cry2Ab2is monoclonal anregulatorytibody was performedowner. by Strategic a document Biosolutions (Newark, DE).any Thesuch purified antibody was stored itsin a buffer (pH 7.2) reproductiondocument containing 0.02 M Na2HPO4 · 7H2ownerO and 0.15 M NaClthis with 0.05%of NaN3 added as a preservative. of rights under this and/or the document to of fall of rights Purified Cry2Ab2 antibodies (lot 7381862) weusere coupled with biotin (Sigma) according the to the manufacturer’sThis instructionsmay anddistribution, assiandgned lot 7381898. The detection reagent was rights owner NeutrAvidin-HRP.subject be the violate 2.4.3. Cry1A.105copy ELISA method of document and may publication,exploitation Goat Itanti-Cry1A.105 capture antibodies were diluted in coating buffer (15 mM Na CO , this 2 3 35 mM NaHCO3, andany 150 mM NaCl, pH 9.6) and immobilized onto 96-well microtiter plates at 5.0 µg/ml followedpermission by incubation in a 4°C refrigerator for >8 h. Prior to each prohibited step in the assay, platesthe were washed with 1X PBS containing 0.05% (v/v) Tween-20 (1X PBST). For graincommercial tissue only, be plates were blocked with the addition of 100 to 150 µl per well of 1X PBST with 9% non-fat dried milk (NFDM) for 30 to 90 minutes at 37oC. Furthermore,any Cry1A.105Consequently, proteinwithout standard or sample extract was added at 100 µl per well and incubated for 1 h at 37°C. The captured Cry1A.105 protein was detected by the addition of 100 µl per well of biotinylated goat anti-Cry1A.105 antibodies and NeutrAvidin-HRP (Pierce). Plates were developed by adding 100 µl per well of HRP substrate, 3,3',5,5'-tetramethyl- benzidine (TMB; Kirkegaard & Perry, Gaithersburg, MD). The enzymatic reaction was terminated by the addition of 100 µl per well of 6 M H3PO4. Quantitation of the Cry1A.105 protein was accomplished by interpolation from a Cry1A.105 protein standard curve that ranged from 0.438 – 14 ng/ml.

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2.4.4. Cry2Ab2 ELISA method

Mouse anti-Cry2Ab2 capture antibody was diluted in coating buffer (15 mM Na2CO3 and 35 mM NaHCO3, pH 9.6) and immobilized onto 96-well microtiter plates at 5.0 µg/ml followed by incubation in a 4°C refrigerator for >8 h. Prior to each step in the assay, plates were washed with 1X PBST. Cry2Ab2 protein standard or sample extract was added at 100 µl per well and incubated for 1 h at 37°C. The captured Cry2Ab2 protein was detected by the addition of 100 µl per well of biotinylated goat anti-Cry2Ab2 antibodies and NeutrAvidin-HRP. 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 H PO . Quantitation of the Cry2Ab2 protein was accomplished by interpolationand from a 3 4 and regime. Cry2Ab2 protein standard curve that ranged fromAG 0.219 – 7 ng/ml.

2.4.5. Sensitivity of the ELISA methods Bayer property publishing protectioncontents The limits of detection (LOD) and limitsof of quantitation (LOQ) for the Cry1A.105 and its Cry2Ab2 protein ELISA methods are summarized in parties.the tabledata below. therefore and/oror

affiliates. property intellectualthird may Cry1A.105its Cry2Ab2 as the of and is 1 regulatory2 1 owner. 2 LOD LOQa documentLOD LOQ Tissue Type any such its (µg/g fwt ) (µg/g fwtreproduction ) (µg/gdocument fwt ) ( µg/g fwt ) owner this of Forage 0.372 0.44 of 0.191 0.44 rights under this Leaf and/or 0.568the 0.66 0.081 0.44 document to of fall of rights Pollen 0.412 1.1use 0.055 0.11 the This Root 0.254may distribution,and0.33 0.056 0.22 rights owner Silksubject 0.275 0.44 0.040 0.22 beGrain 0.262 the 1.1violate 0.123 0.22 copy of 1 and The limitmay of detection document(LOD)publication, was calculated as the mean value plus three SD using the data generated with Itconventional sample extractsexploitation for each tissue type. The LOD value in “ng/ml” was converted to “µg/g fwt” usingthis the respectiveany dilution factor and tissue-to-buffer ratio. 2 The limit of quantitation (LOQ) was calculated based on the lowest standard concentration. The “ng/ml” permission value was converted to “µg/g fwt” usingprohibited the respective dilution factor and tissue-to-buffer ratio. the be commercial Furthermore,any ReferenceConsequently, without

Ritchie, S.W., J.J. Hanway, and G.O. Benson. 1997. How a Corn Plant Develops: Special Report #48. Iowa State University of Science and Technology Cooperative Extension Service, Ames, IA.

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APPENDIX F: Materials and Methods used for Compositional Analysis of MON 89034

1.0. Test, control and reference substances

1.1. Test substance

The test substance was MON 89034. Forage and grain tissues of corn MON 89034 were evaluated in this study.

and 1.2. Control substance and regime. AG The control substance was conventional corn hybrid with genetic background representative of MON 89034. The forage and grain tissues of the control substance Bayer property protectionpublishing were evaluated in this study. of contents its parties.data therefore 1.3. Reference substances and/oror affiliates. property intellectualthird may The reference substances were 15its conventional commercial corn hybrids. A single as replicate of the forage andthe grain of tissues fromand each reference substance was evaluated in is regulatory owner. this study. The following conventional corn hybridsa weredocument analysed: any such its reproductiondocument owner this of Vendor/Hybrid Starting Seed Lot No. Field under of this rightsthe Site documentand/or rights to of fall use of Golden Harvest/ H8751 REF-0404-14931-Sthe IA This may distribution,and Golden Harvest/rights H9231 ownerREF-0404-14932-S IA subject Northrup King/ N60-N2 the REF-0404-14933-Sviolate IA be copy Burrus/ 590 of REF-0404-14934-S IL-1 and may documentpublication, It Mycogen/ 2784 exploitation REF-0404-14935-S IL-1 this Dekalb/ DKC62-15any REF-0404-14936-S IL-1 permission Pfister/ 2730 prohibited REF-0404-14937-S IL-2 the Mycogen/commercial 2E685 be REF-0404-14938-S IL-2 Dekalb/ DKC61-42 REF-0404-14939-S IL-2 Furthermore,any Consequently,Dekalb/without DKC60-15 REF-0404-14940-S NE Mycogen/ 2P682 REF-0404-14941-S NE Mycogen/ 2A791 REF-0404-14942-S NE Seed Consultants / SC1124A REF-0404-14943-S OH Crow’s/ 4908 REF-0404-14944-S OH Asgrow/ RX708 REF-0404-14945-S OH

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2.0. Test, control and reference substance characterization

The identity of the forage and grain samples from each test, control, and reference substance was verified by confirming the chain-of-custody documentation supplied with the forage and grain collected from the plots. The grain from the test, control, and reference substances was further characterized by event-specific PCR analysis of DNA extracted from grain to confirm the presence or absence of each event. The presence or absence of MON 89034 in respective samples of the grain from the test and control substances was confirmed. All forage samples were characterized by the confirmation of chain-of-custody records. regime.and and AG 3.0. Field trial description

Forage and grain of the test, control, andBayer reference property substances protection werepublishing collected at five replicated field sites in the U.S. Seed wasof planted in a randomized completecontents block design its with three replicates per block of each test, control,parties. anddata reference substance.therefore All the samples at the field sites were grown under normal agronomicand/or fieldor conditions for their affiliates. respective geographic regions. property The five U.S.intellectual sitesthird were: Site 1-Jeffersonmay County, IA; Site its 2-Jersey County, IL; Site 3-Warren County,as IL; Site 4-York County, NE; and Site 5- the of and Fayette County, OH. isForage and grain samplesregulatory were harvested fromowner. all plots and a document shipped on dry ice (forage)any or ambientsuch temperature (grain) to Monsantoits Company, St. reproductiondocument Louis, MO, USA. A sub-sample forowner use in compositionalthis analysisof was obtained from each bulk forage and grain sample generatedunder in theof field. Each sub-sample was ground, rightsthe this stored in a -20°Cdocument freezerand/or located at Monsanto Companyrights (St. Louis, MO), and then to of fall use of shipped, overnight, on dry ice to Covance Laboratories,the Inc. (Madison, WI) for analyses. The labelsThis on the samples shippedmay todistribution, Covaandnce Laboratories, Inc. listed the composition rights owner protocol number,subject a unique sample number, line/event number, tissue type, and storage conditions. the violate be copy of and 4.0. Analyticalmay methodsdocumentpublication, It exploitation this A total of 90 groundany forage and grain samples were analyzed by Covance Laboratories, permission Inc. Compositional analyses of prohibitedthe forage samples included proximates (protein, fat, ash, and moisture), ADF, NDF, minerals (calcium and phosphorus), and carbohydrates by the be calculation. Compositionalcommercial analyses of the grain samples included proximates (protein, fat,Furthermore, ash, andany moisture), ADF, NDF, TDF, amino acids, fatty acids, vitamins (B1, B2, B6, E, niacin,Consequently, and folicwithout acid), anti-nutrients (phytic acid and raffinose), secondary metabolites (furfural, ferulic acid, and p-coumaric acid), minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc), and carbohydrates by calculation. The methods used for compositional analyses are summarized below.

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4.1. Acid detergent fiber

The method used was based on an USDA Agriculture Handbook No. 379 (1970) method. The sample was placed in a fritted vessel and washed with an acidic boiling detergent solution that dissolved the protein, carbohydrate, and ash. An acetone wash was used to remove the fats and pigments. The lignocellulose fraction was collected on the frit and determined gravimetrically. The limit of quantitation of this method was 0.1% fw.

4.2. Amino acid composition

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 regime.and asparagine), cystine (including cysteine), glutamic acid (includingand glutamine), glycine, AG 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 requiredBayer a base hydrolysisproperty protectionusing publishingsodium hydroxide. Sulfur-containing amino acids requiredof an oxidation using performiccontents acid prior to its hydrolysis with hydrochloric acid. Analysis ofparties. the dataremaining aminotherefore acids was accomplished through direct hydrolysis with hydrochloric acid.and/or Theor individual amino affiliates. acids were quantitated using anproperty automated intellectual aminothird acid analyzer. The limitmay of quantitation its of this method was 0.1 mg/g fw. Theas reference standards were Beckman K18, 2.5 the of and µmol/mL per constituentis except cystine (1.25 regulatoryµmol/mL), lot numberowner. S407158; Sigma a document L-Tryptophan, 100%, lot numberany such 063K0382; Fluka L-Cysteic Acidits Monohydrate, 100%, reproductiondocument lot number 1157629; Sigma L-Methionineowner Sulfone, 100%,this lot numberof 12H3349. under of rightsthe this 4.3. Ash documentand/or rights to of fall use of the The methodThis used was based onmay AOAC distribution, Internand ational (2000) method 923.03. The sample rights owner was placed in ansubject electric furnace at 550 °C and ignited to drive off volatile organic compounds. The nonvolatile matter remainingthe violate was quantitated gravimetrically and the be copy percent ash was determined by calculation.of The limit of quantitation of this method was and 0.1% fw.may documentpublication, It exploitation this 4.4. Carbohydratesany permission prohibited The method used was based on an USDA Agriculture Handbook No. 74 (1973) method. the be The limit of quantitationcommercial of this method was 0.1% fw. Carbohydrate values were calculatedFurthermore, byany difference using the fresh weight-derived data and the following equation: Consequently,without % carbohydrates = 100% − (% protein + % fat + % ash + % moisture)

4.5. Fat by acid hydrolysis

The method used was based on AOAC International (2000) methods 922.06 and 954.02. The forage sample was hydrolyzed with hydrochloric acid at an elevated temperature. The fat was extracted using diethyl ether followed by hexane. The extract was

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evaporated under nitrogen, re-dissolved in hexane and filtered through a sodium sulfate column. The hexane extract was then evaporated again under nitrogen, dried, and weighed. The limit of quantitation of this method was 0.1% fw.

4.6. Fat by soxhlet extraction

The method used was based on AOAC International (2000) method 960.39. The grain sample was weighed into a cellulose thimble containing sodium sulfate and dried to remove excess moisture. Pentane was dripped through the sample to remove the fat. The extract was evaporated, dried, and weighed. The limit of quantitation of this method was 0.1% fw.

regime.and 4.7. Fatty acids and AG

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 caprylicBayer acid, 10:0property capric acid, protection 12:0publishing lauric acid, 14:0 myristic acid, 14:1 myristoleic acid, 15:0of pentadecanoic acid, 15:1 pentadecenoiccontents acid, its 16:0 palmitic acid, 16:1 palmitoleic acid, 17:0 heptadecanoicparties.data acid, 17:1 heptadecenoictherefore acid, 18:0 stearic acid, 18:1 oleic acid, 18:2 linoleic acid, 18:3and/or linolenic,or 18:3 gamma affiliates. linolenic acid, 20:0 arachidic propertyacid, 20:1 intellectualeicosenoicthird acid, 20:2 eicosadienoicmay acid, 20:3 its eicosatrienoic acid, 20:4 arachidonic acid,as and 22:0 behenic acid. Lipid in grain samples the of and was extracted and saponifiedis with 0.5 N regulatorysodium hydroxide inowner. methanol. The a document saponification mixture anywas suchmethylated with 14% (weight/volume)its boron reproductiondocument trifluoride:methanol. The resulting ownermethyl esters werethis extracted ofwith heptane containing an internal standard. The methyl estersunder of theof fatty acids were analyzed by gas rightsthe this chromatographydocument usingand/or external standards for quantitation.rights The limit of quantitation of to of fall use of this method was 0.003% fw. The reference standards werethe Nu Chek Prep GLC reference standardThis Hazelton no. 1, used mayas 100%, distribution, lotand number D13-0; Nu Chek Prep GLC reference rights owner standard Hazeltonsubject no. 2, used as 100%, lot number M13-0; Nu Chek Prep GLC reference standard Hazelton no. 3, used as 100%,the lot violatenumber MA13-0; Nu Chek Prep GLC be copy reference standard Hazelton no. 4, usedof as 100%, lot number D13-0; Nu Chek Prep and methyl maygamma linolenate,documentpublication, used as 100%, lot number U-63M-MA19-0; and Sigma methyl It exploitation tridecanoate, usedthis as 100%, lot number 035K1392. any permission 4.8. Folic acid prohibited

the be The method usedcommercial was based on AOAC International (2000) methods 960.46 and 992.05 andFurthermore, Methodsany of Analysis for Infant Formulas (1973), Section C-2. The grain sample was hydrolyzedConsequently, in potassiumwithout phosphate buffer with the addition of ascorbic acid to protect the folic acid during autoclaving. Following hydrolysis, the sample was treated with a chicken-pancreas enzyme and incubated approximately 18 hours to liberate the bound folic acid. The amount of folic acid was turbidimetrically determined by comparing the growth response of the bacteria Lactobacillus casei in the sample versus the growth response in folic acid standard. The limit of quantitation of this method was 0.06 µg/g fw. The reference standard was USP folic acid 98%, lot number P.

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4.9. Furaldehyde

The method used was based on a literature method (Albala-Hurtado et al., 1997). The grain sample was extracted with 4% trichloroacetic acid. The level of 2-furaldehyde (furfural) in the extract was determined by HPLC with UV quantitation. The reference standard was ACROS 2-furaldehyde, 99%, lot number A018806701. The quantitation limit of this method was calculated to be 0.5 ppm.

4.10. Minerals/ICP Emission Spectrometry.

The method used was based on AOAC International (2000) methods 984.27 and 985.01 regime.and and a literature method (Dahlquist and Knoll, 1978). Samplesand were dried, precharred, AG and ashed overnight at 500°. Ashed samples were treated with hydrochloric acid, dried, and dissolved in 5% (v/v) hydrochloric acid. The amount of each element was determined at appropriate wavelengths byBayer comparing property the emissionprotectionpublishing of the unknown sample, using inductively coupled plasma,of with the emission of the standardcontents solutions. its The limits of quantitation of this method and Spexparties. CertiPrepdata reference thereforestandards are and/or listed in the table below. or affiliates. property intellectualthird may its Mineral Lot Numbers as Concentration Limit of the of and is regulatory(µg/ml) owner.Quantitation a document any such its (ppm) reproductiondocument Calcium SC5179247, SC5179249owner 201.0,this 1001 of 20.0 under of Copper SC5179247,rights SC5179248the 2.01,this 10.04 0.50 documentand/or rights Iron SC5179247,to of SC5179250fall use 9.99,of 50.2 2.00 Magnesium SC5179247, SC5179248 49.93,the 250.0 20.0 This may distribution,and Manganese SC5179247,rights SC5179248owner 2.01, 10.06 0.30 Phosphorus subject SC5179247, SC5179249 200.7, 1005 20.0 Potassium be SC5179247, SC5179249the violate 199.9, 1007 100 copy of Sodium SC5179247, SC5179249and 201.7, 1007 100 may documentpublication, Zinc It SC5179247,exploitation SC5179248 9.92, 49.82 0.40 this any permission 4.11. Moisture prohibited the commercial be The method used was based on AOAC International (2000) methods 926.08 and 925.09. SamplesFurthermore, wereany dried in a vacuum oven at 100°C to a constant weight. The moisture loss Consequently,without was determined and converted to percent moisture. The limit of quantitation of this method was 0.1% fw.

4.12. Neutral detergent fiber, enzyme method

The method used was based on AACC (1998) method 32.20 and an USDA Agriculture Handbook No. 379 (1970) method. Samples were placed in a fritted vessel and washed with a neutral boiling detergent solution to dissolve the protein, carbohydrate, enzyme,

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and ash. Fats and pigments were removed using an acetone wash. The hemicellulose, cellulose, and lignin fractions were collected on a frit and determined gravimetrically. The limit of quantitation of this method was 0.1% fw.

4.13. Niacin

The method used was based on AOAC International (2000) method 944.13. The grain 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 plantarum 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 H2C121. regime.and and AG 4.14. p-Coumaric and ferulic acids

The method was based on a literature meBayerthod (Hagermanproperty and protection Nicholson,publishing 1982). The grain samples were extracted with methanolof using ultrasonication, and thecontents extracts were its then hydrolyzed using 4N sodium hydroxide, bufferedparties.data using acetic thereforeacid/sodium hydroxide, acidified with 3N hydrochloric acid, and filtered. and/or The orlevels of p-coumaric affiliates. and ferulic acids in the extractsproperty were deintellectualterminedthird by RP-HPLC withmay UV quantitation. its The reference standards were ACROSas p-Hydroxycinnamic acid (p-coumaric acid), the of and 97.9%, lot number A018661301is and ACROSregulatory 4-Hydroxy-3-methoxycinnamic owner. acid a document (ferulic acid), 100%, lot numberany A014010401.such The limit of quantitationits for both analytes reproductiondocument was calculated to be approximately 50owner.0 ppm using thethis following of equation: under of rightsthe this (conc. documentof lowestand/or standard) x (vol) x (dil) / (sample weight)rights = quantitation limit (ppm) to of fall use of the This may distribution,and 4.15. Phytic acid rights owner subject be the violate The method usedcopy was based on two literatuof re methods (Lehrfeld 1989; 1994). Grain samples were extracted using 0.5M HCland with ultrasonication. Purification and may documentpublication, concentrationIt was performedexploitation using a silica-based anion exchange (SAX) column. Sample analysisthis was any conducted using a macroporous polymer HPLC column [PRP-1, 5µm (150 x 4.1 mm)] connected to a refractive index detector. The limit of quantitation permission of this method was approximatelyprohibited 0.1% fw. The reference standard was Aldrich phytic acid, dodecasodium saltthe hydrate, 95%, lot number 01913EC. commercial be

4.16.Furthermore, Proteinany Consequently,without The method used was based on AOAC International (2000) methods 955.04 and 979.09 and two literature methods (Bradstreet, 1965; Kalthoff and Sandell, 1948). Protein and other nitrogenous compounds in the sample were reduced to ammonia by digestion of the sample with sulfuric acid containing a mercury catalyst mixture. The acid digest was made alkaline, and the ammonia was distilled and titrated with a standard acid. The percent nitrogen was determined and converted to percent protein by multiplication with 6.25. The limit of quantitation of this method was 0.1% fw.

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4.17. Pyridoxine/vitamin B6

The method used was based on AOAC International (2000) method 961.15. The grain sample was hydrolyzed with dilute sulfuric acid. The amount of pyridoxine was turbidimetrically determined by comparing the growth response of the yeast Saccharomyces carlsbergensis in the sample with the growth response in a pyridoxine standard. The limit of quantitation of this method was 0.07 µg/g fw. The reference standard was USP pyridoxine, 100%, lot number P.

4.18. Raffinose

regime.and This method was based on two literature methods (Mason and Slover, 1971; Brobst, AG 1972). The grain samples were extracted with deionized water and the extracts treated with an hydroxylamine hydrochloride solution in pyridine containing phenyl-β-D- glucoside as an internal standard. TheBayer resulting property oximes protectionwere publishingconverted to silyl derivatives by treatment with hexamethyldisof ilazane and trifluoracetic acidcontents and analyzed its by gas chromatography using a flame ionization detector.parties.data The reference thereforestandard was Sigma, D(+)-Raffinose Pentahydrate Sigma Ultra, 99%, lot numberand/or 073K0938.or The limit affiliates. of quantitation of this method: propertyThe acceptabl intellectuale rangethird for an 8/2.5 dilutionmay was 0.05-0.9%. its the as of and owner. 4.19. Riboflavin/vitaminis B2 regulatory a document any such its reproductiondocument The method used was based on AOACowner Intern ationalthis (2000) methodof 940.33. The grain sample was hydrolyzed with dilute HClunder and pH adjustedof to remove interferences. The rightsthe this amount of riboflavindocumentand/or was determined by comparing the growthrights response of the bacteria, to of fall use of Lactobacillus casei, in the sample hydrolysate with thethe bacterial growth response in varying Thisamounts of riboflavinmay standard. distribution, and Th e bacterial growth response was measured rights owner turbidimetrically.subject The limit of quantitation of this method was 0.2 µg/g fw. The reference standard was USP riboflavin, 100%,the lotviolate number N0C021. be copy of and 4.20. Thiamin/vitaminmay documentpublication, B1 It exploitation this The method used wasany based on AOAC International (2000) methods 942.23, 953.17, and permission 957.17. The grain sample wasprohibited autoclaved under weak acid conditions to extract the thiamin. The resulting solution was incubated with a buffered enzyme solution to release the be any bound thiamin.commercial The solution was purified on an ion-exchange column. An aliquot wasFurthermore, taken andany reacted with potassium ferricyanide to convert thiamin to thiochrome. The thiochromeConsequently, waswithout extracted into isobutyl alcohol and read on a fluorometer against a known standard. The limit of quantitation of this method was 0.01 mg/100g fw. The reference standard was USP, thiamin, 100%, lot number O.

4.21. Total dietary fiber

The method used was based on AOAC International (2000) method 985.29. Duplicate grain samples were gelatinized with alpha-amylase and digested with enzymes to break

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down starch and protein. Ethanol was added to each sample to precipitate the soluble fiber. The samples were filtered and the residue was rinsed with ethanol and acetone to remove starch and protein degradation products and moisture. Protein content was determined for one of the duplicates; ash content was determined for the other. The total dietary fiber in the sample was calculated using the protein and ash values. The limit of quantitation of this method was approximately 1.0% fw.

4.22. Vitamin E

The method used was based on three literature methods (Cort et al., 1983; McMurray et al., 1980; Speek et al., 1985). Grain samples were saponified to break down fat and release vitamin E. The saponified mixture was extracted with ethyl ether and quantitated regime.and directly by HPLC on a silica column. The limit of quantitationand of this method was AG approximately 0.005 mg/g fw. The reference standard was USP alpha tocopherol, 100%, lot number M. Bayer property protectionpublishing 5.0. Control of bias of contents its parties.data therefore The test, control, and reference substances from each respectiveand/or plotor within the field sites affiliates. were produced under similar agronomicproperty conditionsintellectualthird. To control and/or mayminimize bias, the its samples were analyzed in the orderas spec ified by a computer-generated randomized the of and sample list. The Study Directoris generated the randomizedregulatory sample list andowner. forwarded it to a document Covance Laboratories, Inc. anyprior suchto analysis. its reproductiondocument owner this of under of 6.0. Statistical analysis rightsthe this documentand/or rights to of fall use of The data processing and statistical analysis methodologythe is described in the sections below. This may distribution,and rights owner subject 6.1. Data processing the violate be copy of and After compositionalmay document analysespublication, were performed at Covance Laboratories, Inc., data It exploitation spreadsheets werethis sent to Monsanto Company. The data were reviewed, formatted, and sent to Certus International,any Inc. for statistical analysis. A statistical sub-report was permission generated by Certus and sent toprohibited Monsanto Company. The following formulas were used for re-expression of the data for statistical analysis: the be commercial Furthermore,any Consequently,without

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Component From (X) To Formula Proximates (excluding moisture), % FW % DW X/d Fiber, Raffinose, Phytic Acid Furfural, p-Coumaric Acid, ppm FW ug/g DW X/d Ferulic Acid Calcium, Phosphorus, ppm FW % DW (X/d) X 10-4 Magnesium, Potassium, Sodium Copper, Iron, Manganese, Zinc ppm FW mg/kg DW X/d and Vitamin B1 mg/100g FW mg/kg andDW 10 (X/d)regime. Vitamin E mg/g FWAG mg/kg DW 103 (X/d) Niacin, Folic Acid, Vitamin B2, ug/g FW mg/kg DW X/d Vitamin B6 Amino Acids (AA) Bayermg/g FW property % DW protection publishing X/(10*d) of contents Fatty Acids (FA) (100)Xj/Σ Xj, % FW % Total FA its parties.data for eachtherefore FA j and/oror ‘d’ is the fraction of the sample that is dry matter. affiliates. property intellectualthird may its The following 16 compositional analytesas with >50% of observations below the LOQ of the of and their respective assay wereis excluded from statisticalregulatory analysis: sodium,owner. furfural, raffinose, a document 8:0 caprylic acid, 10:0 capricany acid, such 12:0 lauric acid, 14:0 myristic itsacid, 14:1 myristoleic reproductiondocument acid, 15:0 pentadecanoic acid, 15:1owner pentadecen oic acid,this 17:0 heptadecenoicof acid, 17:1 under of heptadecenoic acid, 18:3rights gammathe linolenic acid, this20:2 eicosadienoic acid, 20:3 documentand/or rights eicosatrienoic acid, and to20:4 arachidonicof fall acid.use of the This may distribution,and The following additionalrights seven observationsowner for forage and grain tissue samples were below the LOQ:subject 16:1 palmitoleic acid (five values in grain); and vitamin E (two values in grain). To includebe a complete data set for thethes eviolate analytes in the statistical analysis, a value copy of equal to half the quantitation limit was assignedand for these seven data points. may documentpublication, It exploitation The data was thisassessed for potential outliers using a studentized PRESS residuals calculation. Two outliersany were identified in the data set: copper (Site 4, Test MON permission 89034, Rep 1) and iron (Site 1, prohibitedReference H8751, Rep 2). The identified copper and iron values were consideredthe outliers be and were removed from further analysis. The outlier test procedure was reappliedcommercial to all remaining copper and iron data to detect potential outliers thatFurthermore, were maskedany in the first analysis. Only one iron value (Site 1, Test MON 89034, RepConsequently, 1) identifiedwithout in the second analysis was considered an outlier and removed from further analysis.

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

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 267 of 323

model analysis of variance. The data from the five replicated sites were analyzed separately and as a combined data set.

Individual replicated site analyses used the model:

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

Combined site analyses used the model:

regime.and Y = U + T + L + B(L) + LT + e , and ijk i j jk ij AGijk where Yijk = unique individual observation, U = overall mean, Ti = hybrid effect, Lj = random location effect, B(L)jk = randomBayer block withinproperty location effect,publishing LTij = random protectioncontents location by hybrid interaction effect, andof eijk = residual error. For each compositional its component, the forage and grain from the testparties. substancedata was comparedtherefore to the conventional control. and/oror affiliates. property intellectualthird may its A range of observed values from theas reference substances was determined for each the of and analytical component. Additionally,is the referenceregulatory substances data wereowner. used to develop a document population tolerance intervals.any Asuch tolerance interval is an interval thatits one can claim, with reproductiondocument a specified degree of confidence, containsowner at least a thisspecified proportioof n, p, of an entire sampled population for the parameterunder measured. of For each compositional component, rightsthe this 99% tolerancedocument intervalsand/or were calculated that are expectedrights to contain, with 95% to of fall use of confidence, 99% of the quantities expressed in the populationthe of commercial references (George Thiset al., 2004; Ridley etmay al., distribution,2002).and Each tolerance interval estimate was based rights owner upon one observationsubject per unique reference substance. Individual substances with multiple observations were summarizedthe with violatein sites to obtain a single estimate for be copy inclusion in tolerance interval calculations.of Because negative quantities are not possible, and ® calculatedmay negative documentlowerpublication, tolerance bounds were set to zero. SAS software was used to It exploitation generate all summarythis statistics and perform all analyses (SAS Software Release 9.1, 2002-2003). Reportany tables present p-values from SAS® as either <0.001 or the actual permission value truncated to three decimalprohibited places. the be commercial ReferencesFurthermore, any Consequently,without AACC. Method 32.20. 1998. Approved Methods of the American Association of Cereal Chemists, 9th Ed.

Albala-Hurtado S., Veciana-Nogues, M. T., Izquierdo-Pulido, M., and Vidal-Carou, M. C. 1997. Determination of Free and Total Furfural Compounds In Infant Milk Formulas By High-Performance Liquid Chromatography. Journal of Agricultural and Food Chemistry. 45:2128-2133.

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AOAC. International Methods 922.06 and 954.02. 2000. Official Methods of Analysis, 17th Ed. Association of Official Analytical Chemists, Gaithersburg, MD.

AOAC. International Methods 923.03. 2000. Official Methods of Analysis, 17th Ed. Association of Official Analytical Chemists, Gaithersburg, MD.

AOAC. International Methods 926.08 and 925.09. 2000. Official Methods of Analysis, 17th Ed. Association of Official Analytical Chemists, Gaithersburg, MD.

AOAC. International Methods 940.33. 2000. Official Methods of Analysis, 17th Ed. Association of Official Analytical Chemists, Gaithersburg, MD. regime.and and AG AOAC. International Methods 942.23, 953.17 and 957.17. 2000. Official Methods of Analysis, 17th Ed. Association of Official Analytical Chemists, Gaithersburg, MD. Bayer property protectionpublishing AOAC. International Methods 944.13.of 2000. Official Methods of Analysis,contents 17 th Ed. its Association of Official Analytical Chemists, Gaithersburg,parties.data MD. therefore and/oror affiliates. AOAC International Methods property955.04 andintellectual 979.09third. 2000. Official Methodsmay of Analysis, th its 17 Ed. Association of Official Analasytical Chemists, Gaithersburg, MD. the of and is regulatory owner. a document th AOAC. International Methodsany 960.39.such 2000. Official Methods ofits Analysis, 17 Ed. reproductiondocument Association of Official Analyticalowner Chemists, Gaithersburg,this MD.of under of this and/orrights the AOAC. Internationaldocument Methods 960.46fall and 992.05. 2000.of rightsOfficial Methods of Analysis, th to of use 17 Ed. Association of Official Analytical Chemists,the Gaithersburg, MD. This may distribution,and rights owner th AOAC. Internationalsubject Methods 961.15. 2000. Official Methods of Analysis, 17 Ed. Association of Official Analytical Chemists,the violate Gaithersburg, MD. be copy of and th AOAC.may International document Methodspublication, 982.30. 2000. Official Methods of Analysis, 17 Ed. It exploitation Associationthis of Official Analytical Chemists, Gaithersburg, MD. any permission AOAC. International Methods prohibited984.27 and 985.01. 2000. Official Methods of Analysis, 17th Ed. Association of Official Analytical Chemists, Gaithersburg, MD. the be commercial th AOAC.Furthermore, Internationalany Methods 985.29. 2000. Official Methods of Analysis, 17 Ed. AssociationConsequently,without of Official Analytical Chemists, Gaithersburg, MD.

AOCS. Method Ce 1-62. 1997. Official Methods and Recommended Practices of the American Oil Chemists Society, 5th Ed. American Oil Chemists Society, Champaign, IL.

Bradstreet, R. B. 1965. The Kjeldahl Method for Organic Nitrogen. Academic Press, New York, NY.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 269 of 323

Brobst, K. M. 1972. Gas-Liquid Chromatography of Trimethylsilyl Derivatives. In Methods in Carbohydrate Chemistry. Vol. 6. Academic Press, New York, NY.

Cort, W. M., T.S. Vincente, E.H. Waysek, and B.D. Williams. 1983. Vitamin E Content of Feedstuffs Determined by High-Performance Liquid Chromatographic Fluorescence. Journal of Agricultural Food Chemistry. 31:1330-1333.

Dahlquist, R.L., and J.W. Knoll. 1978. Inductively Coupled Plasma-Atomic Emission Spectrometry: Analysis of Biological Materials and Soils for Major, Trace, and Ultra Trace Elements. Applied Spectroscopy. 32:1-29.

regime.and George, C., W.P. Ridley, J.C. Obert, M.A. Nemeth, M.L. Breeze,and and J.D. Astwood. AG 2004. Composition of grain and forage from corn rootworm-protected corn event MON 863 is equivalent to that of conventional corn (Zea mays L.). J. Agric. Food Chem. 52:4149-4158. Bayer property protectionpublishing of contents its Hagerman, A. E. and R.L. Nicholson. 1982. High-Performanceparties.data Liquid Chromatographictherefore Determination Of Hydroxycinnamic Acids in Maize Mesocotyl.and/oror J. of Agric. Food affiliates. Chem. 30 (6):1098-1102.property intellectualthird may its as the of and Kalthoff, I.M., and E.B. isSandell. 1948. Quantitativeregulatory Inorganic Analysis.owner. MacMillan, New a document York, NY. any such its reproductiondocument owner this of Lehrfeld, J. 1989. High-performance liquidunder ch romatographyof analysis of phytic acid on a rightsthe this pH-stable,document macroporousand/or polymer column. Cereal Chem.rights 66:510-515. to of fall use of the Lehrfeld,This J. 1994. HPLC separationmay distribution, andand qua ntitation of phytic acid and some inositol rights owner phosphatessubject in foods: problem and solution. J. Agric. Food Chem. 42:2726-2731. the violate be copy Mason, B.S. and H.T. Slover. 1971. A gasof chromatographic method for the determination and ofmay sugars in documentfoods.publication, J. Agric. Food Chem. 19:551-554. It exploitation this McMurray, C. H., anyW.J. Blanchflower, and D.A. Rice. 1980. Influence of extraction permission techniques on determinationprohibited of α -tocopherol in animal feedstuffs. Journal of Association of Official Analytical Chemists. 63:1258-1261. the be commercial MethodsFurthermore, of anyAnalysis for Infant Formulas. Section C-2. 1973. Infant Formula Council, Consequently,Atlanta,without GA.

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. 50:7235-7243.

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Speek, A. J., J. Schrijver, 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. 50:121-124.

USDA. 1970. Forage and Fiber Analysis. Agriculture Handbook No. 379. United States Department of Agriculture, Washington, D.C.

USDA. 1973. Energy value of foods. Pages 2-11 in Agriculture Handbook No. 74. United States Department of Agriculture, Washington, D.C.

and and regime. AG

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APPENDIX G: Compositional Analyses Data for Individual Sites

This appendix contains the compositional analysis tables for the five individual sites as follows:

Site No. (State) Table 1 (IA) G.1-G.7 and 2 (IL) G.8-G.14 and regime. 3 (IL) G.15-G.22AG 4 (NE) G.23-G.30 5 (OH) G.31-G.38 Bayer property protectionpublishing of contents its parties.data therefore and/oror affiliates. property intellectualthird may its as the of and is regulatory owner. a document any such its reproductiondocument owner this of under of rightsthe this documentand/or rights to of fall use of the This may distribution,and rights owner subject be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table G.1. Comparison of the proximates, fiber, and mineral content in forage collected at Site 1 from MON 89034 and conventional corn

and Difference(Test minusand Control) regime. AG Test Mean ± S.E. Control Mean ± S.E. Mean ± S.E. 95% CI Commercial (Range) Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value [99% Tolerance Int.¹] Fiber Bayer property protectionpublishing Acid Detergent Fiber (% DW) 33.94 ± 2.44 32.16 ± 2.44 of 1.78 ± 3.06 -5.28,8.83 0.577contents (26.72 - 38.94) (32.27 - 35.85) (30.00 - 35.59) (0.27 - 2.79) its [16.76,43.76] parties.data therefore and/oror Neutral Detergent Fiber (% DW) 41.26 ± 0.80 42.26 ± 0.80 -1.00 ± 1.13 -3.59,1.59 0.400 (33.70 - 46.74) affiliates. may (39.51 - 42.46) (40.23 - 43.96)property (-2.16intellectual - -0.12)third [25.94,55.67] its as the of and Mineral is regulatory owner. a document Calcium (% DW) 0.24 ± 0.0065 0.26 ± 0.0065any such -0.023 ± 0.0090 -0.044,-0.0024 its 0.033 (0.11 - 0.29) (0.24 - 0.24) (0.25 - 0.28) (-0.036 - -0.014)reproduction document [0.016,0.38] owner this of

under of Phosphorus (% DW) 0.24 ± 0.0048 0.24 ± rights0.0048the 0.0018 ± 0.0039 this -0.0072,0.011 0.654 (0.14 - 0.25) documentand/or rights (0.24 - 0.25) (0.23to - 0.25)of fall(-0.0019 - 0.0041)use of [0.071,0.32] the Proximate This may distribution,and rights owner Ash (% DW) 4.21 ± 0.26 subject4.46 ± 0.26 -0.25 ± 0.36 -1.08,0.59 0.515 (3.40 - 5.45) (3.32 - 4.67) (4.22 - 4.65) (-1.19 - 0.42) [1.93,6.31] be the violate copy of Carbohydrates (% DW) 85.50 ± 0.47 85.51 ± 0.47 -0.013and ± 0.60 -1.40,1.38 0.983 (84.88 - 88.39) may documentpublication, (85.20It - 85.68) (84.51 - 86.46)exploitation (-0 .85 - 1.18) [83.05,90.74] this Moisture (% FW) 74.87 ± 0.44 any 73.83 ± 0.44 1.03 ± 0.51 -0.13,2.20 0.075 (64.90 - 77.40) (74.40 - 75.40) (72.70 - permission74.40) (0.40 - 1.70) [57.62,86.45] prohibited the Protein (% DW) 8.90 ± 0.16 commercial8.49 ± 0.16 be 0.41 ± 0.21 -0.085,0.91 0.092 (6.58 - 8.82) (8.85 - 8.98) (8.24 - 8.87) (0.12 - 0.63) [4.78,10.38] Furthermore, any Total Fat (% DW) Consequently,1.39 ± 0.60 without 1.54 ± 0.60 -0.15 ± 0.66 -1.66,1.36 0.823 (0.58 - 3.11) (0.89 - 2.13) (0.92 - 2.75) (-0.61 - 0.23) [0,4.54] ¹With 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 G.2. Comparison of the of amino acid content in grain collected at Site 1 from MON 89034 and conventional corn

Difference(Test minus Control) Control Mean ± Commercial Test Mean ± S.E. S.E. Mean ± S.E. 95% CI and (Range) and regime. Analytical Component (Range) (Range) (Range) AG (Lower,Upper) p-Value [99% Tolerance Int.¹] Amino Acid (% DW) Alanine (% DW) 0.88 ± 0.017 0.81 ± 0.017 0.064 ± 0.024 0.0078,0.12 0.030 (0.67 - 0.96) (0.87 - 0.88) (0.79 - 0.84) (0.037 - 0.089) [0.48,1.08] Bayer property protectionpublishing of contents Arginine (% DW) 0.51 ± 0.0095 0.46 ± 0.0095 0.050 ± 0.013 0.020,0.081 0.005 (0.37 - 0.49) its (0.50 - 0.52) (0.46 - 0.47) (0.036 - 0.062) parties.data therefore [0.33,0.56] and/oror affiliates. Aspartic acid (% DW) 0.77 ± 0.011 0.71 ± 0.011 property 0.061 intellectual± 0.015 third 0.026,0.097 may0.003 (0.57 - 0.77) (0.77 - 0.78) (0.70 - 0.73) its(0.038 - 0.078) [0.43,0.90] the as of and owner. Cystine (% DW) 0.25 ± 0.0039 0.23 ± is0.0039 0.017 ± 0.0056regulatory 0.0045,0.030 0.014 (0.20 - 0.24) a document (0.24 - 0.26) (0.23 - 0.23)any such(0.011 - 0.023) its [0.18,0.27] reproductiondocument owner this of Glutamic acid (% DW) 2.27 ± 0.039 2.09 ± 0.039 0.18under ± 0.055 of 0.054,0.31 0.011 (1.71 - 2.41) (2.26 - 2.28) (2.03 - 2.16)rights the (0.12 - 0.24) this [1.25,2.75] documentand/or rights to of fall use of Glycine (% DW) 0.41 ± 0.0065 0.38 ± 0.0065 0.026 ± 0.0091 the 0.0052,0.047 0.020 (0.32 - 0.40) (0.40 - 0.41)This (0.37 - 0.39) may distribution,(0.012and - 0.035) [0.28,0.46] rights owner subject Histidine (% DW) 0.34 ± 0.0057 0.32 ± 0.0057 0.023 ± 0.0081 0.0041,0.041 0.022 (0.26 - 0.33) be the violate (0.34 - 0.34) copy (0.31 - 0.32) (0.015of - 0.030) [0.22,0.38]

document and Isoleucine (% DW) 0.39 ± 0.0099may 0.37 ±publication, 0.0099exploitation 0.025 ± 0.014 -0.0075,0.057 0.114 (0.32 - 0.45) (0.39It - 0.40) (0.36 - 0.38) (0.016 - 0.041) [0.23,0.51] this any Leucine (% DW) 1.49 ± 0.034 1.37 ± 0.034 0.12 ± 0.047 0.013,0.23 0.032 (1.14 - 1.68) permission (1.48 - 1.51) (1.33 - 1.41) prohibited (0.098 - 0.16) [0.77,1.92] the be ¹With 95% confidence, interval contains 99% ofcommercial the values expre ssed in the population of commercial lines. Negative limits were set to zero.

Furthermore,any Consequently,without

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Table G.2 (cont.). Comparison of the of amino acid content in grain collected at Site 1 from MON 89034 and conventional corn

Difference(Test minus Control) and Control Mean ± and regime. Commercial Test Mean ± S.E. S.E. Mean ± S.E. AG 95% CI (Range) Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value [99% Tolerance Int.¹]

Lysine (% DW) 0.35 ± 0.0062 0.32 ± 0.0062 0.022 ± 0.0081 0.0029,0.040 0.028 (0.24 - 0.34) Bayer property protectionpublishing (0.33 - 0.36) (0.32 - 0.33) (0.0042of - 0.033) contents [0.20,0.40] its Methionine (% DW) 0.25 ± 0.0043 0.23 ± 0.0043 0.026 ± 0.0061 parties. 0.012,0.040data 0.003therefore (0.17 - 0.22) and/oror (0.25 - 0.27) (0.22 - 0.24) (0.024 - 0.028) [0.14,0.25] affiliates. property intellectual third may Phenylalanine (% DW) 0.58 ± 0.013 0.53 ± 0.013 its 0.050 ± 0.019 0.0066,0.094 0.028 (0.45 - 0.65) as (0.57 - 0.59) (0.52 - 0.54)the of (0.041 - and0.067) [0.32,0.73] is regulatory owner. a document Proline (% DW) 1.05 ± 0.019 0.98 ± 0.019any such0.071 ± 0.027 0.0097,0.13 its 0.028 (0.83 - 1.11) reproductiondocument (1.04 - 1.05) (0.95 - 1.01) owner (0.041 - 0.10) this of [0.68,1.21] under of rightsthe this Serine (% DW) 0.60 ± 0.0085document 0.56and/or ± 0.0085 0.046 ± 0.012 of 0.019,0.074rights 0.004 (0.45 - 0.62) (0.60 - 0.61) (0.55to - 0.57) of fall(0.034 - 0.058)use [0.34,0.71] the Threonine (% DW) 0.37 ± This0.0051 0.34 ± 0.0051may distribution,0.029 ±and 0.0072 0.012,0.046 0.004 (0.29 - 0.37) rights owner (0.37 - 0.37) subject (0.33 - 0.36) (0.016 - 0.039) [0.24,0.41]

be the violate Tryptophan (% DW) 0.062 ± 0.0011 copy 0.061 ± 0.0011 0.0016of ± 0.0015 -0.0018,0.0051 0.311 (0.043 - 0.059) (0.061 - 0.063) (0.058 - 0.063) (-0.0015 - 0.0050) [0.032,0.072] document and may publication, It exploitation Tyrosine (% DW) 0.43 ± 0.012 this 0.36 ± 0.012 0.063 ± 0.018 0.023,0.10 0.006 (0.25 - 0.40) (0.42 - 0.43) any (0.35 - 0.37) (0.052 - 0.072) [0.17,0.52] permission Valine (% DW) 0.53 ± 0.012 0.50 ± 0.012 prohibited 0.035 ± 0.016 -0.0026,0.073 0.063 (0.42 - 0.55) (0.53 - 0.54) (0.48the - 0.51) be (0.020 - 0.055) [0.35,0.62] commercial ¹With 95% confidence, intervalFurthermore, contains 99%any of the values expressed in the population of commercial lines. Negative limits were set to zero. Consequently,without

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Table G.3. Comparison of the fatty acid content in grain collected at Site 1 from MON 89034 and conventional corn

Difference(Test minus Control) Commercial Control Mean ± and (Range) and regime. Test Mean ± S.E. S.E. Mean ± S.E.AG 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Fatty Acid (% Total FA) 16:0 Palmitic (% Total FA) 9.21 ± 0.043 9.23 ± 0.043 -0.023 ± 0.060 -0.16,0.11 0.706 (9.10 - 12.55) Bayer property protectionpublishing (9.12 - 9.31) (9.15 - 9.34) of (-0 .14 - 0.16) contents [6.12,15.67]

its 16:1 Palmitoleic (% Total FA) 0.11 ± 0.0081 0.11 ± 0.0081 -0.0017 ± 0.011 parties. -0.028,0.025data therefore 0.889 (0.050 - 0.19) (0.11 - 0.11) (0.11 - 0.12) (-0.0034 - 0.00039) and/oror [0,0.28] affiliates. property intellectual third may 18:0 Stearic (% Total FA) 1.80 ± 0.016 1.81 ± 0.016 its -0.0070 ± 0.023 -0.061,0.047 0.772 (1.57 - 2.45) (1.79 - 1.83) (1.77the - 1.85) as(-0.055 - 0.063) [0.86,2.98] of and owner. is regulatory a document 18:1 Oleic (% Total FA) 25.08 ± 0.12 24.75any ± 0. 12such 0.34 ± 0.17 -0.057,0.73 its 0.083 (21.17 - 35.33) reproductiondocument (24.87 - 25.36) (24.55 - 24.92) owner (0.099 - 0.81) this of [7.51,46.46] under of 18:2 Linoleic (% Total FA) 61.79 ± 0.12 61.98rights ± 0.12the -0.19 ± 0.17 this -0.58,0.20 0.298 (50.33 - 63.59) documentand/or rights (61.56 - 62.00) (61.74to - 62.18)of fall (-0.45 - use0.25) of [39.41,76.74] the 18:3 Linolenic (% Total FA) 1.21This ± 0.026 1.34 ± 0.may026 distribution, -0.13and ± 0.037 -0.21,-0.040 0.009 (0.93 - 1.52) rights owner (1.20 - 1.23)subject (1.25 - 1.43) (-0.23 - -0.022) [0.63,1.77]

be the violate 20:0 Arachidic (% Total FA) 0.37 ± 0.0057 copy 0.37 ± 0.0057 0.0036of ± 0.0081 -0.015,0.022 0.670 (0.32 - 0.47) (0.36 - 0.39) (0.36 - 0.38) (-0.019 - 0.032) [0.23,0.54] document and may publication, exploitation 20:1 Eicosenoic (% Total FA) It 0.27 ± 0.0050 0.27 ± 0.0050 0.0018 ± 0.0064 -0.013,0.016 0.784 (0.23 - 0.32) this (0.26 - 0.28) any (0.25 - 0.28) (-0.014 - 0.011) [0.15,0.39]

permission 22:0 Behenic (% Total FA) 0.15 ± 0.0035 0.14 ± 0.0035prohibited 0.0030 ± 0.0049 -0.0083,0.014 0.562 (0.12 - 0.19) (0.14 - 0.16) the (0.14 -be 0.15) (-0.0071 - 0.017) [0.081,0.23] commercial 1 With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero. Furthermore,any Consequently,without

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Table G.4. Comparison of the fiber and proximate content in grain collected at Site 1 from MON 89034 and conventional corn

Fiber Bayer property protectionpublishing Acid Detergent Fiber (% DW) 5.39 ± 0.30 5.19 ± 0.30 of 0.20 ± 0.42 -0.76,1.17 contents 0.641 (4.11 - 6.33) (4.76 - 5.80) (4.76 - 5.68) (-0.92 - 1.04) [2.77,7.56] its parties.data therefore Neutral Detergent Fiber (% DW) 11.31 ± 0.28 10.68 ± 0.28 0.63 ± 0.38 -0.25,1.52and/or or 0.136 (8.20 - 11.30) affiliates. (10.78 - 12.08) (9.93 - 11.22)property (-0intellectual.44 - 1.20)third may [5.93,13.63] its Total Dietary Fiber (% DW) 15.37 ± 0.46 14.22the ± 0.46 as 1.15 ± 0.65 -0.34,2.65 0.112 (12.99 - 18.03) of and owner. (14.70 - 16.28) (13.62is - 15.25) (-0.55 - 2.50)regulatory [9.20,20.27] a document Proximate any such its reproductiondocument Ash (% DW) 1.48 ± 0.056 1.44 ± 0.056 owner 0.043 ± 0.063 this -0.10,0.19of 0.513 (1.12 - 1.62) (1.38 - 1.56) (1.35 - 1.49) under(0.021 - 0.076)of [0.74,1.96] rights the this documentand/or rights Carbohydrates (% DW) 83.38 ± 0.23 to 84.52 ±of 0.23 fall -1.13 ±use 0.32 of -1.88,-0.39 0.008 (82.91 - 86.78) (83.29 - 83.55) (84.28 - 84.74) (-1.42 - -0.98) the [81.08,88.80] This may distribution,and rights owner Moisture (% FW) 8.06 ± 0.096subject 8.09 ± 0.096 -0.030 ± 0.14 -0.34,0.28 0.830 (7.60 - 15.30) (7.89 - 8.16) (7.86 - 8.21) (-0.080 - 0.030) [0.45,19.52] be the violate copy of Protein (% DW) 11.89 ± 0.19 10.85 ± 0.19 1.04 ± 0.27 0.41,1.67 0.005 (9.33 - 11.82) document and (11.73may - 11.98) (10.70publication, -exploitation 11.00) (0.87 - 1.28) [7.54,13.13] It this Total Fat (% DW) 3.24 ± 0.047 any 3.19 ± 0.047 0.050 ± 0.067 -0.10,0.20 0.479 (2.66 - 3.71) (3.16 - 3.33) (3.13 - 3.24) (-0.0014 - 0.12) [2.20,4.55] permission prohibited 1 With 95% confidence, interval contains 99% of the valuesthe expressedbe in the population of commercial lines. Negative limits were set to zero. commercial Furthermore,any Consequently,without

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Table G.5. Comparison of the mineral content in grain collected at Site 1 from MON 89034 and conventional corn

Difference(Test minus Control) Control Mean ± and Commercial Test Mean ± S.E. S.E. Mean ± S.E. 95%and CI regime. (Range) Analytical Component (Range) (Range) (Range)AG (Lower,Upper) p-Value [99% Tolerance Int.¹]

Mineral Calcium (% DW) 0.0064 ± 0.00014 0.0058 ± 0.00014 0.00063Bayer ± 0.00020 property 0.00018,0.0011 protection publishing 0.012 (0.0031 - 0.0049) (0.0062 - 0.0066) (0.0056 - 0.0059) (0.00043of - 0.00090) contents [0.0016,0.0059] its parties.data therefore Copper (mg/kg DW) 1.89 ± 0.69 2.82 ± 0.69 -0.92 ± 0.98 -3.17,1.33and/or or 0.372 (1.15 - 3.56) (1.86 - 1.95) (1.68 - 4.54) affiliates. (-2.59 - 0.18) [0,4.20] property intellectual third may its Iron (mg/kg DW) 23.54 ± 1.22 25.49 ± 1.00 as -1.96 ± 1.58 -5.69,1.77 0.254 (18.04 - 29.22) the of and (23.02 - 24.06) (24.07is - 27.02) (-4.01 - -1.33)regulatory owner. [8.88,34.51] a document any such its Magnesium (% DW) 0.13 ± 0.0021 0.12 ± 0.0021 0.0036 ± 0.0030reproduction -0.0034,0.011document of 0.269 (0.099 - 0.14) (0.13 - 0.13) (0.12 - 0.13) owner (-0.0012 - 0.0087) this [0.075,0.17] under of this and/orrights the Manganese (mg/kg DW) 8.34 ± 0.34document to 6.99 ± 0.34 fall 1.35 ± 0.45 of rights 0.30,2.40 0.017 (5.56 - 8.64) (7.62 - 9.32) (6.84 - of7.17) (0.78 -use 2.36) [3.17,9.99] the This may distribution,and Phosphorus (% DW) 0.34 ± 0.0049 rights 0.34 ± 0.0049 0.0050 ±owner 0.0070 -0.011,0.021 0.496 (0.25 - 0.37) (0.34 - 0.35)subject (0.33 - 0.35) (-0.0068 - 0.015) [0.18,0.45] be the violate Potassium (% DW) 0.37 ± 0.0060 copy 0.37 ± 0.0060 -0.of00086 ± 0.0084 -0.020,0.019 0.921 (0.32 - 0.40) (0.36 - 0.38) (0.36 - 0.38) (-0.010and - 0.0055) [0.26,0.46] may documentpublication, It exploitation Zinc (mg/kg DW) 26.50 ± 0.58this any 25.46 ± 0.58 1.04 ± 0.82 -0.85,2.93 0.239 (16.72 - 34.04) (25.91 - 26.89) (24.53 - 26.04) (0.098 - 2.18) [7.16,38.55] permission prohibited 1 With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero. the commercial be Furthermore,any Consequently,without

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Table G.6. Comparison of the vitamin content in grain collected at Site 1 from MON 89034 and conventional corn

Difference(Test minus Control) Control Mean ± and Commercial Test Mean ± S.E. S.E. Mean ± S.E. 95% CIand regime. (Range) Analytical Component (Range) (Range) (Range) AG (Lower,Upper) p-Value [99% Tolerance Int.¹]

Vitamin Folic Acid (mg/kg DW) 0.41 ± 0.048 0.51 ± 0.048 -0.10 ±Bayer 0.059 property -0.24,0.034 protection publishing 0.121 (0.13 - 0.45) (0.39 - 0.43) (0.49 - 0.53) (-0.11of - -0.098) contents [0.012,0.69] its parties.data therefore Niacin (mg/kg DW) 28.14 ± 0.74 29.01 ± 0.74 -0.88 ± 1.04 -3.28,1.52and/or or 0.424 (16.17 - 29.19) (26.27 - 30.05) (27.34 - 29.85) affiliates. (-3.57 - 2.71) [6.97,37.83] property intellectual third may its Vitamin B1 (mg/kg DW) 3.05 ± 0.16 3.23 ± 0.16 -0.18as ± 0.21 -0.67,0.30 0.411 (2.19 - 5.60) the of and (2.39 - 3.38) (3.05is - 3.36) (-0.66 - 0.11)regulatory owner. [0.37,6.35] a document any such its Vitamin B2 (mg/kg DW) 1.45 ± 0.11 1.45 ± 0.11 -0.0076 ± 0.15 reproduction -0.36,0.34document of 0.961 (1.34 - 1.91) (1.37 - 1.52) (1.36 - 1.51) owner (-0.12 - 0.16) this [0.91,2.30] under of this and/orrights the Vitamin B6 (mg/kg DW) 6.74 ± 0.11 document 6.73 to ± 0.11 fall 0.016 ± 0.16 of -0.35,0.38rights 0.922 (5.08 - 7.47) (6.49 - 6.99) (6.67 - 6.80)of (-0.31 - 0.32)use [3.12,9.30] the This may distribution,and Vitamin E (mg/kg DW) 6.33 ± 0.71 4.87rights ± 0.71 1.46 ± 1.01owner -0.86,3.78 0.185 (2.71 - 13.94) (5.73 - 6.70) subject (2.72 - 6.00) (-0.27 - 3.83) [0,20.49] be the violate 1 With 95% confidence, interval contains 99% ofcopy the values expressed in theof population of commercial lines. Negative limits were set to zero. and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table G.7. Comparison of the antinutrient and secondary metabolites content in grain collected at Site 1 from MON 89034 and conventional corn

Antinutrient Bayer property protectionpublishing Phytic Acid (% DW) 0.78 ± 0.039 0.75 ± 0.039 0.028of ± 0.055 -0.099,0.15 contents 0.629 (0.50 - 0.94) (0.77 - 0.80) (0.70 - 0.83) (-0.037 - 0.073) its [0.21,1.22] parties.data therefore and/oror Secondary Metabolite affiliates. Ferulic Acid (µg/g DW) 2458.21 ± 53.43 2571.23 ± 53.43property -113.03 intellectual ± 75.57third -287.29,61.23 may 0.173 (1412.68 - 2297.36) (2366.74 - 2525.31) (2472.77 - 2669.85)its (-303.11 - 9.81) [1136.69,2806.24] as the of and is regulatory owner. p-Coumaric Acid (µg/g DW) 172.95 ± 6.96 172.63 ± 6.96 0.32 ± 9.63a -21.89,22document.53 0.974 (99.30 – 285.75) (166.11-177.48) (167.76-176.90)any such(- 10.80 – 9.73) its [0,378.57] reproductiondocument owner this of 1 With 95% confidence, interval contains 99% of the values expressed in theunder popu lationof 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 G.8. Comparison of the fiber, mineral, and proximates content in forage collected at Site 2 from MON 89034 and conventional corn

regime.and and Difference(TestAG minus Control) Control Mean ± Commercial Test Mean ± S.E. S.E. Mean ± S.E. 95% CI (Range) Analytical Component (Range) (Range) (Range)Bayer (Lower,Upper)property publishingp-Value [99% Tolerance Int.¹] protectioncontents Fiber of Acid Detergent Fiber (% DW) 25.53 ± 1.39 27.40 ± 1.39 -1.87 ± 1.97 -6.42,2.68 its 0.371 (26.72 - 38.94) parties.data therefore (24.18 - 26.38) (24.53 - 32.26) (-6.22 - 1.85) and/oror [16.76,43.76]

affiliates. Neutral Detergent Fiber (% DW) 36.81 ± 1.89 36.96 ± 1.89 property -0.15 intellectual± 2.67 third -6.32,6.02 may 0.956 (33.70 - 46.74) its (33.99 - 39.94) (35.78 - 37.65) (-3as.47 - 4.16) [25.94,55.67] the of and is regulatory owner. Mineral a document any such its Calcium (% DW) 0.17 ± 0.0093 0.17 ± 0.0093 0.0044 ± 0.012 reproduction -0.023,0.031document of 0.716 (0.11 - 0.29) (0.16 - 0.18) (0.15 - 0.19) owner(-0.017 - 0.017) this [0.016,0.38] under of this and/orrights the Phosphorus (% DW) 0.24 ± 0.014document 0.20 ± 0.014 fall 0. 041 ± 0.019 of -0.0036,0.085 rights 0.066 (0.14 - 0.25) (0.23 - 0.24) (0.18to - 0.24)of (0.0048 - 0.064)use [0.071,0.32] the This may distribution,and Proximate rights owner Ash (% DW) 3.55 ± 0.30 subject 3.57 ± 0.30 -0.015 ± 0.43 -1.00,0.97 0.972 (3.40 - 5.45) (3.30 - 3.93)be (2.96 - 4.24) (-0.94the - 0.97) violate [1.93,6.31] copy of Carbohydrates (% DW) 86.92 ± 0.87 88.22 ± 0.87 -1.30 ±and 1.23 -4.13,1.53 0.320 (84.88 - 88.39) may documentpublication, (84.98It - 88.60) (85.87 - 89.57)exploitation (-4.23 - 2.73) [83.05,90.74] this Moisture (% FW) 69.03 ± 0.68 any 66.53 ± 0.68 2.50 ± 0.96 0.29,4.71 0.031 (64.90 - 77.40) (68.50 - 69.40) (65.90 - 67.70)permission (1 .70 - 3.30) [57.62,86.45] prohibited the Protein (% DW) 7.53 ± 0.39 commercial 6.63 ± 0.39 be 0.90 ± 0.56 -0.38,2.18 0.144 (6.58 - 8.82) (6.95 - 8.41) (6.06 - 7.52) (-0.30 - 2.35) [4.78,10.38] Furthermore, any Total Fat (% DW) Consequently,2.00 ± 0.45 without 1.58 ± 0.45 0.42 ± 0.64 -1.05,1.88 0.528 (0.58 - 3.11) (0.88 - 3.17) (1.16 - 2.37) (-1.49 - 1.95) [0,4.54] ¹With 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 G.9. Comparison of the amino acid content in grain collected at Site 2 from MON 89034 and conventional corn

Difference(Test minus Control) Control Mean ± Commercial Test Mean ± S.E. S.E. Mean ± S.E. 95% CI and (Range) and regime. Analytical Component (Range) (Range) (Range) AG (Lower,Upper) p-Value [99% Tolerance Int.¹] Amino Acid (% DW) Alanine (% DW) 0.70 ± 0.027 0.71 ± 0.027 -0.0092 ± 0.033 -0.084,0.066 0.785 (0.67 - 0.96) (0.66 - 0.76) (0.67 - 0.78) (-0.012 - -0.0057) [0.48,1.08] Bayer property protectionpublishing of contents Arginine (% DW) 0.45 ± 0.011 0.45 ± 0.011 0.0049 ± 0.015 -0.031,0.040 0.760 (0.37 - 0.49) its (0.43 - 0.47) (0.44 - 0.47) (-0.0068 - 0.015) parties.data therefore [0.33,0.56] and/oror affiliates. Aspartic acid (% DW) 0.62 ± 0.019 0.62 ± 0.019 property 0.0013 intellectual ± 0.023third -0.052,0.054 may 0.956 (0.57 - 0.77) (0.60 - 0.66) (0.60 - 0.67) its (-0.0033 - 0.0070) [0.43,0.90] the as of and owner. Cystine (% DW) 0.22 ± 0.0058 0.22 ± is0.0058 0.0044 ± 0.0069regulatory -0.011,0.020 0.538 (0.20 - 0.24) a document (0.22 - 0.23) (0.21 - 0.23)any such(-0.0022 - 0.014) its [0.18,0.27] reproductiondocument owner this of Glutamic acid (% DW) 1.78 ± 0.073 1.81 ± 0.073 -0.025under ± 0.085 of -0.22,0.17 0.779 (1.71 - 2.41) (1.69 - 1.95) (1.70 - 2.00)rights the (-0.051 - -0.0056) this [1.25,2.75] documentand/or rights to of fall use of Glycine (% DW) 0.37 ± 0.0084 0.37 ± 0.0084 0.0048 ± 0.011 the -0.020,0.030 0.665 (0.32 - 0.40) (0.36 - 0.39)This (0.36 - 0.38) may (0.0023distribution,and - 0.0062) [0.28,0.46] rights owner subject Histidine (% DW) 0.29 ± 0.0082 0.29 ± 0.0082 0.0011 ± 0.0087 -0.019,0.021 0.898 (0.26 - 0.33) be the violate (0.27 - 0.31) copy (0.28 - 0.30) (-0.012of - 0.011) [0.22,0.38]

document and Isoleucine (% DW) 0.32 ± 0.014may 0.32 ±publication, 0.014exploitation 0.00013 ± 0.017 -0.038,0.038 0.993 (0.32 - 0.45) (0.30It - 0.36) (0.30 - 0.36) (-0.0070 - 0.010) [0.23,0.51] this any Leucine (% DW) 1.15 ± 0.055 1.17 ± 0.055 -0.017 ± 0.064 -0.16,0.13 0.803 (1.14 - 1.68) permission (1.09 - 1.27) (1.08 - 1.33) prohibited(-0.052 - 0.0032) [0.77,1.92] ¹With 95% confidence, interval contains 99% of the valuesthe exprebe ssed in the population of commercial lines. Negative limits were set to zero. commercial Furthermore,any Consequently,without

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Table G.9 (cont.). Comparison of the amino acid content in grain collected at Site 2 from MON 89034 and conventional corn

Bayer property protectionpublishing Lysine (% DW) 0.32 ± 0.0090 0.30 ± 0.0090 0.017 ± 0.012of -0.0093,0.044 0.170contents (0.24 - 0.34) (0.29 - 0.33) (0.29 - 0.31) (0.0028 - 0.031) its [0.20,0.40] parties. data therefore Methionine (% DW) 0.22 ± 0.0056 0.22 ± 0.0056 0.0033 ± 0.0079 -0.015,0.021 and/oror 0.682 (0.17 - 0.22) affiliates. (0.21 - 0.23) (0.20 - 0.23) property(-0.0066 - 0.014)intellectual third may [0.14,0.25] its Phenylalanine (% DW) 0.46 ± 0.019 0.46 ± 0.019 the -0.0020 ±as 0.022 -0.052,0.048 0.929 (0.45 - 0.65) of and owner. (0.44 - 0.50) (0.43 - 0.52)is (-0.018 - 0.0087) regulatory [0.32,0.73] a document any such its reproductiondocument Proline (% DW) 0.89 ± 0.031 0.88 ± 0.031 0.0040owner ± 0.037 -0.082,0.090this of 0.917 (0.83 - 1.11) (0.84 - 0.96) (0.84 - 0.95) (-0.0064 -under 0.011) of [0.68,1.21] rightsthe this documentand/or rights Serine (% DW) 0.48 ± 0.014 0.49 ± 0.014to of -0.0096fall ± 0.018 use -0.051,0.032of 0.607 (0.45 - 0.62) (0.46 - 0.50) (0.46 - 0.52) (-0.021 - 0.0088) the [0.34,0.71] This may distribution, and rights owner Threonine (% DW) 0.31 ± 0.0081 0.31subject ± 0.0081 0.0067 ± 0.0091 -0.014,0.028 0.484 (0.29 - 0.37) (0.30 - 0.33) (0.29 - 0.32) (-0.00043 - 0.011) [0.24,0.41] be the violate copy of Tryptophan (% DW) 0.054 ± 0.0024 0.055 ± 0.0024 -0.00092 ± 0.0026and -0.0070,0.0051 0.735 (0.043 - 0.059) (0.053 - 0.056)may (0.052document - 0.061)publication, (-0.0055 - 0.0014) [0.032,0.072] It exploitation this Tyrosine (% DW) 0.34 ± 0.012 0.35 any± 0.012 -0.0023 ± 0.013 -0.033,0.028 0.863 (0.25 - 0.40) (0.33 - 0.36) (0.32 - 0.38) permission(-0.024 - 0.015) [0.17,0.52] prohibited Valine (% DW) 0.45 ± 0.016 0.45 ± 0.016the be 0.0016 ± 0.020 -0.045,0.048 0.938 (0.42 - 0.55) (0.42 - 0.50) (0.43commercial - 0.49) (-0.011 - 0.0084) [0.35,0.62] ¹With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero. Furthermore,any Consequently,without

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 283 of 323

Table G.10. Comparison of the fatty acid content in grain collected at Site 2 from MON 89034 and conventional corn

Difference(Test minus Control) Control Mean ± Commercial and Test Mean ± S.E. S.E. Mean ± S.E. 95% CI and regime. (Range) Analytical Component (Range) (Range) (Range) AG(Lower,Upper) p-Value [99% Tolerance Int.¹]

Fatty Acid (% Total FA) 16:0 Palmitic (% Total FA) 9.21 ± 0.065 9.08 ± 0.065 0.12 ± 0.089 -0.080,0.33 0.199 (9.10 - 12.55) Bayer property protectionpublishing (9.17 - 9.24) (8.91 - 9.23) (-0.013of - 0.33) contents [6.12,15.67] its parties.data therefore 16:1 Palmitoleic (% Total FA) 0.13 ± 0.0022 0.14 ± 0.0022 -0.0093 ± 0.0029 -0.016,-0.0027and/or or 0.012 (0.050 - 0.19) (0.12 - 0.13) (0.13 - 0.14) (-0.011 - -0.0071) [0,0.28] affiliates. property intellectual third may its 18:0 Stearic (% Total FA) 1.96 ± 0.027 1.82 ± 0.027 0.14as ± 0.024 0.088,0.20 <0.001 (1.57 - 2.45) (1.89 - 2.02) (1.76 - 1.85)the of (0.12 - 0.18)and [0.86,2.98] is regulatory owner. a document any such its 18:1 Oleic (% Total FA) 25.30 ± 0.29 25.78 ± 0.29 -0.48 ± 0.32 reproduction -1.21,0.25document 0.168 (21.17 - 35.33) (25.03 - 25.68) (25.34 - 26.66) owner (-0.98 - -0.15) this of [7.51,46.46] under of rightsthe this 18:2 Linoleic (% Total FA) 61.34 ± 0.22 documentand/or 61.14 ± 0.22 0.20 ± 0.27 of -0.42,0.82rights 0.471 (50.33 - 63.59) (61.02 - 61.54) (60.51to - 61.53)of fall (-0 .051 - 0.51)use [39.41,76.74] the This may distribution,and 18:3 Linolenic (% Total FA) 1.22 ± 0.011 1.21rights ± 0.011 0.014 ± 0.016owner -0.022,0.051 0.390 (0.93 - 1.52) (1.21 - 1.23) subject (1.19 - 1.23) (-0.014 - 0.036) [0.63,1.77]

be the violate 20:0 Arachidic (% Total FA) 0.41 ± 0.0055 copy 0.39 ± 0.0055 0.020of ± 0.0057 0.0073,0.034 0.007 (0.32 - 0.47) (0.40 - 0.42) (0.38 - 0.40) (0.017 - and0.023) [0.23,0.54] may documentpublication, It exploitation 20:1 Eicosenoic (% Total FA) 0.29 ± 0.0052 this 0.29 ± 0.0052 -0.0035 ± 0.0073 -0.020,0.013 0.644 (0.23 - 0.32) (0.28 - 0.29) any (0.29 - 0.29) (-0.0040 - -0.0029) [0.15,0.39] permission 22:0 Behenic (% Total FA) 0.15 ± 0.0042 0.16 ± 0.0042 prohibited -0.013 ± 0.0057 -0.026,0 0.050 (0.12 - 0.19) (0.14 - 0.15) (0.15the - 0.16) be (-0.019 - -0.0013) [0.081,0.23] commercial ¹With 95% confidence, interval containsFurthermore, 99% anyof the values expressed in the population of commercial lines. Negative limits were set to zero. Consequently,without

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Table G.11. Comparison of the fiber and proximate content in grain collected at Site 2 from MON 89034 and conventional corn Difference(Test minus Control) Test Mean ± Commercial S.E. Control Mean ± S.E. Mean ± S.E. 95% CI and (Range) and regime. Analytical Component (Range) (Range) (Range) AG(Lower,Upper) p-Value [99% Tolerance Int.¹]

Fiber Acid Detergent Fiber (% DW) 4.96 ± 0.51 5.55 ± 0.51 -0.59 ± 0.71 -2.24,1.06 0.435 (4.11 - 6.33) Bayer property protectionpublishing (3.82 - 6.05) (4.37 - 7.00) (-3.18of - 1.69) contents [2.77,7.56] its Neutral Detergent Fiber (% DW) 10.00 ± 0.53 10.50 ± 0.53 -0.50 ± 0.75 parties. -2.24,1.24data 0.524 therefore (8.20 - 11.30) (9.83 - 10.11) (9.48 - 11.22) (-1.16 - 0.34) and/oror [5.93,13.63] affiliates. property intellectual third may Total Dietary Fiber (% DW) 14.49 ± 0.70 14.93 ± 0.70 its -0.43 ± 0.99 -2.71,1.84 0.671 (12.99 - 18.03) (13.39 - 15.06) (13.17 - 15.84)the (-2as.45 - 1.88) [9.20,20.27] of and owner. is regulatory a document Proximate any such its reproductiondocument Ash (% DW) 1.40 ± 0.038 1.43 ± 0.038 owner -0.025 ± 0.053 this -0.15,0.097 of 0.648 (1.12 - 1.62) (1.35 - 1.47) (1.34 - 1.48) (-0.11under - 0.046) of [0.74,1.96] rightsthe this documentand/or rights Carbohydrates (% DW) 85.63 ± 0.27 85.67 to± 0.27 of fall-0.044 ± 0.27use of -0.68,0.59 0.875 (82.91 - 86.78) (84.90 - 86.11) (84.94 - 86.22) (-0.11 - 0.012) the [81.08,88.80] This may distribution,and rights owner Moisture (% FW) 8.46 ± 0.22 subject 8.43 ± 0.22 0.030 ± 0.31 -0.69,0.75 0.925 (7.60 - 15.30) (8.15 - 9.02) (8.06 - 9.08) (-0.93 - 0.87) [0.45,19.52] be the violate copy of Protein (% DW) 9.67 ± 0.29 9.67 ± 0.29 0.0034 ± 0.23 -0.53,0.53 0.988 (9.33 - 11.82) document and (9.14 - may10.35) (9.22 -publication, 10.50)exploitation (-0.15 - 0.30) [7.54,13.13] It this Total Fat (% DW) 3.30 ± 0.093 3.23any ± 0.093 0.066 ± 0.11 -0.18,0.31 0.553 (2.66 - 3.71) (3.26 - 3.36) (3.09 - 3.45)permission (-0.20 - 0.21) [2.20,4.55] ¹With 95% confidence, interval contains 99% of the values expressed prohibitedin the population of commercial lines. Negative limits were set to zero. the commercial be Furthermore,any Consequently,without

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 285 of 323

Table G.12. Comparison of the mineral content in grain collected at Site 2 from MON 89034 and conventional corn

Difference (Test minus Control) Control Mean ± andCommercial and regime. Test Mean ± S.E. S.E. Mean ± S.E. AG 95% CI (Range) Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value [99% Tolerance Int.¹] Mineral Calcium (% DW) 0.0048 ± 0.00018 0.0048 ± 0.00018 -0.00004 ± 0.00021 -0.00054,0.00045 0.845 (0.0031 - 0.0049) Bayer property protectionpublishing (0.0046 - 0.0049) (0.0046 - 0.0050) (-0.00006 - of-0.00003) contents [0.0016,0.0059] its Copper (mg/kg DW) 1.76 ± 0.11 1.36 ± 0.11 0.40 ± 0.14 0.071,0.73parties.data 0.023 therefore (1.15 - 3.56) (1.51 - 2.21) (1.26 - 1.43) (0.16 - 0.78) and/oror [0,4.20] affiliates. property intellectualthird may Iron (mg/kg DW) 20.86 ± 0.42 19.48 ± 0.42 its 1.38 ± 0.60 0.012,2.76 0.048 (18.04 - 29.22) (19.23 - 21.79) (19.03 - 19.71)the (-0.47as - 2.75) [8.88,34.51] of and owner. is regulatory a document Magnesium (% DW) 0.12 ± 0.0022 0.11 ± 0.0022 any such 0.0044 ± 0.0029 -0.0023,0.011 its 0.170 (0.099 - 0.14) reproductiondocument (0.11 - 0.12) (0.11 - 0.12) (-0.0066owner - 0.011) this of [0.075,0.17] under of Manganese (mg/kg DW) 6.54 ± 0.17 6.19 ± 0.17 rightsthe 0.35 ± 0.24 -0.20,0.89this 0.180 (5.56 - 8.64) documentand/or rights (6.11 - 6.78) (6.03 - 6.47)to of (-0.36fall - 0.71) use of [3.17,9.99] the Phosphorus (% DW) 0.33 ± 0.0062This 0.33 ± 0.0062 may 0.0070 distribution, ± 0.0088and -0.013,0.027 0.449 (0.25 - 0.37) rights owner (0.31 - 0.35) subject (0.32 - 0.35) (-0.031 - 0.026) [0.18,0.45]

be the violate Potassium (% DW) 0.38 ± 0.0084 0.37copy ± 0.0084 0.011 ±of 0.011 -0.014,0.035 0.345 (0.32 - 0.40) (0.37 - 0.40) (0.36 - 0.40) (-0.030 - 0.035)and [0.26,0.46] may document publication, It exploitation Zinc (mg/kg DW) 20.50 ± 0.52 this 19.26 ± 0.52 1.24 ± 0.74 -0.46,2.94 0.131 (16.72 - 34.04) (18.91 - 22.12) (18.81any - 20.03) (-1.13 - 3.19) [7.16,38.55] permission ¹With 95% confidence, interval contains 99% of the values expressed prohibitedin the population of commercial lines. Negative limits were set to zero. the commercial be Furthermore,any Consequently,without

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 286 of 323

Table G.13. Comparison of the vitamin content in grain collected at Site 2 from MON 89034 and conventional corn

Vitamin Folic Acid (mg/kg DW) 0.30 ± 0.017 0.33 ± 0.017 -0.030 ± 0.024 -0.086,0.026 0.249 (0.13 - 0.45) Bayer property protectionpublishing (0.27 - 0.33) (0.30 - 0.36) (-0.058of - 0.0038) contents [0.012,0.69]

its Niacin (mg/kg DW) 32.34 ± 1.44 32.70 ± 1.44 -0.36 ± 2.04 parties. -5.06,4.34data 0.865therefore (16.17 - 29.19) (30.61 - 34.84) (31.03 - 35.75) (-4.17 - 3.81) and/oror [6.97,37.83] affiliates. property intellectual third may Vitamin B1 (mg/kg DW) 3.17 ± 0.18 2.84 ± 0.18 its 0.33 ± 0.26 -0.27,0.93 0.242 (2.19 - 5.60) (3.05 - 3.27) (2.39 - 3.16)the (0.030as - 0.66) [0.37,6.35] of and owner. is regulatory a document Vitamin B2 (mg/kg DW) 1.46 ± 0.069 1.53 ± 0.069any such-0.072 ± 0.098 -0.30,0.15 its 0.484 (1.34 - 1.91) reproductiondocument (1.35 - 1.65) (1.45 - 1.61) owner (-0.22 - 0.20) this of [0.91,2.30] under of Vitamin B6 (mg/kg DW) 6.49 ± 0.14 6.53 ± 0.14rights the -0.042 ± 0.16 this -0.41,0.33 0.800 (5.08 - 7.47) documentand/or rights (6.27 - 6.64) (6.45to - 6.63)of fall (-0.37 - 0.20)use of [3.12,9.30] the Vitamin E (mg/kg DW) 7.16 ±This 0.26 6.96 ± 0.26 may distribution, 0.20and ± 0.35 -0.62,1.01 0.591 (2.71 - 13.94) rights owner (6.47 - 7.91) subject (6.65 - 7.40) (-0.93 - 1.08) [0,20.49]

be the violate ¹With 95% confidence, interval contains 99% ofcopy the values expressed in theof population of commercial lines. Negative limits were set to zero. and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table G.14. Comparison of the antinutrient and secondary metabolites content in grain collected at Site 2 from MON 89034 and conventional corn

Bayer property protectionpublishing Antinutrient of contents Phytic Acid (% DW) 0.86 ± 0.041 0.78 ± 0.041 0.081 ± 0.059 -0.055,0.22 its 0.206 (0.50 - 0.94) parties.data therefore (0.83 - 0.87) (0.69 - 0.85) (-0.013 - 0.18) and/oror [0.21,1.22]

affiliates. Secondary Metabolite property intellectualthird may Ferulic Acid (µg/g DW) 2057.02 ± 106.60 2184.45 ± 106.60 its -127.43 ± 150.76 -475.07,220.22 0.422 (1412.68 - 2297.36) as (1923.50 - 2298.73) (2033.94the - 2265.73) of (-330.17and - 264.79) [1136.69,2806.24] is regulatory owner. a document p-Coumaric Acid (µg/g DW) 196.97 ± 7.65 195.82any ± 7.65 such 1.15 ± 10.81 -23.78,26.08 its 0.917 (99.30 – 285.75) reproductiondocument (185.76 – 214.62) (188.08- 210.13) owner (-24.37 – 25.37) this of [0,378.57] under of rightsthe this ¹With 95% confidence, interval contains 99%document of theand/or values expressed in the population of commercialrights lines. Negative limits were set to zero. 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 G.15. Comparison of the fiber and mineral content in forage collected at Site 3 from MON 89034 and conventional corn

and Difference (Test minusand Control)regime. AG Commercial Test Mean ± Control Mean ± (Range) S.E. S.E. Mean ± S.E. 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range)Bayer property(Lower,Upper) protection publishing p-Value Int.¹] of contents Fiber its parties.data therefore Acid Detergent Fiber (% DW) 29.76 ± 1.78 28.84 ± 1.78 0.92 ± 2.27 -4.32,6.15and/or or 0.696 (26.72 - 38.94) (26.56 - 33.83) (25.00 - 31.08) affiliates. (-3.89 - 3.89) [16.76,43.76] property intellectual third may its Neutral Detergent Fiber (% DW) 39.23 ± 2.36 37.97 ± 2.36 as 1.26 ± 2.13 -3.66,6.19 0.570 (33.70 - 46.74) (34.41 - 42.19) (35.41the - 42.21) of (-1.11and - 5.89) [25.94,55.67] is regulatory owner. a document any such its Mineral reproductiondocument of Calcium (% DW) 0.22 ± 0.016 0.21 ± 0.016 owner 0.011 ± 0.022 this -0.038,0.061 0.615 (0.11 - 0.29) (0.22 - 0.22) (0.19 - 0.23) (-0.014under - 0.034)of [0.016,0.38] rightsthe this and/or document to fall of rights Phosphorus (% DW) 0.22 ± 0.0097 0.20 ± 0.0097of 0.026 ± 0.014use -0.0052,0.058 0.090 (0.14 - 0.25) (0.22 - 0.23) (0.19 - 0.21) (0.016 - 0.033) the [0.071,0.32] This may distribution,and rights owner ¹With 95% confidence, interval contains 99% subjectof the values 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 G.16. Comparison of the proximates content in forage collected at Site 3 from MON 89034 and conventional corn

Difference (Test minus Control) Test Mean ± S.E. Control Mean ± S.E. Mean ± S.E. 95% CI regime.andCommercial (Range) Analytical Component (Range) (Range) (Range) (Lower,Upper)and p-Value [99% Tolerance Int.¹] Proximate AG Ash (% DW) 4.36 ± 0.24 4.13 ± 0.24 0.23 ± 0.34 -0.55,1.01 0.522 (3.40 - 5.45) (3.99 - 4.57) (3.78 - 4.47) (-0.48 - 0.74) [1.93,6.31] Bayer property publishing protection contents Carbohydrates (% DW) 86.08 ± 0.54 86.94 ± 0.54 -0.85of ± 0.76 -2.60,0.89 0.292 (84.88 - 88.39) its (84.93 - 86.69) (86.69 - 87.13) (-2.05 - -0.060) parties.data therefore [83.05,90.74] and/oror Moisture (% FW) 74.03 ± 0.82 75.13 ± 0.82 affiliates. -1.10 ± 0.99 -3.38,1.18 0.297 (64.90 - 77.40) property intellectualthird may (73.00 - 74.70) (73.00 - 76.80) its (-2.40 - 0) [57.62,86.45] the as of and owner. Protein (% DW) 8.15 ± 0.36 7.85is ± 0.36 0.30 ± 0.51 regulatory -0.88,1.48 0.574 (6.58 - 8.82) a document (7.59 - 8.75) (7.54 - 8.07)any such (-0.33 - 1.21) its [4.78,10.38] reproductiondocument owner this of Total Fat (% DW) 1.41 ± 0.19 1.08 ± 0.19 0.under32 ± 0.27 of this -0.30,0.95 0.262 (0.58 - 3.11) (1.20 - 1.75) and/or (0.77 - rights1.31) the (0.037 - 0.51) [0,4.54] document fall of rights ¹With 95% confidence, interval contains 99% of the values expreto ssedof in the populationuse of commercial 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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Table G.17. Comparison of the amino acid content in grain collected at Site 3 from MON 89034 and conventional corn

Difference (Test minus Control) Commercial Control Mean ± and (Range) and regime. Test Mean ± S.E. S.E. Mean ± S.E. AG 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Amino Acid (% DW) Alanine (% DW) 0.69 ± 0.020 0.71 ± 0.020 -0.028 ± 0.025 -0.085,0.028 0.282 (0.67 - 0.96) Bayer property protectionpublishing (0.67 - 0.71) (0.67 - 0.74) (-0.070of - 0.0025) contents [0.48,1.08]

its Arginine (% DW) 0.47 ± 0.012 0.45 ± 0.012 0.017 ± 0.017 parties. -0.022,0.057data 0.342therefore (0.37 - 0.49) (0.44 - 0.49) (0.41 - 0.47) (-0.023 - 0.056) and/oror [0.33,0.56] affiliates. property intellectual third may Aspartic acid (% DW) 0.63 ± 0.014 0.64 ± 0.014 its-0.0031 ± 0.019 -0.047,0.041 0.875 (0.57 - 0.77) (0.60 - 0.66) (0.61 - 0.66)the (-0.040as - 0.028) [0.43,0.90] of and owner. is regulatory a document Cystine (% DW) 0.21 ± 0.0055 0.22 ± 0.0055any such -0.0091 ± 0.0078 -0.027,0.0088 its 0.274 (0.20 - 0.24) reproductiondocument (0.20 - 0.21) (0.21 - 0.23) (-0.022owner - 0.0052) this of [0.18,0.27] under of Glutamic acid (% DW) 1.77 ± 0.053 1.84 ± 0.053rights the -0.077 ± 0.068 this -0.23,0.079 0.286 (1.71 - 2.41) documentand/or rights (1.71 - 1.82) (1.73 to- 1.90) of fall (-0.19 - 0.040)use of [1.25,2.75] the Glycine (% DW) 0.38 ± 0.0066This 0.37 ± 0.0066 may 0.0089distribution, and± 0.0094 -0.013,0.031 0.372 (0.32 - 0.40) rights owner (0.36 - 0.39) subject (0.36 - 0.38) (-0.0046 - 0.017) [0.28,0.46]

be the violate Histidine (% DW) 0.29 ± 0.0061 copy 0.29 ± 0.0061 0.0017of ± 0.0083 -0.017,0.021 0.841 (0.26 - 0.33) (0.28 - 0.30) (0.28 - 0.30) (-0.0026 - 0.0066) [0.22,0.38] document and may publication, exploitation Isoleucine (% DW) 0.34 ±It 0.0097 0.34 ± 0.0097 -0.0078 ± 0.014 -0.039,0.024 0.587 (0.32 - 0.45) this (0.32 - 0.34) any (0.33 - 0.36) (-0.018 - 0.012) [0.23,0.51]

permission Leucine (% DW) 1.15 ± 0.041 1.21 ± 0.041 prohibited -0.064 ± 0.051 -0.18,0.053 0.243 (1.14 - 1.68) (1.12 - 1.18) (1.13the - 1.26) be (-0.13 - 0.015) [0.77,1.92] commercial ¹With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero. Furthermore,any Consequently,without

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Table G.17 (cont.). Comparison of the amino acid content in grain collected at Site 3 from MON 89034 and conventional corn

Difference (Test minus Control) Commercial Control Mean ± regime.and (Range) Test Mean ± S.E. S.E. Mean ± S.E. 95% CIand [99% Tolerance Analytical Component (Range) (Range) (Range) AG (Lower,Upper) p-Value Int.] Amino Acid (% DW) Lysine (% DW) 0.33 ± 0.0098 0.31 ± 0.0098 0.021 ± 0.014 -0.011,0.053 0.163 (0.24 - 0.34) Bayer property protectionpublishing (0.31 - 0.35) (0.29 - 0.32) (0.017of - 0.023) contents [0.20,0.40]

its Methionine (% DW) 0.20 ± 0.0056 0.21 ± 0.0056 -0.012 ± 0.0071 parties. -0.029,0.0039data 0.117therefore (0.17 - 0.22) (0.20 - 0.20) (0.20 - 0.22) (-0.017 - -0.0048) and/oror [0.14,0.25] affiliates. property intellectualthird may Phenylalanine (% DW) 0.46 ± 0.015 0.48 ± 0.015 its -0.016 ± 0.019 -0.060,0.027 0.414 (0.45 - 0.65) as (0.45 - 0.48) (0.45 - 0.50)the of (-0.039 -and 0.016) [0.32,0.73] is regulatory owner. a document Proline (% DW) 0.88 ± 0.020 0.88 ± 0.020any such 0.00035 ± 0.025 -0.057,0.057 its 0.989 (0.83 - 1.11) reproductiondocument (0.87 - 0.91) (0.83 - 0.91) owner(-0.035 - 0.035) this of [0.68,1.21] under of Serine (% DW) 0.46 ± 0.011 0.48 ± 0.011rights the -0.019 ± 0.014 -0.051,0.014this 0.227 (0.45 - 0.62) documentand/or rights (0.45 - 0.49) (0.46 to- 0.50) of fall (-0.053 - 0)use of [0.34,0.71] the Threonine (% DW) 0.31 ± 0.0085This 0.31 ± 0.0085 may 0.distribution,00064and ± 0.010 -0.023,0.024 0.951 (0.29 - 0.37) rights owner (0.30 - 0.32) subject (0.30 - 0.32) (-0.015 - 0.013) [0.24,0.41]

the violate Tryptophan (% DW) 0.050 ± 0.0025 be copy 0.050 ± 0.0025 -0.00022 ± 0.0032 -0.0075,0.0071 0.946 (0.043 - 0.059) (0.048 - 0.052) (0.045 - 0.054) (-0.0051of - 0.0068) [0.032,0.072] document and may publication, exploitation Tyrosine (% DW) 0.36 It± 0.024 0.32 ± 0.024 0.034 ± 0.021 -0.015,0.082 0.150 (0.25 - 0.40) (0.35 - 0.36) this any (0.24 - 0.38) (-0.014 - 0.11) [0.17,0.52]

permission Valine (% DW) 0.46 ± 0.011 0.46 ± 0.011 prohibited -0.00051 ± 0.016 -0.036,0.035 0.974 (0.42 - 0.55) (0.45 - 0.48) (0.45the - 0.48) be (-0.019 - 0.021) [0.35,0.62] commercial ¹With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero. Furthermore,any Consequently,without

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Table G.18. Comparison of the fatty acid content in grain collected at Site 3 from MON 89034 and conventional corn

Difference(Test minus Control) and and regime. Commercial Control Mean ± AG (Range) Test Mean ± S.E. S.E. Mean ± S.E. 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Fatty Acid (% Total FA) Bayer property protectionpublishing 16:0 Palmitic (% Total FA) 9.29 ± 0.069 9.10 ± 0.069 of 0.19 ± 0.092 -0.019,0.41 contents 0.068 (9.10 - 12.55) (9.12 - 9.46) (9.06 - 9.16) (0.050 - 0.30) its [6.12,15.67] parties.data therefore and/oror 16:1 Palmitoleic (% Total FA) 0.12 ± 0.0018 0.13 ± 0.0018 affiliates. -0.0055 ± 0.0026 -0.011,0.00046 may 0.066 (0.050 - 0.19) (0.12 - 0.13) (0.12 - 0.13)property (-0.012 intellectual - -0.0013)third [0,0.28] its as the of and 18:0 Stearic (% Total FA) 1.98 ± 0.024 1.82is ± 0.024 0.16 ± 0.032regulatory 0.090,0.24 owner. <0.001 (1.57 - 2.45) (1.93 - 2.03) (1.79 - 1.85) (0.13 - a0.18) document [0.86,2.98] any such its reproductiondocument of 18:1 Oleic (% Total FA) 24.75 ± 0.18 23.82 ± 0.18 owner 0.94 ± 0.23 this 0.40,1.47 0.003 (21.17 - 35.33) (24.14 - 25.25) (23.62 - 24.11) under (0.52 - 1.15) of this [7.51,46.46] and/orrights the document to of fall of rights 18:2 Linoleic (% Total FA) 61.87 ± 0.23 63.17 ± 0.23 -1.30 ±use 0.28 -1.95,-0.66 0.001 (50.33 - 63.59) the (61.19This - 62.42) (62.80 - 63.41)may distribution, (-1and.62 - -1.00) [39.41,76.74] rights owner 18:3 Linolenic (% Total FA) 1.17 ± 0.014subject 1.18 ± 0.014 -0.013 ± 0.018 -0.054,0.029 0.505 (0.93 - 1.52) (1.12 - 1.22)be (1.15 - 1.21) (-0.033the - 0.024)violate [0.63,1.77] copy of 20:0 Arachidic (% Total FA) 0.39 ± 0.0063 0.37 ± 0.0063 0.025and ± 0.0088 0.0047,0.045 0.021 (0.32 - 0.47) may documentpublication, It(0.38 - 0.40) (0.36 - exploitation0.37) (0.015 - 0.032) [0.23,0.54] this any 20:1 Eicosenoic (% Total FA) 0.28 ± 0.0034 0.27 ± 0.0034 0.0022 ± 0.0046 -0.0083,0.013 0.644 (0.23 - 0.32) (0.27 - 0.28) (0.27permission - 0.28) (-0.0050 - 0.0073) [0.15,0.39] prohibited

the 22:0 Behenic (% Total FA) 0.14 ± 0.0053commercial 0.14 ± 0.be0053 0.0017 ± 0.0075 -0.016,0.019 0.830 (0.12 - 0.19) (0.13 - 0.15) (0.14 - 0.15) (-0.012 - 0.011) [0.081,0.23] Furthermore, any ¹With 95% confidence, interval Consequently,contains 99%without of the values expressed in the population of commercial lines. Negative limits were set to zero.

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Table G.19. Comparison of the fiber and proximates content in grain collected at Site 3 from MON 89034 and conventional corn

and and regime. AG Difference (Test minus Control) Commercial Bayer property publishing Control Mean ± protectioncontents (Range) Test Mean ± S.E. S.E. Meanof ± S.E. 95% CI [99% Tolerance its Analytical Component (Range) (Range) (Range) parties.(Lower,Upper)data p-Valuetherefore Int.¹] Fiber and/oror Acid Detergent Fiber (% DW) 5.53 ± 0.37 4.95 ± 0.37 affiliates. 0.58 ± 0.50 -0.57,1.73 0.277 (4.11 - 6.33) property intellectualthird may (4.53 - 6.10) (4.54 - 5.46) its (-0.32 - 1.42) [2.77,7.56] the as of and owner. Neutral Detergent Fiber (% DW) 8.98 ± 0.84 9.63is ± 0.84 -0.65 ± 1.19regulatory -3.40,2.10 0.601 (8.20 - 11.30) a document (8.59 - 9.49) (8.48 - 11.75)any such (-2.26 - 0.20) its [5.93,13.63] reproductiondocument owner this of Total Dietary Fiber (% DW) 15.11 ± 0.90 14.75 ± 0.90 0.36under ± 1.25 of -2.53,3.24 0.782 (12.99 - 18.03) rightsthe this (14.02 - 17.02)document and/or (12.82 - 17.62) (-3.61 - 4.20) of rights [9.20,20.27] to of fall use Proximate the This may distribution,and Ash (% DW) 1.38 ± 0.092 1.31rights ± 0.092 0.069 ± 0.13owner -0.23,0.37 0.607 (1.12 - 1.62) (1.35 - 1.44) subject (1.28 - 1.35) (0.0050 - 0.13) [0.74,1.96] the violate be copy Carbohydrates (% DW) 85.86 ± 0.31 85.68 ± 0.31 0.18of ± 0.44 -0.85,1.20 0.699 (82.91 - 86.78) (85.08 - 86.52) (85.53 - 85.84) (-0.44and - 0.84) [81.08,88.80] may documentpublication, It exploitation Moisture (% FW) 9.79 ± 0.16 this any 9.60 ± 0.16 0.19 ± 0.23 -0.34,0.72 0.441 (7.60 - 15.30) (9.51 - 10.10) (9.51 - 9.77) (0 - 0.33) [0.45,19.52] permission prohibited Protein (% DW) 9.11 ± 0.27 9.58the ± 0.27 -0.47 ± 0.38 -1.34,0.41 0.252 (9.33 - 11.82) (8.54 - 9.67) commercial (9.38 - 9.80) be (-1.26 - 0.29) [7.54,13.13]

Total Fat (% DW) Furthermore,3.65 ± 0.099any 3.43 ± 0.099 0.22 ± 0.13 -0.079,0.52 0.127 (2.66 - 3.71) Consequently,(3.50 - 3.89) without (3.22 - 3.75) (0.15 - 0.29) [2.20,4.55]

¹With 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 G.20. Comparison of the mineral content in grain collected at Site 3 from MON 89034 and conventional corn

and and regime. Difference(TestAG minus Control) Commercial Control Mean ± (Range) Test Mean ± S.E. S.E. Mean ± S.E. 95% CI [99% Tolerance Bayer property protectionpublishing Analytical Component (Range) (Range) of (Range) (Lower,Upper) contentsp-Value Int.¹] its parties.data therefore Mineral and/oror Calcium (% DW) 0.0040 ± 0.00009 0.0041 ± 0.00009 affiliates. -0.00014 ± 0.00011 -0.00039,0.00010 0.216 (0.0031 - 0.0049) (0.0038 - 0.0042) (0.0040 - 0.0044)property (-0.00027 intellectual - -0.00001)third may [0.0016,0.0059] its as Copper (mg/kg DW) 1.61 ± 0.57 1.81the ± 0.57of -0and.19 ± 0.81 -2.06,1.68 0.818 (1.15 - 3.56) is regulatory owner. (1.50 - 1.72) (1.61 - 1.93) (-0.43a - 0.10) document [0,4.20] any such its reproductiondocument of Iron (mg/kg DW) 19.62 ± 0.27 20.28 ± 0.27 owner -0. 66 ± 0.39 this -1.55,0.23 0.126 (18.04 - 29.22) under of (19.23 - 19.91) (19.34rights - 20.89)the (-1.66 - 0.38) this [8.88,34.51] documentand/or rights to of fall use of Magnesium (% DW) 0.11 ± 0.0026 0.11 ± 0.0026 -0.00043 ± 0.0027 -0.0067,0.0058 0.879 (0.099 - 0.14) the (0.11This - 0.12) (0.11 - 0.12)may distribution, (-0.0055and - 0.0051) [0.075,0.17] rights owner subject Manganese (mg/kg DW) 5.65 ± 0.14 5.74 ± 0.14 -0.084the ± 0.13 -0.39,0.22 0.540 (5.56 - 8.64) (5.43 -be 5.80) (5.57 - 5.85) (-0.36 - violate0.15) [3.17,9.99] copy of and Phosphorus (% DW) 0.32may ± 0.0077 documentpublication, 0.32 ± 0.0077 0.0021 ± 0.0097 -0.020,0.025 0.831 (0.25 - 0.37) It exploitation (0.31 - 0.32)this (0.30 - 0.34) (-0.015 - 0.013) [0.18,0.45] any Potassium (% DW) 0.37 ± 0.0075 0.36permission ± 0.0075 0.011 ± 0.011 -0.014,0.035 0.342 (0.32 - 0.40) (0.37 - 0.38) (0.35 - 0.38)prohibited (-0.0066 - 0.022) [0.26,0.46] the commercial be Zinc (mg/kg DW) 19.73 ± 0.33 20.13 ± 0.33 -0.40 ± 0.44 -1.42,0.61 0.387 (16.72 - 34.04) Furthermore,(19.35 any- 20.00) (19.39 - 20.67) (-0.66 - -0.040) [7.16,38.55] Consequently, without ¹With 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 G.21. Comparison of the vitamin content in grain collected at Site 3 from MON 89034 and conventional corn

Difference(Test minus Control) and and regime. Commercial Control Mean ± AG (Range) Test Mean ± S.E. S.E. Mean ± S.E. 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Vitamin Bayer property protectionpublishing Folic Acid (mg/kg DW) 0.37 ± 0.0059 0.32 ± 0.0059 0.045of ± 0.0084 0.025,0.064 contents <0.001 (0.13 - 0.45) (0.35 - 0.38) (0.32 - 0.33) (0.028 - 0.057) its [0.012,0.69] parties.data therefore and/oror Niacin (mg/kg DW) 31.66 ± 1.26 30.16 ± 1.26 affiliates. 1.49 ± 1.78 -2.62,5.61 may 0.426 (16.17 - 29.19) (27.14 - 34.70) (29.06 - 31.59)property (-4 .44intellectual - 5.64)third [6.97,37.83] its as the of and Vitamin B1 (mg/kg DW) 2.81 ± 0.23 2.65is ± 0.23 0.15 ± 0.33 regulatory -0.63,0.94 owner. 0.657 (2.19 - 5.60) (2.66 - 2.98) (2.55 - 2.76) (-0.10 - 0.33)a document [0.37,6.35] any such its reproductiondocument of Vitamin B2 (mg/kg DW) 1.43 ± 0.060 1.28 ± 0.060 owner 0.15 ± 0.085 this -0.046,0.35 0.115 (1.34 - 1.91) (1.38 - 1.51) (1.20 - 1.39) (0.0051under - 0.27) of this [0.91,2.30] and/orrights the document to of fall of rights Vitamin B6 (mg/kg DW) 5.82 ± 0.17 5.89 ± 0.17 -0.070 ± 0.25use -0.64,0.50 0.784 (5.08 - 7.47) the (5.65 -This 6.13) (5.67 - 6.07)may distribution, (-0.38and - 0.46) [3.12,9.30] rights owner Vitamin E (mg/kg DW) 6.66 ± 0.18 subject 6.38 ± 0.18 0.28 ± 0.19 -0.16,0.72 0.182 (2.71 - 13.94) (6.43 - 6.98)be (6.05 - 6.82) (-0.39the - 0.71) violate [0,20.49] copy of and ¹With 95% confidence, interval containsmay 99% of thedocument valuespublication, expre ssed 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 G.22. Comparison of the antinutrient and secondary metabolite content in grain collected at the Site 3 from MON 89034 and conventional corn

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Table G.23. Comparison of the fiber and mineral content in forage collected at Site 4 from MON 89034 and conventional corn

Difference(Test minus Control) Commercial

Control Mean ± and (Range) Test Mean ± S.E. S.E. Mean ± S.E. 95% CI and regime. [99% Tolerance Analytical Component (Range) (Range) (Range) AG(Lower,Upper) p-Value Int.¹] Fiber Acid Detergent Fiber (% DW) 24.19 ± 1.74 24.29 ± 1.74 -0.11 ± 2.47 -5.80,5.58 0.965 (26.72 - 38.94) (22.60 - 27.08) (19.93 - 26.90) (-4.03 -Bayer 7.15) property protectionpublishing [16.76,43.76] of contents Neutral Detergent Fiber (% DW) 37.93 ± 2.41 32.96 ± 2.41 4.97 ± 3.41 -2.89,12.84 its 0.182 (33.70 - 46.74) parties.data therefore (35.64 - 39.24) (31.44 - 34.62) (2.83 - 7.47) and/oror [25.94,55.67] affiliates. Mineral property intellectualthird may its Calcium (% DW) 0.17 ± 0.0084 0.16 ± 0.0084 0.010as ± 0.012 -0.017,0.038 0.415 (0.11 - 0.29) the of and (0.16 - 0.18) (0.13is - 0.17) (-0.017 - 0.049)regulatory owner. [0.016,0.38] a document any such its Phosphorus (% DW) 0.25 ± 0.023 0.17 ± 0.023 0.080 ± 0.032 reproduction 0.0064,0.15document of 0.036 (0.14 - 0.25) (0.23 - 0.28) (0.15 - 0.21) owner(0.024 - 0.13) this [0.071,0.32] under of rightsthe this ¹With 95% confidence, interval contains 99% ofdocument the valuesand/or expre ssed in the population of commercialof rights lines. Negative limits were set to zero. 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

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Table G.24. Comparison of the proximates content in forage collected at Site 4 from MON 89034 and conventional corn

Difference (Test minus Control) Commercial Control Mean ± and (Range) Test Mean ± S.E. S.E. Mean ± S.E. 95%and CI regime. [99% Tolerance Analytical Component (Range) (Range) (Range)AG (Lower,Upper) p-Value Int.¹] Proximate Ash (% DW) 3.20 ± 0.29 4.39 ± 0.29 -1.19 ± 0.42 -2.15,-0.23 0.021 (3.40 - 5.45) (2.93 - 3.38) (3.30 - 5.10) (-1Bayer.72 - -0.37) property protectionpublishing [1.93,6.31] of contents Carbohydrates (% DW) 88.16 ± 0.65 84.98 ± 0.65 3.18 ± 0.82 1.29,5.07 its 0.004 (84.88 - 88.39) parties.data therefore (86.86 - 88.84) (84.36 - 85.29) (1.57 - 4.41) and/or or [83.05,90.74] affiliates. may Moisture (% FW) 71.73 ± 1.01 72.23 ± property1.01 -0intellectual.50 ± 1.31third -3.51,2.51 0.711 (64.90 - 77.40) its (69.70 - 74.30) (70.10 - 74.70) as (-3.50 - 4.20) [57.62,86.45] the of and is regulatory owner. Protein (% DW) 7.03 ± 0.38 8.02 ± 0.38 -0.99 a± 0.54 document -2.23,0.25 0.104 (6.58 - 8.82) any such its (6.34 - 7.52) (7.63 – 8.66) (-2.32 - -0.23)reproduction document of [4.78,10.38] owner this Total Fat (% DW) 1.61 ± 0.43 2.62 ± 0.43 under -1.00 ± 0.46of this -2.05,0.049 0.059 (0.58 - 3.11) and/orrights the (0.63 - document2.33) to (2.18 -of 2.91) fall (-2.28 - 0.15) of rights [0,4.54] use the ¹With 95% confidence, interval containsThis 99% of the values expremayssed indistribution, the populationand of commercial lines. Negative limits were set to zero. 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 G.25. Comparison of the amino acid content in grain collected at the Site 4 from MON 89034 and conventional corn

and Difference(Test minusand Control) regime. AG Commercial Control Mean ± (Range) Test Mean ± S.E. S.E. Mean ± S.E. 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Bayer property protectionpublishing Amino Acid (% DW) of contents Alanine (% DW) 0.73 ± 0.023 0.77 ± 0.023 -0.043 ± 0.033 -0.12,0.032 its 0.223 (0.67 - 0.96) parties.data therefore (0.64 - 0.78) (0.76 - 0.78) (-0.13 - 0.0071) and/oror [0.48,1.08]

affiliates. Arginine (% DW) 0.44 ± 0.018 0.47 ± 0.018 property -0.028 intellectual± 0.025third -0.085,0.029 may 0.289 (0.37 - 0.49) its (0.38 - 0.48) (0.46 - 0.48) (-0.090as - 0.010) [0.33,0.56] the of and is regulatory owner. Aspartic acid (% DW) 0.63 ± 0.020 0.67 ± 0.020 -0.041 ± 0.028a -0.11,0.024document 0.182 (0.57 - 0.77) any such its (0.56 - 0.67) (0.66 - 0.68) (-0.11 - -0.0031) reproductiondocument [0.43,0.90] owner this of Cystine (% DW) 0.24 ± 0.0042 0.24 ± 0.0042 0.00021under ± 0.0060 of -0.014,0.014 0.973 (0.20 - 0.24) rightsthe this (0.23 - 0.25) document (0.23and/or - 0.25) (-0.012 - 0.012) of rights [0.18,0.27] to of fall use Glutamic acid (% DW) 1.86 ± 0.062 1.99 ± 0.062 -0.12 ± 0.088 the -0.33,0.080 0.200 (1.71 - 2.41) This may distribution,and (1.63 - 2.01) (1.96rights - 2.00) (-0.33 - 0.011)owner [1.25,2.75] subject Glycine (% DW) 0.36 ± 0.012 be 0.38 ± 0.012 -0.023 the± 0.017 violate -0.061,0.016 0.212 (0.32 - 0.40) (0.32 - 0.39) copy (0.38 - 0.39) (-0.067of - 0.00013) [0.28,0.46] and may documentpublication, Histidine (% DW) 0.29 ±It 0.0094 0.31 ± 0.0094exploitation -0.016 ± 0.013 -0.046,0.015 0.266 (0.26 - 0.33) (0.25 - 0.32) this (0.30 - 0.31) (-0.050 - 0.0033) [0.22,0.38] any Isoleucine (% DW) 0.34 ± 0.012 0.36 ± 0.012permission -0.019 ± 0.017 -0.057,0.020 0.294 (0.32 - 0.45) (0.30 - 0.37) (0.36 - 0.36) prohibited(-0.056 - 0.0027) [0.23,0.51] the commercial be Leucine (% DW) 1.25 ± 0.040 1.33 ± 0.040 -0.080 ± 0.057 -0.21,0.051 0.195 (1.14 - 1.68) Furthermore,(1.09 - 1.33) any (1.30 - 1.35) (-0.21 - -0.015) [0.77,1.92] Consequently,without ¹With 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 G.25 (cont.). Comparison of the amino acid content in grain collected at the Site 4 from MON 89034 and conventional corn

Difference (Test minus Control) and and regime. Commercial Control Mean ± AG (Range) Test Mean ± S.E. S.E. Mean ± S.E. 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Amino Acid (% DW) Bayer property protectionpublishing Lysine (% DW) 0.30 ± 0.013 0.31 ± 0.013 -0.018of ± 0.018 -0.060,0.024 contents0.349 (0.24 - 0.34) (0.26 - 0.32) (0.31 - 0.32) (-0.056 - 0.0089) its [0.20,0.40] parties.data therefore and/oror Methionine (% DW) 0.23 ± 0.0038 0.23 ± 0.0038 -0.0036 ± 0.0054 -0.016,0.0090 0.528 (0.17 - 0.22) affiliates. (0.22 - 0.24) (0.22 - 0.24) property(-0.014 intellectual- 0.0072)third may [0.14,0.25] its as Phenylalanine (% DW) 0.49 ± 0.016 0.52 ± 0.016the of -0.030 ±and 0.023 -0.083,0.023 0.231 (0.45 - 0.65) is regulatory owner. (0.43 - 0.52) (0.51 - 0.53) (-0.080 - -0.0037)a document [0.32,0.73] any such its reproductiondocument Proline (% DW) 0.90 ± 0.028 0.94 ± 0.028 owner -0.043 ± 0.040 this -0.14,0.049 of 0.314 (0.83 - 1.11) (0.79 - 0.97) (0.93 - 0.96) (-0.15under - 0.012) of [0.68,1.21] rightsthe this documentand/or of rights Serine (% DW) 0.49 ± 0.014 0.52 ±to 0.014 of fall -0 .031 ± 0.020use -0.077,0.015 0.160 (0.45 - 0.62) (0.44 - 0.54) (0.52 - 0.53) (-0.087 - 0.0053) the [0.34,0.71] This may distribution,and rights owner Threonine (% DW) 0.31 ± 0.011 subject 0.34 ± 0.011 -0.024 ± 0.015 -0.058,0.011 0.155 (0.29 - 0.37) (0.27 - 0.34) (0.33 - 0.35) (-0.052 - -0.00037) [0.24,0.41] be the violate copy of Tryptophan (% DW) 0.054 ± 0.0020 0.055document ± 0.0020 -0.00057 ±and 0.0029 -0.0072,0.0060 0.846 (0.043 - 0.059) (0.051 - may0.056) (0.052 publication,- 0.057) (-0 .0032 - 0.0035) [0.032,0.072] It exploitation this Tyrosine (% DW) 0.36 ± 0.011 any 0.39 ± 0.011 -0.029 ± 0.016 -0.065,0.0073 0.103 (0.25 - 0.40) (0.32 - 0.38) (0.38 - 0.40)permission (-0.066 - -0.0014) [0.17,0.52] prohibited Valine (% DW) 0.46 ± 0.016 0.48 the± 0.016 be -0.026 ± 0.022 -0.078,0.026 0.277 (0.42 - 0.55) (0.40 - 0.49) commercial (0.48 - 0.49) (-0.084 - 0.0093) [0.35,0.62] Furthermore,any ¹With 95% confidence, interval Consequently,contains 99%without of the values expressed in the population of commercial lines. Negative limits were set to zero.

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Table G.26. Comparison of the fatty acid content in grain collected at the Site 4 from MON 89034 and conventional corn

Difference (Test minus Control) Commercial Control Mean ± and (Range) and regime. Test Mean ± S.E. S.E. Mean ± S.E.AG 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Fatty Acid (% Total FA) 16:0 Palmitic (% Total FA) 9.26 ± 0.050 9.23 ± 0.050 0.037 ± 0.071 -0.13,0.20 0.619 (9.10 - 12.55) Bayer property protectionpublishing (9.14 - 9.35) (9.19 - 9.29) (-0of .051 - 0.14) contents [6.12,15.67]

its 16:1 Palmitoleic (% Total FA) 0.14 ± 0.010 0.14 ± 0.010 0.00086 ± 0.015 parties. -0.033,0.035data therefore 0.954 (0.050 - 0.19) (0.13 - 0.14) (0.13 - 0.14) (-0.00036 - 0.0019) and/oror [0,0.28] affiliates. property intellectual third may 18:0 Stearic (% Total FA) 1.88 ± 0.019 1.85 ± 0.019 its 0.029 ± 0.027 -0.033,0.090 0.318 (1.57 - 2.45) (1.86 - 1.89) (1.82the - 1.87) as(0.021 - 0.034) [0.86,2.98] of and owner. is regulatory a document 18:1 Oleic (% Total FA) 25.60 ± 0.23 25.59any ± 0. 23such 0.0060 ± 0.28 -0.63,0.64 its 0.983 (21.17 - 35.33) reproductiondocument (25.42 - 25.75) (24.96 - 25.98) owner (-0 .41 - 0.66) this of [7.51,46.46] under of 18:2 Linoleic (% Total FA) 61.12 ± 0.24 61.19rights ± 0.24the -0.071 ± 0.32 this -0.82,0.67 0.830 (50.33 - 63.59) documentand/or rights (60.85 - 61.27) (60.77to - 61.91)of fall (-0.64 - use0.35) of [39.41,76.74] the 18:3 Linolenic (% Total FA) 1.18This ± 0.020 1.19 ± 0.may020 distribution, -0.0096and ± 0.022 -0.059,0.040 0.669 (0.93 - 1.52) rights owner (1.17 - 1.18)subject (1.16 - 1.20) (-0.031 - 0.019) [0.63,1.77]

be the violate 20:0 Arachidic (% Total FA) 0.38 ± 0.0020 copy 0.38 ± 0.0020 0.0039of ± 0.0023 -0.0014,0.0091 0.130 (0.32 - 0.47) (0.38 - 0.39) (0.38 - 0.39) (0.00019 - 0.0078) [0.23,0.54] document and may publication, exploitation 20:1 Eicosenoic (% Total FA) It 0.28 ± 0.0012 0.29 ± 0.0012 -0.0043 ± 0.0016 -0.0081,-0.00052 0.030 (0.23 - 0.32) this (0.28 - 0.28) any (0.28 - 0.29) (-0.0071 - -0.0018) [0.15,0.39]

permission 22:0 Behenic (% Total FA) 0.17 ± 0.016 0.16 ± 0.016prohibited 0.0092 ± 0.018 -0.031,0.050 0.612 (0.12 - 0.19) (0.15 - 0.18) the (0.13 -be 0.18) (-0.0098 - 0.022) [0.081,0.23] commercial ¹With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero. Furthermore,any Consequently,without

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Table G.27. Comparison of the fiber and proximates content in grain collected at the Site 4 from MON 89034 and conventional corn

Difference (Test minus Control) and and regime. Commercial Control Mean ± AG (Range) Test Mean ± S.E. S.E. Mean ± S.E. 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Fiber Bayer property protectionpublishing Acid Detergent Fiber (% DW) 5.55 ± 0.41 5.41 ± 0.41 of 0.14 ± 0.58 -1.19,1.47 contents 0.817 (4.11 - 6.33) (5.06 - 5.94) (5.28 - 5.49) (-0.43 - 0.49) its [2.77,7.56] parties.data therefore and/or or Neutral Detergent Fiber (% DW) 10.52 ± 0.42 9.05 ± 0.42 1.47 ± 0.55 0.20,2.75 0.028 (8.20 - 11.30) affiliates. (10.43 - 10.69) (8.64 - 9.38)property (1intellectual.07 - 2.05)third may [5.93,13.63] its as Total Dietary Fiber (% DW) 16.51 ± 0.66 15.63the ± 0.66of 0.88and ± 0.93 -1.26,3.03 0.368 (12.99 - 18.03) is regulatory owner. (16.27 - 16.76) (15.07 - 16.69) (-0.17a - 1.63) document [9.20,20.27] any such its reproductiondocument Proximate owner this of Ash (% DW) 1.34 ± 0.042 1.35 ± 0.042 under -0.013 ± 0.059of -0.15,0.12 0.826 (1.12 - 1.62) rightsthe this (1.25 - document1.38) and/or (1.30 - 1.40) (-0.043 - 0.030) of rights [0.74,1.96] to of fall use Carbohydrates (% DW) 85.11 ± 0.22 85.11 ± 0.22 -0.0055 ± 0.31 the -0.73,0.72 0.986 (82.91 - 86.78) (84.99This - 85.29) (84.75 - 85.31)may distribution, (-0and.28 - 0.28) [81.08,88.80] rights owner subject Moisture (% FW) 12.40 ± 0.27 12.77 ± 0.27 -0.37 ± 0.38 -1.23,0.50 0.357 (7.60 - 15.30) be the violate (12.10 - 12.80)copy (12.10 - 13.10) of (-1.00 - 0.20) [0.45,19.52] document and Protein (% DW) may10.31 ± 0.15 publication, 10.39 ± 0.15 -0.078 ± 0.21 -0.57,0.42 0.725 (9.33 - 11.82) It exploitation (10.26 - 10.35)this (10.33 - 10.49) (-0.19 - 0.019) [7.54,13.13] any Total Fat (% DW) 3.25 ± 0.12 3.15permission ± 0.12 0.097 ± 0.16 -0.27,0.47 0.562 (2.66 - 3.71) (3.19 - 3.28) (3.05 - 3.35)prohibited (-0.069 - 0.23) [2.20,4.55] the commercial be ¹With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero. Furthermore,any Consequently,without

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Table G.28. Comparison of the mineral content in grain collected at the Site 4 from MON 89034 and conventional corn

Difference(Test minus Control) Commercial Control Mean ± and (Range) and regime. Test Mean ± S.E. S.E. Mean ± S.E. AG 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Mineral Calcium (% DW) 0.0050 ± 0.00016 0.0050 ± 0.00016 0.00008 ± 0.00023 -0.00044,0.00061 0.722 (0.0031 - 0.0049) Bayer property protectionpublishing (0.0048 - 0.0054) (0.0047 - 0.0051) (-0.00024of - 0.00064) contents [0.0016,0.0059]

its Copper (mg/kg DW) 2.15 ± 0.13 1.67 ± 0.11 0.48 ± 0.17 parties. 0.086,0.87data therefore0.023 (1.15 - 3.56) (1.92 - 2.38) (1.54 - 1.75) (0.38 - 0.63) and/oror [0,4.20] affiliates. property intellectual third may Iron (mg/kg DW) 21.47 ± 0.93 20.02 ± 0.93 its 1.45 ± 1.32 -1.60,4.49 0.304 (18.04 - 29.22) (19.45 - 25.23) (19.33 - 20.71)the (-0as.98 - 5.90) [8.88,34.51] of and owner. is regulatory a document Magnesium (% DW) 0.10 ± 0.0038 0.11 ± 0.0038any such -0. 0075 ± 0.0047 -0.018,0.0032 its 0.145 (0.099 - 0.14) reproductiondocument (0.10 - 0.11) (0.11 - 0.12) owner(-0.018 - 0.0036) this of [0.075,0.17] under of Manganese (mg/kg DW) 6.85 ± 0.33 7.11 ± 0.33rights the -0.26 ± 0.47 this -1.35,0.82 0.590 (5.56 - 8.64) documentand/or rights (6.45 - 7.45) (6.66to - 8.00)of fall (-1.54 - 0.78)use of [3.17,9.99] the Phosphorus (% DW) 0.29 ± This0.010 0.30 ± 0.010 may distribution, -0.011and ± 0.012 -0.038,0.016 0.357 (0.25 - 0.37) rights owner (0.27 - 0.31) subject (0.29 - 0.31) (-0.038 - 0.016) [0.18,0.45]

be the violate Potassium (% DW) 0.34 ± 0.0082 copy 0.35 ± 0.0082 -0.0086of ± 0.0087 -0.029,0.012 0.352 (0.32 - 0.40) (0.32 - 0.37) (0.34 - 0.35) (-0.029 - 0.014) [0.26,0.46] document and may publication, exploitation Zinc (mg/kg DW) 21.39It ± 0.80 22.46 ± 0.80 -1.07 ± 1.14 -3.69,1.54 0.371 (16.72 - 34.04) this (20.07 - 23.74) any (21.75 - 23.44) (-3.37 - 1.53) [7.16,38.55]

permission ¹With 95% confidence, interval contains 99% of the values expreprohibitedssed in the population of commercial lines. Negative limits were set to zero. the commercial be Furthermore,any Consequently,without

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Table G.29. Comparison of the vitamin content in grain collected at the Site 4 from MON 89034 and conventional corn

Difference (Test minus Control) Commercial Control Mean ± and (Range) and regime. Test Mean ± S.E. S.E. Mean ± S.E. AG 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Vitamin Folic Acid (mg/kg DW) 0.27 ± 0.017 0.26 ± 0.017 0.0069 ± 0.020 -0.040,0.053 0.740 (0.13 - 0.45) Bayer property protectionpublishing (0.26 - 0.28) (0.23 - 0.29) (-0.012of - 0.045) contents [0.012,0.69]

its Niacin (mg/kg DW) 31.47 ± 0.86 30.38 ± 0.86 1.09 ± 1.21 parties. -1.70,3.88data therefore 0.394 (16.17 - 29.19) (30.39 - 33.52) (30.26 - 30.49) (-0.10 - 3.15) and/oror [6.97,37.83] affiliates. property intellectual third may Vitamin B1 (mg/kg DW) 3.20 ± 0.13 2.98 ± 0.13 its 0.22 ± 0.18 -0.21,0.64 0.274 (2.19 - 5.60) (3.07 - 3.44) (2.76 - 3.22)the (-0.15as - 0.68) [0.37,6.35] of and owner. is regulatory a document Vitamin B2 (mg/kg DW) 1.25 ± 0.065 1.45 ± 0.065any such -0.20 ± 0.092 -0.41,0.017 its 0.066 (1.34 - 1.91) reproductiondocument (1.24 - 1.26) (1.30 - 1.55) owner (-0.30 - -0.049) this of [0.91,2.30] under of Vitamin B6 (mg/kg DW) 6.36 ± 0.29 6.02 ± rights0.29 the 0.34 ± 0.39 this -0.57,1.25 0.412 (5.08 - 7.47) documentand/or rights (6.15 - 6.47) (5.37to - 6.44)of fall (-0.088 - 1.10)use of [3.12,9.30] the Vitamin E (mg/kg DW) 6.88 ±This 0.55 6.95 ± 0.55 may distribution,-0.074and ± 0.74 -1.78,1.63 0.923 (2.71 - 13.94) rights owner (6.19 - 7.28) subject (6.73 - 7.23) (-1.04 - 0.55) [0,20.49]

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Table G.30. Comparison of the antinutrient and secondary metabolites content in grain collected at Site 4 from MON 89034 and conventional corn

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Table G.31. Comparison of the fiber and mineral content in forage collected at the Site 5 from MON 89034 and conventional corn

Difference(Test minus Control) regime.and Commercial Control Mean ± and (Range) Test Mean ± S.E. S.E. Mean ± S.E. AG 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Fiber Bayer property publishing Acid Detergent Fiber (% DW) 31.31 ± 1.70 23.58 ± 1.70 7.73 ± 2.40 2.20,13.26 protection 0.012contents (26.72 - 38.94) (26.92 - 34.93) (23.06 - 24.48) (3.72of - 10.45) [16.76,43.76] its parties.data therefore Neutral Detergent Fiber (% DW) 43.21 ± 2.11 37.87 ± 2.11 5.34 ± 1.98 0.78,9.90 and/oror 0.027 (33.70 - 46.74) (40.07 - 46.82) (35.06 - 41.38) affiliates. (5.00 - 5.58) [25.94,55.67] property intellectualthird may its Mineral as the of and Calcium (% DW) 0.18 ± 0.014 0.15 is± 0.014 0.030 ± 0.018 regulatory -0.011,0.071 owner. 0.131 (0.11 - 0.29) a document (0.17 - 0.21) (0.14 - 0.17)any such(-0.0023 - 0.063) its [0.016,0.38] reproduction document owner this of Phosphorus (% DW) 0.27 ± 0.019 0.22 ± 0.019 0.049 ± 0.027 -0.012,0.11 0.102 (0.14 - 0.25) under of (0.24 - 0.32) (0.22 - 0.23)rights the (0.0094 - 0.10) this [0.071,0.32] documentand/or of rights to of fall use ¹With 95% confidence, interval contains 99% of the values expressed in the population of commercial 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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Table G.32. Comparison of the proximates content in forage collected at the Site 5 from MON 89034 and conventional corn

Difference(Test minus Control)

regime.and Commercial Control Mean ± and (Range) Test Mean ± S.E. S.E. Mean ± S.E. AG 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Proximate Bayer property publishing Ash (% DW) 3.19 ± 0.29 2.97 ± 0.29 0.22 ± 0.39 -0.67,1.12 protection0.582 (3.40 - 5.45) (2.51 - 3.61) (2.59 - 3.38) (-0.87of - 0.86) contents [1.93,6.31] its parties.data therefore Carbohydrates (% DW) 87.86 ± 0.91 87.82 ± 0.91 0.037 ± 0.85 -1.93,2.00 and/oror 0.966 (84.88 - 88.39) (85.66 - 89.13) (86.94 - 88.77) (-1affiliates..28 - 1.03) [83.05,90.74] property intellectualthird may its Moisture (% FW) 71.33 ± 1.21 69.93 ± 1.21 1.40as ± 1.40 -1.84,4.64 0.348 (64.90 - 77.40) the of and (70.10 - 73.10) (69.20is - 71.00) (-0.90 - 3.50) regulatory owner. [57.62,86.45] a document any such its Protein (% DW) 7.50 ± 0.49 7.50 ± 0.49 0.0046 ± 0.67 reproduction -1.53,1.54document 0.994 (6.58 - 8.82) owner this of (6.65 - 8.49) (7.30 - 7.79) (-0.65 - 0.70) [4.78,10.38] under of this and/orrights the Total Fat (% DW) 1.45 ± 0.42 document 1.71 ± 0.42 -0.26 ± 0.38 -1.14,0.61rights 0.507 (0.58 - 3.11) to of fall use of (0.77 - 2.23) (1.34 - 2.32) (-0.57 - -0.093) [0,4.54] the This may distribution,and ¹With 95% confidence, interval contains 99% of the values rightsexpressed in the populationowner of commercial lines. Negative limits were set to zero. subject

be the violate copy of and may documentpublication, It exploitation this any permission prohibited the commercial be Furthermore,any Consequently,without

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 308 of 323

Table G.33. Comparison of the amino acid content in grain collected at the Site 5 from MON 89034 and conventional corn

Difference (Test minus Control) regime.and Commercial Control Mean ± and (Range) Test Mean ± S.E. S.E. Mean ± S.E. AG 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Amino Acid (% DW) Alanine (% DW) 0.86 ± 0.023 0.87 ± 0.023 -0.018 ± 0.027Bayer -0.081,0.045property protectionpublishing0.534 (0.67 - 0.96) (0.82 - 0.89) (0.86 - 0.89) (-0.069 - 0.023)of contents [0.48,1.08] its parties. data therefore Arginine (% DW) 0.51 ± 0.010 0.50 ± 0.010 0.011 ± 0.014 -0.021,0.044 and/oror 0.443 (0.37 - 0.49) (0.50 - 0.52) (0.49 - 0.51) (0.0046affiliates. - 0.016) [0.33,0.56] property intellectualthird may its Aspartic acid (% DW) 0.74 ± 0.019 0.74 ± 0.019 0.00048 as± 0.026 -0.060,0.061 0.985 (0.57 - 0.77) the of and (0.73 - 0.76) (0.72 - 0.76)is (-0.038 - 0.039) regulatory owner. [0.43,0.90] a documentits any such reproduction Cystine (% DW) 0.24 ± 0.0060 0.24 ± 0.0060 -0.0014 ± 0.0059 -0.015,0.012this document of 0.817 (0.20 - 0.24) (0.23 - 0.25) (0.24 - 0.25) (-0.014owner - 0.0076) [0.18,0.27] under of rightsthe this Glutamic acid (% DW) 2.20 ± 0.063 document 2.21 ±and/or 0.063 -0.015 ± 0.078 -0.19,0.16rights 0.852 (1.71 - 2.41) to of fall use of (2.10 - 2.29) (2.18 - 2.26) (-0.16 - 0.10) [1.25,2.75] the This may distribution, and Glycine (% DW) 0.41 ± 0.0072 0.40 ± 0.0072rights 0.0035 ± 0.010 owner -0.020,0.027 0.741 (0.32 - 0.40) (0.40 - 0.41) subject (0.40 - 0.41) (-0.0074 - 0.015) [0.28,0.46] the violate be copy Histidine (% DW) 0.34 ± 0.0072 0.33 ± 0.0072 0.0036 ± of0.0080 -0.015,0.022 0.664 (0.26 - 0.33) and (0.33 - 0.35)may (0.33document - 0.34)publication, (-0.0083 - 0.014) [0.22,0.38] It exploitation Isoleucine (% DW) 0.42 ± 0.011 this 0.42 any± 0.011 0.0013 ± 0.014 -0.032,0.035 0.930 (0.32 - 0.45) (0.41 - 0.43) (0.41 - 0.42) (-0.0052 - 0.0063) [0.23,0.51] permission prohibited Leucine (% DW) 1.50 ± 0.050 1.53 ± 0.050the -0.032 ± 0.055 -0.16,0.096 0.582 (1.14 - 1.68) (1.41 - 1.57) (1.51commercial - 1.55) be (-0.13 - 0.044) [0.77,1.92]

¹With 95% confidence, interval containsFurthermore, 99% anyof the values expressed in the population of commercial lines. Negative limits were set to zero. Consequently,without

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Table G.33 (cont.). Comparison of the amino acid content in grain collected at the Site 5 from MON 89034 and conventional corn

Difference (Test minus Control) and and regime. Commercial Control Mean ± AG (Range) Test Mean ± S.E. S.E. Mean ± S.E. 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Amino Acid (% DW) Bayer property protectionpublishing Lysine (% DW) 0.35 ± 0.0073 0.35 ± 0.0073 0.0018of ± 0.010 -0.022,0.026 contents0.864 (0.24 - 0.34) (0.35 - 0.35) (0.34 - 0.36) (-0.012 - 0.011) its [0.20,0.40] parties.data therefore and/oror Methionine (% DW) 0.24 ± 0.0066 0.23 ± 0.0066 0.0062 ± 0.0054 -0.0063,0.019 0.284 (0.17 - 0.22) affiliates. (0.23 - 0.24) (0.23 - 0.24) property(-0.0037 intellectual - 0.017)third may [0.14,0.25] its as Phenylalanine (% DW) 0.58 ± 0.018 0.59 ± 0.018the of -0.0084 ±and 0.020 -0.055,0.039 0.692 (0.45 - 0.65) is regulatory owner. (0.55 - 0.61) (0.58 - 0.60) (-0.054 - 0.032)a document [0.32,0.73] any such its reproductiondocument Proline (% DW) 0.95 ± 0.026 0.97 ± 0.026 owner -0.016 ± 0.028 this -0.081,0.050 of 0.598 (0.83 - 1.11) (0.90 - 1.00) (0.96 - 0.98) (-0.064under - 0.026) of [0.68,1.21] rightsthe this documentand/or of rights Serine (% DW) 0.56 ± 0.016 0.57 ±to 0.016 of fall -0 .010 ± 0.023use -0.063,0.043 0.664 (0.45 - 0.62) (0.54 - 0.59) (0.55 - 0.60) (-0.052 - 0.034) the [0.34,0.71] This may distribution,and rights owner Threonine (% DW) 0.33 ± 0.012 subject 0.34 ± 0.012 -0.0095 ± 0.014 -0.042,0.023 0.524 (0.29 - 0.37) (0.30 - 0.35) (0.33 - 0.35) (-0.032 - 0.012) [0.24,0.41] be the violate copy of Tryptophan (% DW) 0.060 ± 0.0015 0.058document ± 0.0015 0.0016 ±and 0.0021 -0.0031,0.0064 0.449 (0.043 - 0.059) (0.055 - may0.064) (0.057 publication,- 0.059) (-0 .0038 - 0.0072) [0.032,0.072] It exploitation this Tyrosine (% DW) 0.35 ± 0.051 any 0.37 ± 0.051 -0.022 ± 0.071 -0.19,0.14 0.764 (0.25 - 0.40) (0.22 - 0.42) (0.25 - 0.42)permission (-0.21 - 0.14) [0.17,0.52] prohibited Valine (% DW) 0.54 ± 0.013 0.54 the± 0.013 be 0.0068 ± 0.016 -0.030,0.044 0.682 (0.42 - 0.55) (0.53 - 0.55) commercial (0.53 - 0.55) (-0.015 - 0.021) [0.35,0.62] Furthermore,any ¹With 95% confidence, interval Consequently,contains 99%without of the values expressed in the population of commercial lines. Negative limits were set to zero.

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 310 of 323

Table G.34. Comparison of the fatty acid content in grain collected at the Site 5 from MON 89034 and conventional corn

Difference (Test minus Control) Commercial Control Mean ± and (Range) and regime. Test Mean ± S.E. S.E. Mean ± S.E.AG 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Fatty Acid (% Total FA) 16:0 Palmitic (% Total FA) 9.00 ± 0.026 8.97 ± 0.026 0.022 ± 0.037 -0.065,0.11 0.575 (9.10 - 12.55) Bayer property protectionpublishing (8.98 - 9.03) (8.94 - 9.01) (-0of.032 - 0.064) contents [6.12,15.67]

its 16:1 Palmitoleic (% Total FA) 0.13 ± 0.014 0.11 ± 0.014 0.026 ± 0.018 parties. -0.015,0.067data therefore 0.177 (0.050 - 0.19) (0.13 - 0.14) (0.048 - 0.14) (-0.0012 - 0.079) and/oror [0,0.28] affiliates. property intellectual third may 18:0 Stearic (% Total FA) 1.84 ± 0.012 1.81 ± 0.012 its 0.031 ± 0.016 -0.0058,0.068 0.087 (1.57 - 2.45) (1.82 - 1.86) (1.80the - 1.82) as(0.017 - 0.059) [0.86,2.98] of and owner. is regulatory a document 18:1 Oleic (% Total FA) 24.07 ± 0.29 24.28any ± 0.29 such -0.22 ± 0.38 -1.10,0.67 its 0.588 (21.17 - 35.33) reproductiondocument (23.38 - 24.53) (23.98 - 24.85) owner (-1 .48 - 0.51) this of [7.51,46.46] under of 18:2 Linoleic (% Total FA) 62.98 ± 0.30 62.86rights ± 0.30the 0.12 ± 0.37 this -0.74,0.98 0.762 (50.33 - 63.59) documentand/or rights (62.56 - 63.61) (62.37to - 63.16)of fall (-0.60 - use1.24) of [39.41,76.74] the 18:3 Linolenic (% Total FA) 1.15This ± 0.012 1.16 ± 0.may012 distribution, -0.0082and ± 0.015 -0.042,0.026 0.596 (0.93 - 1.52) rights owner (1.13 - 1.17)subject (1.15 - 1.18) (-0.049 - 0.016) [0.63,1.77]

be the violate 20:0 Arachidic (% Total FA) 0.38 ± 0.0033 copy 0.37 ± 0.0033 of 0.012 ± 0.0046 0.00099,0.022 0.035 (0.32 - 0.47) (0.38 - 0.39) (0.37 - 0.38) (0.0058 - 0.022) [0.23,0.54] document and may publication, exploitation 20:1 Eicosenoic (% Total FA) It 0.28 ± 0.0021 0.28 ± 0.0021 0.0038 ± 0.0029 -0.0027,0.010 0.215 (0.23 - 0.32) this (0.28 - 0.28) any (0.27 - 0.28) (0.0022 - 0.0049) [0.15,0.39]

permission 22:0 Behenic (% Total FA) 0.17 ± 0.012 0.16 ± 0.012prohibited 0.013 ± 0.012 -0.015,0.040 0.311 (0.12 - 0.19) (0.15 - 0.20) the (0.14 -be 0.17) (-0.0020 - 0.029) [0.081,0.23] commercial ¹With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero. Furthermore,any Consequently,without

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Table G.35. Comparison of the fiber and proximates content in grain collected at the Site 5 from MON 89034 and conventional corn

Difference (Test minus Control) and and regime. Commercial Control Mean ± AG (Range) Test Mean ± S.E. S.E. Mean ± S.E. 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Fiber Bayer property protectionpublishing Acid Detergent Fiber (% DW) 5.99 ± 0.52 5.27 ± 0.52 of 0.72 ± 0.74 -0.98,2.42 contents 0.358 (4.11 - 6.33) (5.33 - 7.24) (4.17 - 6.22) (-0.84 - 3.07) its [2.77,7.56] parties.data therefore and/or or Neutral Detergent Fiber (% DW) 9.48 ± 0.42 8.87 ± 0.42 0.61 ± 0.59 -0.75,1.96 0.334 (8.20 - 11.30) affiliates. (8.87 - 9.79) (8.57 - 9.44)property (0intellectual.26 - 1.22)third may [5.93,13.63] its as Total Dietary Fiber (% DW) 14.34 ± 0.34 13.82the ± 0.34of 0.52and ± 0.24 -0.035,1.07 0.062 (12.99 - 18.03) is regulatory owner. (13.80 - 14.94) (13.50 - 14.32) (0.30 a- 0.65) document [9.20,20.27] any such its reproductiondocument Proximate owner this of Ash (% DW) 1.43 ± 0.048 1.44 ± 0.048 under -0.0025 ± 0.055of -0.13,0.12 0.964 (1.12 - 1.62) rightsthe this (1.37 - document1.53) and/or (1.31 - 1.51) (-0.099 - 0.063) of rights [0.74,1.96] to of fall use Carbohydrates (% DW) 84.26 ± 0.19 83.80 ± 0.19 0.46 ± 0.14 the 0.15,0.78 0.009 (82.91 - 86.78) (83.99This - 84.59) (83.58 - may84.03) distribution,and (0.41 - 0.56) [81.08,88.80] rights owner subject Moisture (% FW) 8.89 ± 0.10 8.60 ± 0.10 0.29 ± 0.14 -0.039,0.61 0.076 (7.60 - 15.30) be the violate (8.71 - 9.01)copy (8.36 - 8.89) of (-0.18 - 0.65) [0.45,19.52] document and Protein (% DW) may11.15 ± 0.20 publication, 11.31 ± 0.20 -0.15 ± 0.25 -0.72,0.42 0.558 (9.33 - 11.82) It exploitation (10.83 - 11.43)this (11.15 - 11.52) (-0.69 - 0.19) [7.54,13.13] any Total Fat (% DW) 3.15 ± 0.098 3.46permission ± 0.098 -0.31 ± 0.14 -0.63,0.0082 0.054 (2.66 - 3.71) (3.05 - 3.21) (3.14 - 3.68)prohibited (-0.50 - 0.070) [2.20,4.55] the commercial be ¹With 95% confidence, interval contains 99% of the values expressed in the population of commercial lines. Negative limits were set to zero. Furthermore,any Consequently,without

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Table G.36. Comparison of the mineral content in grain collected at the Site 5 from MON 89034 and conventional corn

Difference (Test minus Control) Commercial Control Mean ± and (Range) and regime. Test Mean ± S.E. S.E. Mean ± S.E. AG 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Mineral Calcium (% DW) 0.0049 ± 0.00016 0.0046 ± 0.00016 0.00026 ± 0.00023 -0.00027,0.00079 0.288 (0.0031 - 0.0049) Bayer property protectionpublishing (0.0048 - 0.0049) (0.0045 - 0.0047) (0.00013of - 0.00037) contents [0.0016,0.0059]

its Copper (mg/kg DW) 1.35 ± 0.65 2.68 ± 0.65 -1.32 ± 0.86 parties. -3.31,0.66data therefore0.163 (1.15 - 3.56) (1.33 - 1.39) (1.84 - 4.29) (-2.96 - -0.51) and/oror [0,4.20] affiliates. property intellectual third may Iron (mg/kg DW) 21.37 ± 0.84 25.74 ± 0.84 its -4.38 ± 1.19 -7.12,-1.63 0.006 (18.04 - 29.22) (20.59 - 21.76) (22.83 - 28.26)the (-6as.50 - -2.24) [8.88,34.51] of and owner. is regulatory a document Magnesium (% DW) 0.13 ± 0.0024 0.13 ± 0.0024any such -0. 0014 ± 0.0028 -0.0080,0.0052 its 0.636 (0.099 - 0.14) reproductiondocument (0.13 - 0.14) (0.13 - 0.14) (-0.0046owner - 0.00096) this of [0.075,0.17] under of Manganese (mg/kg DW) 6.56 ± 0.17 6.52 ± 0.17rights the 0.039 ± 0.17 this -0.34,0.42 0.817 (5.56 - 8.64) documentand/or rights (6.09 - 6.85) (6.38to - 6.66)of fall (-0.29 - 0.32)use of [3.17,9.99] the Phosphorus (% DW) 0.35 ± 0.0055This 0.35 ± 0.0055may -0.distribution,00071and ± 0.0067 -0.016,0.015 0.917 (0.25 - 0.37) rights owner (0.34 - 0.36) subject (0.35 - 0.36) (-0.011 - 0.0058) [0.18,0.45]

be the violate Potassium (% DW) 0.36 ± 0.0041 copy 0.35 ± 0.0041 0.0040of ± 0.0058 -0.0092,0.017 0.503 (0.32 - 0.40) (0.35 - 0.36) (0.35 - 0.35) (-0.0029 - 0.011) [0.26,0.46] document and may publication, exploitation Zinc (mg/kg DW) 22.13It ± 0.55 22.25 ± 0.55 -0.11 ± 0.78 -1.90,1.68 0.889 (16.72 - 34.04) this (21.25 - 22.95) any (21.76 - 22.92) (-0.81 - 1.19) [7.16,38.55]

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Table G.37. Comparison of the vitamin content in grain collected at the Site 5 from MON 89034 and conventional corn

Difference (Test minus Control) Commercial Control Mean ± and (Range) and regime. Test Mean ± S.E. S.E. Mean ± S.E. AG 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range) (Lower,Upper) p-Value Int.¹] Vitamin Folic Acid (mg/kg DW) 0.39 ± 0.025 0.35 ± 0.025 0.041 ± 0.035 -0.040,0.12 0.276 (0.13 - 0.45) Bayer property protectionpublishing (0.32 - 0.48) (0.32 - 0.37) (-0.043of - 0.11) contents [0.012,0.69]

its Niacin (mg/kg DW) 26.78 ± 0.70 25.71 ± 0.70 1.07 ± 0.90 parties. -1.00,3.14data therefore 0.268 (16.17 - 29.19) (25.72 - 28.00) (24.93 - 26.19) (-0.47 - 3.07) and/oror [6.97,37.83] affiliates. property intellectual third may Vitamin B1 (mg/kg DW) 3.11 ± 0.49 2.99 ± 0.49 its 0.12 ± 0.69 -1.47,1.71 0.866 (2.19 - 5.60) (2.96 - 3.40) (2.85 - 3.06)the (-0.088as - 0.34) [0.37,6.35] of and owner. is regulatory a document Vitamin B2 (mg/kg DW) 1.52 ± 0.089 1.39 ± 0.any089 such 0.13 ± 0.13 -0.16,0.42 its 0.322 (1.34 - 1.91) reproductiondocument (1.44 - 1.61) (1.16 - 1.52) owner (-0.038 - 0.45) this of [0.91,2.30] under of Vitamin B6 (mg/kg DW) 5.68 ± 0.30 6.11 ± rights0.30 the -0.43 ± 0.43 this -1.42,0.56 0.348 (5.08 - 7.47) documentand/or rights (5.28 - 6.00) (5.95to - 6.38)of fall (-0 .72 - -0.19)use of [3.12,9.30] the Vitamin E (mg/kg DW) 6.83 ±This 0.81 8.01 ± 0.81 may distribution,-1.18and ± 1.15 -3.82,1.46 0.331 (2.71 - 13.94) rights owner (5.55 - 8.62) subject (6.35 - 9.02) (-2.35 - -0.40) [0,20.49]

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Table G.38. Comparison of the antinutrients and secondary metabolites content in grain collected at the Site 5 from MON 89034 and conventional corn

and Difference(Test minusand Control)regime. AG Commercial Control Mean ± (Range) Test Mean ± S.E. S.E. Mean ± S.E. 95% CI [99% Tolerance Analytical Component (Range) (Range) (Range)Bayer property(Lower,Upper) protection publishing p-Value Int.¹] Antinutrient of contents Phytic Acid (% DW) 0.85 ± 0.018 0.86 ± 0.018 -0.010 ± 0.023 -0.064,0.044 its 0.676 (0.50 - 0.94) parties.data therefore (0.85 - 0.86) (0.83 - 0.88) (-0.031 - 0.025) and/oror [0.21,1.22] affiliates. Secondary Metabolite property intellectualthird may its Ferulic Acid (µg/g DW) 2026.73 ± 69.44 1932.90 ± 69.44 as 93.82 ± 91.31 -116.73,304.37 0.334 (1412.68 - 2297.36) (1954.76 - 2092.23) (1898.73the - 1979.22) of (-24.47and - 171.48) [1136.69,2806.24] is regulatory owner. a document any such its p-Coumaric Acid (µg/g DW) 218.38 ± 10.54 185.63 ± 10.54 32.75 ± 12.71reproduction 3.44,62document .07of 0.032 (99.30 - 285.75) (187.79 - 253.04) (182.20 - 189.17) owner(-1.39 - 70.84) this [0,378.57] under of rightsthe this ¹With 95% confidence, interval contains 99%document of theand/or values expressed in the population of commercialrights lines. Negative limits were set to zero. 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 FDA BNF No. 00105 / Monsanto 06-CR-161F Page 315 of 323

APPENDIX H: Supplementary Compositional Analysis Data

The purpose of this Appendix is to provide statistical summaries of re-expressed amino acid and fatty acid corn composition data for the combined sites.

The grain fatty acid, total fat, amino acid, protein and moisture data from MON 89034, and the conventional control, LH198 x LH172, and 15 unique commercial reference corn hybrids were selected from the data set. The corn test, control and references were grown in U.S. field trials during 2004. The test and control substances were grown in single plots randomly assigned within each of three replication blocks at each of five sites (1-5).

Analytes with greater than fifty percent of observations below the assay’s limit of quantitation (LOQ) were excluded from summaries and analysis. Otherwise,and results and regime. below the quantitation limit were assigned a valueAG equal to half the quantitation limit. The following analytes of interest were assigned values:

Bayer Obs. Below LOQproperty protectionpublishing of contents Total its Value parties.data therefore Analyte Units N (%) and/orN or LOQ Assigned Cottonseed Fatty Acid affiliates. property intellectualthird may 16:1 Palmitoleic % FWits 6 6.7 90 0.0030 0.0015 as 22:0 Behenic the %of FW and1 1.1 90 0.0030 0.0015 is regulatory owner. a document any such its The following formulas were used for re-expressionreproduction of aminodocument acid and fatty acid data: owner this of

under of Component rightsFromthe To this Formula1 documentand/or of rights to of (X)fall use % Total Proteinthe X/(10*fp) ThisAmino Acids (AA) mg/gmay FW distribution, and rights %owner Total AA (100)Xj/Σ Xj, for each AA j subject % Total Fat X/(ff) Fatty Acid (FA) % FW the violate be copy % DW X/(d) 1 of fp is the protein fraction of fresh weightand obtained by proximate analysis may documentpublication, It = (% protein /100); exploitationff is the total fat fraction of fresh weight obtained by proximate analysis = (% total fat / 100); d is the fraction of the sample that is dry matter. this any

permission prohibited 2. Statistical Approachthe be commercial SummaryFurthermore, statisticsany for the analytes of interest were generated using SAS®1 software. Consequently,without 3. Results Discussion

Statistical results of re-expression of amino acid and fatty acid components are summarized in Tables H.1-H.4. For each amino acid and fatty acid component re- expression, the overall mean, standard error (S.E.), and the range of observed values are

Monsanto Company FDA BNF No. 00105 / Monsanto 06-CR-161F Page 316 of 323

presented. In addition, the overall range of observed values for commercial references is presented in the desired unit.

and and regime. AG

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Table H.1. Combined-Site Statistical Summary for Amino Acid Content as Percent Total Amino Acid in the Grain of MON 89034, Control and Commercial Corn

MON 89034 Control Mean (S.E.) Mean (S.E.) Commercial Component [Range] [Range] [Range]

Alanine (% Total AA) 7.48 (0.041) 7.56 (0.037) [7.31 - 8.04] [7.18 - 7.75] [7.34 - 7.84]

and Arginine (% Total AA) 4.66 (0.061) 4.55 (0.045) and [3.85 - regime.4.83] [4.28 - 5.01] AG [4.25 - 4.87]

Aspartic acid (% Total AA) 6.62 (0.033) 6.59 (0.024) [6.17 - 6.82] Bayer [6.43 - 6.82] [6.45property - 6.76] protectionpublishing of contents its parties.data therefore Cystine (% Total AA) 2.27 (0.052) 2.25 (0.027) and/oror [1.97 - 2.48] [2.07 - 2.89] [2.09 - 2.45] affiliates. may property intellectualthird its as Glutamic acid (% Total AA) the 19.21of (0.11) and 19.38 (0.073) [18.68 - 20.26] is regulatory owner. [18.57 - 19.77] a [18.86 - document19.82] any such its reproductiondocument of Glycine (% Total AA) 3.76 owner(0.050) 3.72this (0.039) [3.39 - 3.95] under of rights [3.45the - 4.03] [3.53this - 3.98] documentand/or rights to of fall use of Histidine (% Total AA) 3.03 (0.018) 3.01the (0.019) [2.64 - 3.24] This may [2.93 distribution,- 3.16]and [2.91 - 3.16] rights owner subject the violate Isoleucine (% beTotal copy AA) 3.53 (0.034) 3.54 (0.029) [3.40 - 3.80] [3.36 - 3.87]of [3.35 - 3.79] and may documentpublication, It exploitation Leucine (% Totalthis AA) 12.71 (0.13) 12.87 (0.12) [12.38 - 14.14] any [11.95 - 13.54] [11.98 - 13.81] permission prohibited the commercial be Furthermore,any Consequently,without

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Table H.1 (contd.). Combined-Site Statistical Summary for Amino Acid Content as Percent Total Amino Acid in the Grain of MON 89034, Control and Commercial Corn

MON 89034 Control Mean (S.E.) Mean (S.E.) Commercial Componenta [Range] [Range] [Range]

Lysine (% Total AA) 3.20 (0.056) 3.12 (0.038) [2.66 - 3.21] [2.90 - 3.57] [2.92 - 3.41]

regime.and Methionine (% Total AA) 2.23 (0.044) 2.19 (0.026) and [1.71 - 2.21] [2.08 - 2.75] AG [2.01 - 2.42]

Phenylalanine (% Total AA) 5.01 (0.036) Bayer 5.03property (0.038) protection [4.97publishing - 5.48] [4.82 - 5.28]of [4.74 - 5.27] contents its parties. data therefore Proline (% Total AA) 9.12 (0.086) 9.10 (0.072) and/or [8.73or - 9.65] affiliates. property [8.43 - 9.51] intellectual third [8.36 - 9.38] may its as the of and Serine (% Total AA) is 5.06 (0.038) regulatory 5.12 (0.032) [4.74owner. - 5.44] a document any [4.78such - 5.29] [4.90 - 5.32] its reproductiondocument owner this of Threonine (% Total AA) 3.20 (0.036)under 3.20of (0.028)this [2.91 - 3.34] and/orrights [2.83the - 3.35] [2.90 - 3.32] document to fall of rights of use the TryptophanThis (% Total AA) may 0.55 (0.0094)distribution,and 0.54 (0.0086) [0.40 - 0.60] rights owner subject [0.49 - 0.64] [0.50 - 0.59]

be the violate Tyrosine (% Totalcopy AA) 3.58 (0.11)of 3.49 (0.11) [2.36 - 3.80] [2.04 - 3.84] and [2.29 - 3.85] may documentpublication, It exploitation Valine (% Totalthis AA) any 4.78 (0.034) 4.75 (0.019) [4.40 - 4.96] permission [4.58 - 4.99] [4.65 - 4.94] prohibited a the AA = amino acid.commercial be

Furthermore,any Consequently,without

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Table H.2. Combined-Site Statistical Summary for Amino Acid Content as Percent Total Protein in the Grain of MON 89034, Control and Commercial Corn

MON 89034 Control Mean (S.E.) Mean (S.E.) Commercial Component [Range] [Range] [Range]

Alanine (% Total Protein) 7.37 (0.12) 7.48 (0.052) [7.18 - 8.08] [6.17 - 8.33] [7.19 - 7.77]

Arginine (% Total Protein) 4.60 (0.11) 4.50 (0.052) [3.87 - 4.82] [3.66 - 5.72] [4.21 - 4.85] regime.and and Aspartic acid (% Total Protein) 6.52 (0.12) AG 6.52 (0.030) [6.01 - 6.88] [5.38 - 7.72] [6.34 - 6.72]

Bayer property protectionpublishing Cystine (% Total Protein) 2.23 (0.032)of 2.22 (0.026) [1.91contents - 2.45] its [2.00 - 2.42] [2.10parties. - 2.40]data therefore and/oror affiliates. Glutamic acid (% Total Protein) property 18.93 (0.31) intellectual third 19.16 (0.12) [18.26may - 20.37] its [15.80 as- 21.35] [18.41 - 19.91] the of and is regulatory owner. a document Glycine (% Total Protein) any such 3.71 (0.088) 3.68 (0.037) its [3.32 - 3.99] reproductiondocument [3.07owner - 4.59] [3.44this - 3.90] of

under of this Histidine (% Total Protein)and/or rights the 2.99 (0.055) 2.97 (0.016) [2.56 - 3.16] document to fall of rights of [2.47 - 3.53] use [2.87 - 3.08] the This may distribution, and Isoleucine (% Total Protein)rights 3.48 (0.068) owner 3.50 (0.036) [3.37 - 3.77] subject [2.91 - 4.01] [3.28 - 3.73] be the violate copy of Leucine (% Total Protein) 12.53 (0.22)and 12.73 (0.15) [12.17 - 14.22] may documentpublication, It exploitation [10.61 - 13.84] [11.75 - 13.59] this any permission prohibited the commercial be Furthermore,any Consequently,without

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Table H.2 (contd.). Combined-Site Statistical Summary for Amino Acid Content as Percent Total Protein in the Grain of MON 89034, Control and Commercial Corn

MON 89034 Control Mean (S.E.) Mean (S.E.) Commercial Component [Range] [Range] [Range] Lysine (% Total Protein) 3.16 (0.090) 3.08 (0.035) [2.57 - 3.27] [2.48 - 4.06] [2.86 - 3.32]

Methionine (% Total Protein) 2.19 (0.025) 2.16 (0.025) [1.75 - 2.15] regime.and [2.03 - 2.37] [2.01 - 2.37]and AG Phenylalanine (% Total Protein) 4.93 (0.085) 4.97 (0.051) [4.82 - 5.49] [4.15 - 5.56] [4.65 - 5.30] Bayer property protectionpublishing of contents its Proline (% Total Protein) 8.99 (0.17) 8.99parties. (0.073)data [8.44 -therefore 9.50] [7.63 - 10.69] [8.34 - 9.46]and/or or affiliates. property intellectualthird may Serine (% Total Protein) 4.99its (0.081) 5.06 (0.029) [4.62 - 5.41] as the of[4.23 - 5.71]and [4.92 - 5.25] is regulatory owner. a document any such its reproductiondocument Threonine (% Total Protein) 3.15owner (0.064) 3.16this (0.030) of [2.82 - 3.41] [2.66 - 3.79] [2.90 - 3.35] under of this and/orrights the document fall of rights Tryptophan (% Total Protein)to of 0.54 (0.0053) use 0.54 (0.0078) [0.39 - 0.59] the This may[0.50 distribution, - 0.58]and [0.48 - 0.58] rights owner subject Tyrosine (% Total Protein) 3.53 (0.13) 3.45 (0.12) [2.38 - 3.95] be the violate copy [2.00 - 4.24]of [2.25 - 3.89] and may documentpublication, Valine (%It Total Protein) exploitation 4.71 (0.092) 4.70 (0.026) [4.37 - 4.90] this any [3.87 - 5.56] [4.52 - 4.88] permission prohibited the commercial be Furthermore,any Consequently,without

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Table H.3. Combined-Site Statistical Summary for Fatty Acid Content as Percent Dry Weight in the Grain of MON 89034, Control and Commercial Corn

MON 89034 Control Mean (S.E.) Mean (S.E.) Commercial Component [Range] [Range] [Range]

16:0 Palmitic (% DW) 0.29 (0.0050) 0.29 (0.0052) [0.28 - 0.42] [0.26 - 0.34] [0.26 - 0.33]

16:1 Palmitoleic (% DW) 0.0040 (0.00009) 0.0039 (0.00019) [0.0016 - 0.0064]and and regime. [0.0033 - 0.0044] AG [0.0016 - 0.0047]

18:0 Stearic (% DW) 0.060 (0.0014) 0.057 (0.0011) [0.044 - 0.084] [0.053 - 0.073]Bayer [0.052property - 0.067] protection publishing of contents

its 18:1 Oleic (% DW) 0.79 (0.013) parties. 0.78 (0.014)data [0.58 therefore- 1.18] and/oror [0.68 - 0.91] [0.72 - 0.87] affiliates. property intellectualthird may its 18:2 Linoleic (% DW) 1.95 (0.026)as 1.95 (0.041) [1.57 - 2.14] the of and is [1.85 - 2.20] regulatory [1.77 - 2.27] owner. a document any such its reproductiondocument 18:3 Linolenic (% DW) 0.037owner (0.00051) 0.038this (0.00069)of [0.031 - 0.045] under of rightsthe [0.034 - 0.041] [0.034this - 0.042] documentand/or rights to of fall use of 20:0 Arachidic (% DW) 0.012 (0.00025) 0.012the (0.00023) [0.0096 - 0.017] This may[0.011 distribution, - 0.014]and [0.011 - 0.014] rights owner subject 20:1 Eicosenoic (% DW) 0.0088 (0.00015)the violate 0.0087 (0.00018) [0.0066 - 0.011] be copy [0.0078 of- 0.010] [0.0074 - 0.010] and may documentpublication, It exploitation 22:0 Behenic (% DW)this 0.0049 (0.00015) 0.0048 (0.00018) [0.0040 - 0.0060] any [0.0042 - 0.0061] [0.0037 - 0.0061] permission prohibited the commercial be Furthermore,any Consequently,without

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Table H.4. Combined-Site Statistical Summary for Fatty Acid Content as Percent Dry Weight in the Grain of MON 89034, Control and Commercial Corn

MON 89034 Control Mean (S.E.) Mean (S.E.) Commercial Component [Range] [Range] [Range]

16:0 Palmitic (% Total Fat) 8.74 (0.051) 8.69 (0.030) [8.75 - 12.18] [8.51 - 9.29] [8.55 - 8.90]

16:1 Palmitoleic (% Total Fat) 0.12 (0.0027) 0.12 (0.0057) [0.046 - 0.18] and [0.11 - 0.14] [0.046 - 0.13]and regime. AG 18:0 Stearic (% Total Fat) 1.80 (0.020) 1.73 (0.0087) [1.44 - 2.35] [1.68 - 1.92] [1.69 - 1.81] Bayer property protectionpublishing of contents 18:1 Oleic (% Total Fat) 23.73 (0.21) 23.68 (0.23) [20.18its - 33.87] parties.data therefore [22.34 - 25.36] [22.52 - 25.60]and/or or affiliates. property intellectualthird may 18:2 Linoleic (% Total Fat) 58.78its (0.34) 59.17 (0.33) [48.24 - 61.46] as the [56.57of - 61.07]and [56.90 - 61.17] is regulatory owner. a document any such its reproductiondocument 18:3 Linolenic (% Total Fat) 1.13 owner(0.0090) 1.16this (0.016) of [0.85 - 1.42] [1.04 - 1.18] [1.10 - 1.34] under of this and/orrights the document fall of rights 20:0 Arachidic (% Total Fat)to of 0.37 (0.0042) use 0.36 (0.0027) [0.29 - 0.46] the This may [0.34 distribution, - 0.40]and [0.34 - 0.37] rights owner subject 20:1 Eicosenoic (% Total Fat) 0.26 (0.0024) 0.27 (0.0026) [0.22 - 0.31] be the violate copy [0.25 - 0.28]of [0.24 - 0.28] and may documentpublication, 22:0 BehenicIt (% Total Fat) exploitation 0.15 (0.0048) 0.15 (0.0038) [0.12 - 0.18] this any [0.12 - 0.19] [0.12 - 0.17] permission prohibited the commercial be

Furthermore,any Consequently,without

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