Submission to the French Comimission du Genie Biomoleculaire Application to Place on the Market Genetically Modified Higher Plants: Insect-Protected Maize (MON 810) by Monsanto Company represented by Monsanto Europe S.A. List of Abbreviations 2,4-D (2,4-dichlorophenoxy)acetic acid APHIS Animal Plant Health Inspection Service bp, Kb Base pairs, kilobase pairs B. t.h. Bacillus thuringiensis subsp. kurstaki CaMV Cauliflower mosaic virus CaMVV E35S 35S promoter with enhancer sequence from CaMV CFR U S. Code of federal regulations CP4 EPSPS EPSPS from Agrobacterium sp. strain CP4 cry JA(b) C"lass I (Lepidoptera-specific) crystal protein gene CTP Chloroplast transit peptide E. coli Escherichica colt ECB European corn borer ELISA Enzyme-linked immunosorbent assay EPA U.S. Environmental Protection Agency EPSPS 5-enolpyruvylshikimate-3-phosphate synthase FDA U.S. Food and Drug Administration GLP Good Laboratory Practices GMO Genetically Modified Organism gox Gene for glyphosate oxidoreductase GOX Glyphosate oxidoreductase hsp70 Intron sequence from maize heat-shock protein 70 IPM Integrated Pest Management kD Kilodaltons N.A. Not analysed N.D. Not detected NOS 3' 3' transcriptional termination sequence from nopaline synthase NPTII Neomycin phosphotransferase II np tll Gene for neomycin phosphotransferase II 0 ri-p UC Bacterial origin of replication from the pUC plasmid PCR Polymerase chain reaction ppm parts per million SSUIA small subunit gene of ribulose-1,5-bisphosphate carboxylase USDA United States Department of Agriculture W/W weight/weight 2 Table of Contents PART A: GENERAL INFORMATION 1. Details of notification ................ .. 7 2. Notifier/manufacturer/importer........ ........ 7 3. Characterization of the GMOs contained in the product...... 8 4. General description of the product...... ......... 9 5. Has the combination of GMOs contained in the product been noified under part B of Directive 901220fEEC? .. ..... 13 6 Is the product being simultaneously notified to another M'vember State?7 ........ .... ........ 13 7. Has another product, with the same combinations of GM.1s been placed on the EC market by another notifier? . ......... 13 8. Information on releases of the same GMOs or of the same combi- nation of GMOs previously or currently notified and/or carried out by the notifier either inside or outside the Community.......13 9. Specify instructions and/or recommendations for storage and handling....... ...... ...... .. .... 14 10. Proposed packaging........... ....... ... 14 11. Proposed labelling......... ............ 15 12. Measures to take in case of unintended release or misuse..... 15 13. Measures for waste disposal and treatment .......... 15 PART B: NATURE OF THE GMOS CONTAINED IN THE PRODUCT INFORMATION RELATING TO THE RECIPIENT OR PARENTAL ORGANISM(S) FROM WHICH THE GMO IS DERIVED 14. Scientific name and other names........... ..... 16 15. Phenotypic and genetic traits ................ 16 16. Geographic distribution and natural habitat of the organisms......16 17. Genetic stability of the organism and factors affecting it....... 17 18S. Potential for genetic transfer and exchange with other organisms .. 17 19. Information concerning reproduction and factors affecting it ...... 19 20. Information on survival and factors affecting it ......... 19 21. Ways of dissemination and factors affecting it ......... 20 22. Interactions with the environment .. ..... ..... 20 23 (a) Detection techniques .......... .... ....... 20 (b) Identification techniques ......... ........ 20 24. Classification under existing Community rules concerning the protection of human health and/or the environment........ 20 25 (a) Pathogenic characteristics ......... .I..... .. 20 (b) Other harmful characteristics of the organism living or dead, including its extracellular products..............20 26. Nature and description of known extrachromosomal genetic elements 21 27. History of previous genetic modifications .......... .21 3 INFORMATION RELATING TO THE GENETIC MODIFICATION 28. Methods used for the genetic modification............21 29. Characteristics of the vector.................22 30. Information on the insert .................. 29 INFORMATION ON THE ORGANISM(S) FROM WHICH THE INSERT IS DERIVED (DONOR) 31. Scientific and other names..................31 32 (a) Pathogenic characteristics of the donor organism........31 (b) Other harmful characteristics of the organism living or dead, including its extracellular products ............. 31 33. If the donor organism has any pathogenic or harmful characteristics, indicate whether the donated sequences are in any way involved in them ..................... 31 34. Classification under existing Community rules relating to the protection of human health and the environment ......... 32 35. Potential for natural exchange of genetic material between the donor(s) and recipient organism................32 INFORMATION RELATING TO THE GMO(S) CONTAINED IN THE PRODUCT 36. Description of genetic traits or phenotypic characteristics and in particular any new traits and characteristics which may be expressed or no longer expressed ............... 32 37. Genetic stability of the GMO.................33 38. Rate and level of expression of the new genetic material ...... 36 39. Activity of the expressed proteins...............40 40 (a) Description of detection techniques for the GMO in the environm ent .. 40 40 (b) Description of identification techniques............41 41. Health considerations ................... 41 INTERACTIONS OF THE GMO WITH THE ENVIRONMENT 42. Survival, multiplication and dissemination of the GMO(s) in the environment .. .42 43. Interactions of the GMOs with the environment ......... 44 44. Environmental impacts of the GMO(s) ............. 44 4 PART C: PREDICTED BEHAVIOUR OF THE PRODUCT 1. Environmental impact of the product...............45 a. Honey bee larvae and adults................45 b. Green lacewing.....................46 c. Parasitic hymenoptera..................46 d. Ladybird beetles .......... .......... 46 e. Resistance management ..... ........ .... 46 2. Human health effects of the product...............47 a. Assessment of the safety of the CryIA(b) protein .. ...... 47 b. Toxicity assessment of CryIA(b) protein........ ... 48 c. Assessment of the allergenic potential of the CryIA(b) protein . .. 51 d. Compositional analyses of Insect-Protected maize... ...... 54 1. Compositional analyses of grain from line MON 810 plants grown in 1994 US trials ................ 54 1 . Compositional analyses of grain from line MON 810 plants grown in 1995 European field trials............58 ill.Compositional analyses of forage from line MON 810 plants grown in 1995 European field trials .. .... .... 61 iv. Compositional analyses of progeny of MON 810 .. ..... 62 List of Supporting Information Figures Figure B. 1 Plasmid map of PV-ZMBKO7..............22 Figure B.2 Plasmid map of PV-ZMGT10..............24 Figure B.3 Deduced amino acid sequence of the CryIA(b) protein as encoded by vector PV-ZMBKO7............27 Figure B.4 Southern blot analysis of genetic stability of Insect-Protected maize line MON 810 .. ........ 35 Tables Table A. 1 Maize Area Distribution in the European Union...... 10 Table A. 2 Maize Production and Imports within the E.U....... 12 Table A. 3 Insect-Protected Notifications in the European Union.....1 3 Table AA4 Ex-E.U. Insect-Protected Field Trials .... .. ..... 14 Table B.1I World Maize Production 1994-95 . ...... ... .. ..... 17 Table B. 2 Summary of DNA Elements in the Plasmid PV-ZMBKO7 ... 23 Table B3.3 Summary of DNA Elements in the Plasmid PV-ZMGT10 ... 25 Table B.4 Summary of Maize Line MON 810 Molecular Analysis .... 30 Table B3.5 Segregation Data and Analysis of Progeny of Insect-Protected Maize Line MON 810.... .. ..... 33 Table B.6 Stability of Gene Transfer Based on Segregation Data for Backcross Derivatives of Insect-Protected Maize Line MON 810 in Two Unrelated Inbred Lines (B73 and Mo 17) .. .. 34 Table B3.7 Summary of Specific Protein Levels in Tissues of Maize Line MON 810 from the 1994 U.S. Field Trials.....37 5 Table B.8 Summary of Specific Protein Levels in Tissues of Maize Line MON 810 from the 1995 European Field Trials.......38 Table B.9 Summary of CryIA(b) Protein Levels in Tissues of Progeny from Maize Line MON 810 Grown in the 1995 E.U. Field Trials .39 Table B. 10 Field Germination Results for Insect-Protected Maize Line MON 810 and Control ................ 42 Table C. 1 Summary of the Results of the Acute Gavage Study Performed with the CryIA(b) Protein. ......... 50 Table C.2 Characteristics of Known Allergenic Proteins........53 Table C 3 Summary of Proximate Analysis of Grain from Maize Line MON 810 .... ............ 55 Table CA4 Amino Acid Composition of Maize Grain ......... 56 Table C. 5 Fatty Acid C omposition of Maize Grain ......... 57 Table C.6 Summary of Proximate Analysis of Grain from Maize Line MON 810 Harvested from France Field Trials.......58 Table C. 7 Amino Acid Composition of Maize Line MON 810 Grain Harvested from France Field Trials.........59 Table C.8 Fatty Acid Composition of Maize Line MON 810 Grain Harvested from France Field Trials.........60 Table C.9 Compositional Analyses on Forage Samples of Maize Line MON 810 from France Field Trials ........... 61 Table C.lO Proximate Analyses on Grain from Progeny of Maize Line MON 810.................63 Table C. 11 Amino Acid Composition of Grain from Progeny of Maize Line MON 810.................64 Table C. 12 Fatty Acid Profile of Grain from Progeny of Maize Line