TRANSMITTAL DOCUMENT

SUBMITTER: Monsanto Company 800 North Lindbergh Blvd. Saint Louis, Missouri 63167

COMPANY OFFICIAL: Debbie Mahadeo, B.S. Regulatory Affairs Manager.

COMPANY CONTACT: Daniel J. Jenkins, J.D., M.S. (202) 383-2851 daniel.j.jenkins@monsanto.com Fax Number: (202) 789-1748

REGULATORY ACTION IN SUPPORT OF WHICH THIS PACKAGE IS SUBMITTED Application for a New Product Registration of the Plant-Incorporated Protectant, Bacillus thuringiensis Cryl A.105 and Cry2Ab2 Proteins and the Genetic Material (Vector PVGMIR13196) Necessary for their Production in MON 87751 Soybean

(EPA File. No.: 524-ARO)

LIST OF SUBMITTED DOCUMENTS Volume 20: Petition to Establish a Tolerance Exemption from the Requirement of a

Tolerance for Bacillus thuringiensis Cryl A.105 protein When Used as Plantincorporated Protectant in all Plants and in all Food and Feed Commodities - SY258-14E1-1

MRID NumberMonsanto Company SY258-14E1-1 Page 1 of 31 Title Petition to Establish a Tolerance Exemption from the Requirement of a Tolerance for Bacillus thuringiensis Cry1A.105 protein When Used as Plant-incorporated Protectant in all Plants and in all Food and Feed Commodities

Data Requirement 40 CFR Parts 172 and 180

Author Debbie Mahadeo, B.S.

Report Date April 14, 2014 Test Guideline Not applicable Report Generated By Monsanto Company Registrant Submitting Monsanto Company 800 North Lindbergh Blvd. St. Louis, MO 63167 Report ID SY258-14E1-1

Page Count
1 of 31

# Z 20

Sponsor Chloe Pavely, M.S. Monsanto Company Oilseeds Regulatory Affairs Lead Date GLP Compliance Statement This report does not meet the U.S. EPA Good Laboratory Practice requirements as specified in 40 CFR Part 160. This volume contains the administrative materials for an application to register MON 87751 Soybean and therefore does not meet the definition of a study under 40 CFR §160.3.



### 29 2614 Date Submitter Debbie Mahadeo, B.S. Monsanto Company Regulatory Affairs Manager

Monsanto Company SY258-14E1-1 Page 3 of 32Monsanto Company SY258-14E1-1 Page 4 of 31 Copyright Information Page © 2014 Monsanto Company. All Rights Reserved. This document is protected under national and international copyright law and treaties. This document and any accompanying material are for use only by the regulatory authority to which it has been submitted by Monsanto Company and its affiliates, collectively “Monsanto Company”, and only in support of actions requested by Monsanto Company. Any other use, access to, or transmission, including internet posting, of this document and the materials described in or accompanying this document, without prior consent of Monsanto Company, is strictly prohibited; except that Monsanto Company hereby grants such consent to the regulatory authority where required under applicable law or regulation. Intellectual property, information and materials described in or accompanying this document are owned by Monsanto Company, which has filed for or been granted patents on those materials. By submitting this document and any accompanying materials, Monsanto Company does not grant any party or entity any right or license to the information, material or intellectual property described or contained in this submission.Monsanto Company SY258-14E1-1 Page 5 of 31 Table of Contents
Title 1 STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS 2 GLP Compliance Statement 3 Copyright Information Page 4 Table of Contents 5 Introduction 6 Section A: The Name, Chemical Identity, and Composition of the Plant-incorporated Protectant .........................................................................................................................................8 Section B: The Amount, Frequency, and Time of Application of the Plant-incorporated Protectant .........................................................................................................................................9 Section C: Reports Made with Regards to the Safety of the Plant-incorporated Protectant..........10 Section D: Reports with Regard to the Nature and Amount of Plant-incorporated Protectant Remaining In or On the Food, Including a Description of Analytical Methods Used ..................15 Section E: Practical Methods for Removing Plant-incorporated Protectant that Exceeds any Proposed Tolerance........................................................................................................................16 Section F: Proposed Tolerances for the Plant-incorporated Protectant if Tolerances are Proposed.........................................................................................................................................17 Section G: Practical Methods for Removing Residues That Exceed Proposed Tolerance...........18 Section H: Practical Method for Detecting and Measuring the Levels of the Pesticide Chemical Residue ..........................................................................................................................19 Section I: Proposed Tolerances for Processed Food......................................................................20 Section J: Such Information as the Administrator May Require to Make This Determination.....21 Section K: Information Regarding Potential Endocrine Effects of Residues on Humans.............22 Section L: Information Regarding Exposure to the Pesticide Chemical Residue Due to Any Tolerance or Exemption Already Granted.....................................................................................23 Section M: Information Concerning MRLs Established By Codex Alimentarius ........................24 Section N: Reasonable Grounds in Support of the Petition...........................................................25 Attachment I. Petitioner’s Notice of Filing Summary 26Monsanto Company SY258-14E1-1 Page 6 of 31 Introduction Monsanto Company has developed biotechnology-derived soybean MON 87751 (OECD unique identifier MON-87751-7) that provides protection from feeding damage caused by targeted lepidopteran insect pests. MON 87751 produces the CrylA.105 and Cry2Ab2 insecticidal (Cry) proteins from Bacillus thuringiensis . Cry1A.105 protein is a chimeric protein comprised of domains I, II and C-terminal from the Cry1Ac (subsp. kurstaki ), and domain III from Cry1F (subsp. aizawai ). Domains I and II of the Cry1A.105 share 100% amino acid sequence homology with the domains I and II of the Cry1Ac and Cry1Ab proteins. Amino acid sequence of domain III of the Cry1A.105 protein is 99% identical to the corresponding domain of Cry1F. The overall amino acid sequence identity to the Cry1Ab, Cry1Ac, and Cry1F proteins is 90.0%, 93.6%, and 76.7% respectively. The Cry1A.105 and Cry2Ab2 proteins belong to a family of Cry proteins from B. thuringiensis that has been used commercially in the U.S. to produce microbial-derived products with insecticidal activity. Microbial pesticides that contain Cry1Ac/Cry1F chimeric protein have been used for control of lepidopteran pests since 1997 (Baum 1998; Baum, et al. 1999). Cry1A.105 and Cry2Ab2 are expressed in MON 89034 corn, which was reviewed and approved by regulatory agencies around the world and has been commercially available in the United States since 2009. The Cry1A.105 and Cry2Ab2 proteins expressed in MON 87751 have greater than 99% and 97% amino acid identity, respectively, to the Cry1A.105 and Cry2Ab2 expressed proteins in commercially approved MON 89034. The protease-resistant core domains of the Cry1A.105 and Cry2Ab2 proteins expressed in MON 87751 share 100% amino acid identity to MON 89034 expressed Cry1A.105 and Cry2Ab2 proteins. These domains are responsible for target organism specificity and efficacy (Gill, et al. 1992; Widner and Whiteley 1989). On July 2, 2008, U.S.EPA established an exemption from the requirement of a tolerance for residues of the plantincorporated protectant Cry2Ab2 in or on corn or cotton when used as a plant-incorporated protectant in the food and feed commodities of corn or cotton (40 CFR 174.519). On July 16, 2008, U.S. EPA established an exemption from the requirement of a tolerance for residues of the plant-incorporated protectant Cry1A.105 in or on corn when used as a plant-incorporated protectant in the food and feed commodities of corn (40 CFR 174.502). U.S. EPA reviewed and concluded on the safety of both proteins in its Biopesticide Registration Action Document for MON 89034 (U.S. EPA 2010). Therefore, MON 87751, though a new transformation event in soybean, contains the proteins and associated modes of action that have already received U.S. EPA tolerance exemptions and registrations for other crops. The safety data provided for the Cry1A.105 and Cry2Ab2 proteins demonstrate that neither protein exhibits amino acid sequence similarities to known allergenic or toxic proteins, both proteins are degraded in simulated gastric fluid, and both proteins lose functional activity after heating. In previous acute toxicity assays, neither protein was acutely toxic to mammals at the highest doses tested (>2,000 mg/kg body weight in mice) and based on estimated consumption and protein levels large margins of exposure (MOE) have been demonstrated for the Cry1A.105 and Cry2Ab2 proteins. These data support the safety of the Cry1A.105 and Cry2Ab2 proteins when used as plant-incorporated protectant (PIP) which is applicable across crops as well as food an d f e e d s o ur c e s

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m o r e , d u e t o t h e l a c k o f m am m a l i an t o x i c i t y f o r t h e δ

- e n d o t o x i n s tested at very high doses, traditional pesticide residue studies are not considered necessary,Monsanto Company SY258-14E1-1 Page 7 of 31

therefore microbial B. thuringiensis pesticides that are registered in countries that require tolerances (Maximum Residue Levels) have been given an exemption from the requirement for setting a numerical tolerance (MRL) (OECD 2007). By the way of this petition, Monsanto is requesting EPA to amend 40 CFR 174.502 to extend the exemption from the requirement of a tolerance for the PIP, Bacillus thuringiensis Cry1A.105 protein and genetic material necessary for its production to all plants and to all food and feed commodities. Monsanto consents to publication of this summary as part of the Agency’s notice of filing in the Federal Register and as a part of a proposed regulation.Monsanto Company SY258-14E1-1 Page 8 of 31

Section A: The Name, Chemical Identity, and Composition of the Plant-incorporated Protectant The Bacillus thuringiensis protein, Cry1A.105, when used as a plant-incorporated protectant, is the subject of this petition requesting an exemption from the requirement of a tolerance. This exemption would include the genetic material necessary for the production of Cry1A.105 in all plants and when used as plant-incorporated protectant in all food and feed commodities. Reports describing characterization of the Cry1A.105 are referenced by MRID below. MacRae, T.C., C.R. Brown, and S.L. Levine. 2006. Spectrum of Insecticidal Activity of Bacillus thuringiensis CrylA.105 Protein. Monsanto Technical Report MSL-20230. MRID 46951413 Garnaat, C. L., Nemali, A., Robinson, K., Arackal, S. M., Skottke, K., Yan, Y., Kovalic, D. 2013 Amended Report for MSL0024773: Molecular Characterization of Insect Protected Soybean (MON 87751). Monsanto technical report MSL0025312 . MRID 49332003 Chen, B., Mueller, G. M. 2013 Characterization of the Cry1A.105 Protein Purified from the Soybean Seed of MON 87751 and Comparison of the Physicochemical and Functional Properties of the Plant Produced and Escherichia coli Produced Cry1A.105 Proteins. Monsanto technical report MSL0025088 MRID 49332005 Mueller, G. M., Uffman J., Levine, S.L. 2013 Comparison of the Functional Activity of two E. coli -Produced Cry1A.105 Protein Lots (10000776 and 11349124) Against a Susceptible Lepidopteran Species. Monsanto technical report MSL0025113 MRID 49332007 Kang, H. T., Silvanovich, A. 2013. Bioinformatics Evaluation of the Cry1A.105 Protein in MON 87751 Utilizing the AD_2013, TOX_2013 and PRT_2013 Databases. Monsanto technical report MSL0024925 MRID 49332013 Rice, J.F., B.J. Wolff, J.R, Groat, N.K. Scanlon, J.C. Jennings, and J.D. Masucci. 2006. Amended Report for MSL-20072: Molecular Analysis of Corn MON 89034. Monsanto Technical Report MSL-20311. MRID 46951402 Supplemental Information for MRID No. 46951402 “Amended Report for MSL-20072: Molecular analysis of Corn MON 89034”. MRID 47127503 Rice, J.F., B.J. Wolff, J.C. Jennings, and J.D. Masucci. 2005. Summary of Southern Blot Analysis of MON 89034 and MON 89597 Corn. Monsanto Technical Report MSL-20068 MRID 46694501 Goertz, B., T. Ganguly, J. Lee, T. Lee, and E.A. Rice. 2005. Characterization of the Cry1A.105 Protein Purified from the Corn Grain of MON 89034 and Comparison of the Physicochemical and Functional Properties of the Plant-Produced and E.coli -Produced Cry1A.105 Proteins. Monsanto Technical Report MSL19960 MRID 46694604Monsanto Company SY258-14E1-1 Page 9 of 31

Section B: The Amount, Frequency, and Time of Application of the Plant-incorporated Protectant The Cry1A.105 insecticidal protein is a plant-incorporated protectant, therefore it is not applied to plants. The protein has been found to be present in corn and soybean plants throughout their development and in all tested tissues, with the exception of soybean root tissue which was below the limit of detection. Reports describing protein levels in corn and soybean tissues are referenced in Section D of this petition.Monsanto Company SY258-14E1-1 Page 10 of 31

Section C: Reports Made with Regards to the Safety of the Plant-incorporated Protectant Bacillus thuringiensis Cry proteins are widely used as topical bioinsecticides and in biotechnology-derived crops. Cry1A.105 protein is a chimeric protein comprised of domains I, II and C-terminal from the Cry1Ac (subsp. kurstaki ), and domain III from Cry1F (subsp. aizawai ). Domains I and II of the Cry1A.105 share 100% amino acid sequence homology with the domains I and II of the Cry1Ac and Cry1Ab proteins. Amino acid sequence of domain III of the Cry1A.105 protein is 99% identical to the corresponding domain of Cry1F. The overall amino acid sequence identity to the Cry1Ab, Cry1Ac, and Cry1F proteins is 90.0%, 93.6%, and 76.7% respectively. Microbial pesticides that contain Cry1Ac/Cry1F chimeric protein have been used for control of lepidopteran pests since 1997 (Baum 1998; Baum, et al. 1999). Cry1A.105 is expressed in MON 89034 corn, which was reviewed and approved by regulatory agencies around the world and has been commercially available in the United States since 2009. The consumption of agricultural food crops sprayed with commercial B. thuringiensis microbial pesticides have a 50- year history of safe use and crops expressing proteins derived from B. thuringiensis have been commercially cultivated since 1996 (Hammond 2004; Hammond and Koch 2012; OECD 2007; Perlak, et al. 2001). Microbial pesticides containing B. thuringiensis Cry proteins as well as individual Cry proteins have been subjected to extensive toxicity testing showing no adverse effects to human health (Baum et al. 1999; Betz et al. 2000; Federici and Siegel 2008; Hammond 2004; McClintock et al. 1995; Mendelsohn et al. 2003; Siegel 2001; U.S. EPA 2001; 2005). Additionally, there are no confirmed cases of allergic reactions to Cry proteins in microbial-derived B. thuringiensis products during more than 50 years of use (IPCS 1999; McClintock, et al. 1995; OECD 2007). Similarly, for the millions of tons of crops produced since the 1990s, that express proteins derived from B. thuringiensis, there have been no reports of allergenic reactions in those that handle or consume the grain/seed (Hammond and Koch 2012). The mode-of-action of Bt proteins has been extensively studied and is well-documented (Gill, et al. 1992; OECD 2007; Schnepf, et al. 1998; Vachon, et al. 2012). The specificity of Cry proteins is dependent upon their binding to specific receptors present in the insect mid-gut (OECD 2007; Pigott and Ellar 2007). These specific receptors have not been found in taxa outside of insects (OECD 2007; Schnepf et al. 1998). Consistent with this observation, no adverse effects have been reported in mammals, birds, amphibians or reptiles (OECD 2007; Schnepf et al. 1998). Additional considerations that support the dietary safety of Cry proteins for humans and animals include the acidic environment of the mammalian stomach that does not favor solubilization and activation of the Cry proteins; and the rapid degradation of the Cry proteins by proteases in the mammalian gastrointestinal tract (Hammond 2004). The Cry1A.105 and Cry2Ab2 proteins expressed in MON 87751 have greater than 99% and 97% amino acid identity, respectively, to the Cry1A.105 and Cry2Ab2 expressed proteins in commercially approved MON 89034. The protease-resistant core domains of the Cry1A.105 and Cry2Ab2 proteins expressed in MON 87751 share 100% amino acid identity to MON 89034 expressed Cry1A.105 and Cry2Ab2 proteins. These domains are responsible for target organism specificity and efficacy (Gill, et al. 1992; Widner and Whiteley 1989). On July 2, 2008, U.S.EPA established an exemption from the requirement of a tolerance for residues of the plantincorporated protectant Cry2Ab2 in or on corn or cotton when used as a plant-incorporated protectant in the food and feed commodities of corn or cotton (40 CFR 174.519). On July 16,Monsanto Company SY258-14E1-1 Page 11 of 31

2008, U.S. EPA established an exemption from the requirement of a tolerance for residues of the plant-incorporated protectant Cry1A.105 in or on corn when used as a plant-incorporated protectant in the food and feed commodities of corn (40 CFR 174.502). U.S. EPA reviewed and concluded on the safety of both proteins in its Biopesticide Registration Action Document for MON 89034 (U.S. EPA 2010). Therefore, MON 87751, though a new transformation event in soybean, contains the proteins and associated modes of action that have already received U.S. EPA tolerance exemptions and registrations for other crops. The safety data provided for the Cry1A.105 and Cry2Ab2 proteins demonstrate that neither protein exhibits amino acid sequence similarities to known allergenic or toxic proteins, both proteins are degraded in simulated gastric fluid, and both proteins lose functional activity after heating. In previous acute toxicity assays, neither protein was acutely toxic to mammals at the highest doses tested (>2,000 mg/kg body weight in mice) and based on estimated consumption and protein levels large margins of exposure (MOE) have been demonstrated for the Cry1A.105 and Cry2Ab2 proteins. These data support the safety of the Cry1A.105 and Cry2Ab2 proteins when used as Plant-incorporated Protectant which is applicable across crops as well as food and f e e d s o ur c e s

. F urth e r

m o r e , d u e t o th e l a c k o f m a

m m a l i an t o x i c i t y f

o r t h e δ

- e n d o t o x i n s t e s t e d a t very high doses, traditional pesticide residue studies are not considered necessary, therefore microbial B. thuringiensis pesticides that are registered in countries that require tolerances (Maximum Residue Levels) have been given an exemption from the requirement for setting a numerical tolerance (MRL) (OECD 2007). Safety studies for the Cry1A.105 protein are referenced by MRID below. Bonnette, K.L. 2005. An Acute Oral Toxicity Study in Mice with Cry1A.105 protein. Monsanto study number CRO-2005-050. MRlD 46694603 Gallagher, S.P. and J.B. Beavers. 2006. Evaluation of Potential Dietary Effects of MON 89034 with the Northern Bobwhite: an Eight-day Dietary Study with Corn Grain. Monsanto Technical Report WL-2005-012. MRID 46951427 Davis, S.W. 2006. Comparison of Broiler Performance and Carcass Parameters When Fed Diets Containing MON 89034, Control or Commercial Corn. Monsanto Study 05-01-50-13, Amended Report. MRID 46951412 MacRae, T.C., C.R. Brown, S.L. Levine. 2005. Evaluation of the Potential for Interactions Between the Bacillus Thuringiensis Proteins Cry1A.105 and Cry2Ab2. Monsanto Technical Report MSL-19859. MRID 46694602 Huesing, J.E., J.J. Duan, and S.L. Levine. 2006. Endangered Species Risk Assessment for Corn MON 89034. Monsanto Technical Report MSL0020394. MRID 46951429 Bogdanova, N.N. 2006. Human Health and Environmental Assessment of the Plant-Incorporated Protectant Bacillus thuringiensis Cry1A.105 and Cry2Ab2 Proteins Produced in Corn MON 89034. MRID 46951401Monsanto Company SY258-14E1-1 Page 12 of 31

Webb, Elizabeth. 2013. Acute Dietary Risk Assessment of Cry1A.105 and Cry2Ab2 Protein from Consumption of MON 87751 Soybean in the U.S. Monsanto technical report MSL0024993 MRID 49332001 Bachman, P.M., P.D. Jensen, S.L. Levine. 2014. Environmental Risk Assessment for Cry1A.105 and Cry2Ab2 Proteins as Expressed in MON 87751 Soybean. Monsanto technical report MSL0025537 MRID 49332002 Barlow, R., Wang, R., Hernan, R. 2013. Assessment of the in vitro Digestibility of Cry1A.105 Protein in Simulated Gastric and Simulated Intestinal Fluids. Monsanto technical report MSL0024977 MRID 49332009 Gu, X., Mueller, G. 2013. Effect of Heat Treatment on the Functional Activity of Escherichia coli ( E. coli )-Produced MON 87751 Cry1A.105 Protein. Monsanto technical report MSL0025092 MRID 49332011 Kang, H. T., Silvanovich, A. 2013. Bioinformatics Evaluation of the Cry1A.105 Protein in MON 87751 Utilizing the AD_2013, TOX_2013 and PRT_2013 Databases. Monsanto technical report MSL0024925 MRID 49332013 Kang, H. T., Silvanovich, A. 2013 Bioinformatics Evaluation of the Transfer DNA Insert in MON 87751 Utilizing the AD_2013, TOX_2013 and PRT_2013 Databases. Monsanto technical report MSL0024971 MRID 49332016 Mozaffar, S. 2013 Amended Report for MSL0024805: Assessment of Cry1A.105 and Cry2Ab2 Protein Levels in Soybean Tissues Collected from MON 87751 Produced in U.S. Field Trials during 2012. Monsanto technical report MSL0025199 MRID 49332004 Hartmann, A.J., K.E. Niemeyer, and A. Silvanovich. 2006. Assessment of the Cry1A.105 and Cry2Ab2 Protein Levels in Tissues of Insect-Protected Corn MON 89034 Produced in 2005 U.S. Field Trials. Monsanto Technical Report MSL-20285. MRID 46951403 Supplemental Information for MRID No. 46951403 “Assessment of the Cry1A.105 and Cry2Ab2 Protein Levels in Tissues of Insect-Protected Corn MON 89034 Produced in 2005 U.S. Field Trials” MRID 47127505 Kapadia, S. and E.A. Rice. 2005. Assessment of the in vitro Digestibility of the Cry1A.105 Protein in Simulated Intestinal Fluid. Monsanto Technical Report MSL-19930 MRID 46951408 Kapadia, S.A. and E.A. Rice. 2005. Assessment of the in vitro Digestibility of the Cry1A.105 Protein in Simulated Gastric Fluid. Monsanto Technical Report MSL-19929 MRID 46694606 Goley, M.E. and J.J. Thorp. 2005. Immunodetection of Cry2Ab2 and Cry1A.105 Proteins inCorn Grain from MON 89034 Following Heat Treatment. Monsanto Technical Report MSL19899. MRID 46694607Monsanto Company SY258-14E1-1 Page 13 of 31

References : Baum, J.A. 1998. Transgenic Bacillus thuringiensis . Phytoprotection 79:127-130. Baum, J.A., T.B. Johnson and B.C. Carlton. 1999. Bacillus thuringiensis : Natural and recombinant bioinsecticide products. Pages 189-209 in Methods in Biotechnology: Biopesticides: Use and Delivery. Volume 5. F.R. Hall and J.J. Menn (eds.). Humana Press Inc., Totowa, New Jersey. Betz, F.S., B.G. Hammond and R.L. Fuchs. 2000. Safety and advantages of Bacillus thuringiensis -protected plants to control insect pests. Regulatory Toxicology and Pharmacology 32:156-173. Federici, B.A. and J.P. Siegel. 2008. Safety assessment of Bacillus thuringiensis and Bt crops used in insect control. Pages 45-102 in Food Safety of proteins in Agricultural Biotechnology. B.G. Hammond (ed.). Taylor & Francis, Boca Raton, Florida. Gill, S.S., E.A. Cowles and P.V. Pietrantonio. 1992. The mode of action of Bacillus thuringiensis endotoxins. Annual Review of Entomology 37:615-636. Hammond, B. 2004. A review of the food/feed safety and benefits of Bacillus thuringiensis protein containing insect-protected crops. Pages 103-123 in ACS Symposium, American Chemical Society, Washington, D.C. Hammond, B.G. and S.K. Koch. 2012. A review of the food safety of bt crops. Bacillus thuringiensis Biotechnology pages 305-325 E. Sansinenea (ed.). Springer Science+Business Media, Heidelberg London, New York. IPCS. 1999. Environmental health criteria 217: Bacillus thuringiensis . World Health Organization, International Programme on Chemical Safety, Geneva, Switzerland. http://www.inchem.org/documents/ehc/ehc/ehc217.htm [Accessed May 14, 2009]. McClintock, J.T., C.R. Schaffer and R.D. Sjoblad. 1995. A comparative review of the mammalian toxicity of Bacillus thuringiensis -based pesticides. Pesticide Science 45:95-105. Mendelsohn, M., J. Kough, Z. Vaituzis and K. Matthews. 2003. Are Bt crops safe Nature Biotechology 21:1003-1009. OECD. 2007. Consensus document on safety information on transgenic plants expressing Bacillus thuringiensis -derived insect control proteins. ENV/JM/FOOD(2007)14. Organisation for Economic Co-operation and Development, Paris, France. Perlak, F.J., M. Oppenhuizen, K. Gustafson, R. Voth, S. Sivasupramaniam, D. Heering, B. Carey, R.A. Ihrig and J.K. Roberts. 2001. Development and commercial use of Bollgard ® cotton in the USA - Early promises versus today's reality. The Plant Journal 27:489-501.Monsanto Company SY258-14E1-1 Page 14 of 31

Pigott, C.R. and D.J. Ellar. 2007. Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiology and Molecular Biology Reviews 71:255-281. Schnepf, E., N. Crickmore, J. van Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Zeigler and D.H. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews 62:775-806. Siegel, J.P. 2001. The mammalian safety of Bacillus thuringiensis -based insecticides. Journal of Invertebrate Pathology 77:13-21. U.S. EPA. 2001. Biopesticides registration action document - Bacillus thuringiensis plantincorporated protectants. U.S. Environmental Protection Agency, Washington, D.C. http://www.epa.gov/pesticides/biopesticides/pips/bt_brad.htm [Accessed July 24, 2013]. U.S. EPA. 2005. Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary for its production (Vector ZMIR13L) in event MON 863 corn & Bacillus thuringiensis Cry1Ab delta-endotoxin and the genetic material necessary for its Monsanto Company SY258-13F1 102 of 141 production in corn (006430, 006484) fact sheet. U.S. Environmental Protection Agency, Washington, D.C. http://www.epa.gov/pesticides/biopesticides/ingredients_keep/factsheets/factsheet_006430- 006484.htm [Accessed September 23, 2013]. U.S. EPA. 2010. Biopesticides registration action document: Bacillus thuringiensis Cry1A.105 and Cry2Ab2 insecticidal proteins and the genetic material necessary for their production in corn [PC Codes 006515 (Cry2Ab2), 006514 (Cry1A.105)]. U.S. Environmental Protection Agency, Office of Pesticide Programs, Biopesticides and Pollution Prevention Division, Washington, D.C. Vachon, V., R. Laprade and J.-L. Schwartz. 2012. Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: A critical review. Journal of Invertebrate Pathology 111:1-12. Widner, W.R. and H.R. Whiteley. 1989. Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities. Journal of Bacteriology 171:965-974.Monsanto Company SY258-14E1-1 Page 15 of 31

Section D: Reports with Regard to the Nature and Amount of Plant-incorporated Protectant Remaining In or On the Food, Including a Description of Analytical Methods Used When used as a PIP, Cry1A.105 protein has been found to be present at a range of concentrations in tissues of corn and soybean plants. Results from studies to quantify concentrations of Cry1A.105 in MON 87751 Soybean is referenced below, this report also describes the method for a validated ELISA to detect the Cry1A.105 protein. In addition a quantitative PCR detection method is to detect MON 87751 referenced below and provided with this submission as a separate volume. EPA has reviewed results from studies to quantify concentrations of Cry1A.105 in MON 89034 corn, in addition a validated ELISA method developed to detect Cry1A.l05 protein is described in the report MRlD 46951403. Mozaffar, S. 2013 Amended Report for MSL0024805: Assessment of Cry1A.105 and Cry2Ab2 Protein Levels in Soybean Tissues Collected from MON 87751 Produced in U.S. Field Trials during 2012. Monsanto technical report MSL0025199 MRID 49332004 Smith, H. 2014 A Recommended Procedure for Real-Time Quantitative TaqMan PCR for MON 87751 Monsanto Report Hartmann, A.J., K.E. Niemeyer, and A. Silvanovich. 2006. Assessment of the Cry1A.105 and Cry2Ab2 Protein Levels in Tissues of Insect-Protected Corn MON 89034 Produced in 2005 U.S. Field Trials. Monsanto Technical Report MSL-20285. MRID 46951403 Supplemental Information for MRID No. 46951403 “Assessment of the Cry1A.105 and Cry2Ab2 Protein Levels in Tissues of Insect-Protected Corn MON 89034 Produced in 2005 U.S. Field Trials” MRID 47127505Monsanto Company SY258-14E1-1 Page 16 of 31

Section E: Practical Methods for Removing Plant-incorporated Protectant that Exceeds any Proposed Tolerance Such methods are not required because an exemption from the requirement of a tolerance is being sought.Monsanto Company SY258-14E1-1 Page 17 of 31

Section F: Proposed Tolerances for the Plant-incorporated Protectant if Tolerances are Proposed On July 16, 2008, U.S. EPA established an exemption from the requirement of a tolerance forresidues of the plant-incorporated protectant Cry1A.105 in or on corn when used as a plantincorporated protectant in the food and feed commodities of corn (40 CFR 174.502). A permanent exemption from the requirement of a tolerance for Cry1A.105 protein and the genetic material necessary for its production in all plants and when used as plant-incorporated protectant in all food and feed commodities is being sought. Monsanto proposes an amendment to 40 CFR174.502 to extend the exemption from the requirement of a tolerance to include the plantincorporated protectant, Bacillus thuringiensis Cry1A.105 protein and genetic material necessary for its production in all plants and when used as plant-incorporated protectant in all food and feed commodities.Monsanto Company SY258-14E1-1 Page 18 of 31

Section G: Practical Methods for Removing Residues That Exceed Proposed Tolerance A residue removal method is not necessary because an exemption from the requirement of a tolerance for Cry1A.105 protein residues is being sought.Monsanto Company SY258-14E1-1 Page 19 of 31

Section H: Practical Method for Detecting and Measuring the Levels of the Pesticide Chemical Residue A validated ELISA method developed to detect Cry1A.105 protein is described in the reports MRID 49332004 and MRlD 46951403, additionally a quantitative PCR detection method to detect MON 87751 is referenced below and provided with this submission as a separate volume. Mozaffar, S. 2013 Amended Report for MSL0024805: Assessment of Cry1A.105 and Cry2Ab2 Protein Levels in Soybean Tissues Collected from MON 87751 Produced in U.S. Field Trials during 2012. Monsanto technical report MSL0025199 MRID 49332004 Smith, H. 2014 A Recommended Procedure for Real-Time Quantitative TaqMan PCR for MON 87751 Monsanto Report Hartmann, A.J., K.E. Niemeyer, and A. Silvanovich. 2006. Assessment of the Cry1A.105 and Cry2Ab2 Protein Levels in Tissues of Insect-Protected Corn MON 89034 Produced in 2005 U.S. Field Trials. Monsanto Technical Report MSL-20285. MRID 46951403 Supplemental Information for MRID No. 46951403 “Assessment of the Cry1A.105 and Cry2Ab2 Protein Levels in Tissues of Insect-Protected Corn MON 89034 Produced in 2005 U.S. Field Trials” MRID 47127505Monsanto Company SY258-14E1-1 Page 20 of 31

Section I: Proposed Tolerances for Processed Food This section is not relevant as there are no regulated processed food items in the raw agricultural commodities for which tolerances are being proposed.Monsanto Company SY258-14E1-1 Page 21 of 31

Section J: Such Information as the Administrator May Require to Make This Determination All relevant information needed to make this determination has been provided and/or referencedin this petition and in the application for Section 3 New Product Registration of the PlantIncorporated Protectant, Bacillus thuringiensis Cry1A.105 and Cry2Ab2 Proteins and the Genetic Material (Vector PV-GMIR13196) Necessary for their Production in MON 87751 Soybean (EPA Reg. No.: 524-ARO) MRID 49332000.Monsanto Company SY258-14E1-1 Page 22 of 31

Section K: Information Regarding Potential Endocrine Effects of Residues on Humans The results of available toxicology studies do not reveal any potential for adverse endocrine effects associated with exposure to Cry1A.105 protein.Monsanto Company SY258-14E1-1 Page 23 of 31

Section L: Information Regarding Exposure to the Pesticide Chemical Residue Due to Any Tolerance or Exemption Already Granted On July 16, 2008, U.S. EPA established an exemption from the requirement of a tolerance forresidues of the plant-incorporated protectant Cry1A.105 in or on corn when used as a plantincorporated protectant in the food and feed commodities of corn (40 CFR 174.502). There are no tolerances granted for Cry1A.105 protein beyond 40 CFR 174.502.Monsanto Company SY258-14E1-1 Page 24 of 31

Section M: Information Concerning MRLs Established By Codex Alimentarius No maximum residue levels have been established by Codex Alimentarius for Cry1A.105 residues.Monsanto Company SY258-14E1-1 Page 25 of 31

Section N: Reasonable Grounds in Support of the Petition Support for this petition for an exemption from the requirement of a tolerance for Bacillus thuringiensis Cry1A.105 protein and the genetic material necessary for its production in all plants and in or on the food and feed commodities of all plants when used as a plant-incorporated protectant are presented in this petition and in the application for a Section 3 New Product Registration of the Plant-Incorporated Protectant, Bacillus thuringiensis Cry1A.105 and Cry2Ab2 Proteins and the Genetic Material (Vector PV-GMIR13196) Necessary for their Production in MON 87751 Soybean (EPA Reg. No.: 524-ARO) MRID 49332000

. On July 16, 2008, U.S. EPA established an exemption from the requirement of a tolerance for residues of the plant-incorporated protectant Cry1A.105 in or on corn when used as a plant-incorporated protectant in the food and feed commodities of corn (40 CFR 174.502). A permanent exemption from the requirement of a tolerance for Cry1A.105 protein and the genetic material necessary for its production in all plants and when used as plant-incorporated protectant in all food and feed commodities is being sought.Monsanto Company SY258-14E1-1 Page 26 of 31 Attachment I. Petitioner’s Notice of Filing Summary A summary of this pesticide petition for an exemption from the requirement of a tolerance for residues of the plant-incorporated protectant, Bacillus thuringiensis Cry1A.105 protein, in all plants and when used as plant-incorporated protectant in all food and feed commodities is appended in the following pages. Monsanto consents to the use of all or part of this summary by the Agency in preparing the notice of filing and the proposed or final regulation. This summary is intended for inclusion in the Federal Register Notice of Filing for this tolerance extension petition. An electronic copy of this petition summary in MS-Word format is also being provided on a CD-ROM, and Monsanto authorizes the public disclosure of this summary as part of the notice of filing of the petition.Monsanto Company SY258-14E1-1 Page 27 of 31

EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION COMPANY NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL REGISTER EPA Biopesticides and Pollution Prevention Division contact: : [insert name and telephone number with area code] Monsanto Company [Insert petition number] EPA has received a pesticide petition ([insert petition number]) from Monsanto Company proposing, pursuant to section 408(d) of the Federal Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 346a(d), to amend 40 CFR part 174 1. NA-Remove 2. NA-Remove 3. to establish an amendment/expansion of an existing tolerance exemption for the 1. NA-Remove 2. NA-Remove 3. plant-pesticide Bacillus thuringiensis Cry1A.105 protein in or on all plants. Pursuant to section 408(d)(2)(A)(i) of the FFDCA, as amended, Monsanto Company, 800 North Lindbergh Blvd., Saint Louis, MO 63198 has submitted the following summary of information, data, and arguments in support of their pesticide petition. This summary was prepared by Monsanto and EPA has not fully evaluated the merits of the pesticide petition. The summary may have been edited by EPA if the terminology used was unclear, the summary contained extraneous material, or the summary unintentionally made the reader conclude that the findings reflected EPA’s position and not the position of the petitioner.

I. Monsanto Petition Summary [Insert petition number] A. Product Name and Proposed Use Practices This notice of filing summarizes the information submitted by Monsanto Company in support of a request for an exemption from the requirement of a tolerance for the Bacillus thuringiensis Cry1A.105 protein and the genetic material necessary for its production in all plants. Monsanto Company has developed biotechnology-derived soybean MON 87751 (OECD unique identifier MON-87751-7) that provides protection from feeding damage caused by targeted lepidopteran insect pests. MON 87751 produces the CrylA.105 and Cry2Ab2 insecticidal (Cry) proteins derived from Bacillus thuringiensis . The Cry1A.105 protein is closely related to the Cry1A class of proteins, which has well-established human and animal safety. BothMonsanto Company SY258-14E1-1 Page 28 of 31

the Cry1A.105 and Cry2Ab2 proteins have been fully characterized and no adverse effects were observed for human health and environmental safety. B. Product Identity/Chemistry 1. Identity of the pesticide and corresponding residues. Cry1A.105 protein is a chimeric protein comprised of domains I, II and C-terminal from the Cry1Ac (subsp. kurstaki ), and domain III from Cry1F (subsp. aizawai ). Domains I and II of the Cry1A.105 share 100% amino acid sequence homology with the domains I and II of the Cry1Ac and Cry1Ab proteins. Amino acid sequence of domain III of the Cry1A.105 protein is 99% identical to the corresponding domain of Cry1F. The overall amino acid sequence identity to the Cry1Ab, Cry1Ac, and Cry1F proteins is 90.0%, 93.6%, and 76.7% respectively. The Cry1A.105 protein has been previously produced in corn (MON 89034); the protein's safety has been fully characterized (U.S. EPA 2010). The Cry1A.105 protein has been incorporated to provide protection from feeding damage caused by targeted lepidopteran insect pests.

2. Magnitude of residues at the time of harvest and method used to determine the residue. A determination of the magnitude of residues at harvest is not required for residues that are exempt from tolerances.

3. A statement of why an analytical method of detecting and measuring the levels of the pesticide residue are not needed. This petition is an exemption from the requirement of a tolerance without numerical limitation, thus an analytical detection method should not be required. However, validated ELISA methods are developed to detect Cry1A.105 protein in MON 89034 and MON 87751 as well as a quantitative PCR method for detection of MON 87751 soybean is available. C. Mammalian Toxicological Profile The Cry1A.105 protein that is produced has an insecticidal mode-of-action that is well understood. B. thuringiensis proteins have been used as safe and effective pest control agents in microbial formulations for many years. The numerous toxicology studies conducted with these microbial products show no significant adverse effects and demonstrate that the products are nontoxic to mammals. The specificity of the Bt Crystal (Cry) proteins activity is dependent upon their binding to specific receptors present in the insect mid-gut (OECD, 2007; Pigott and Ellar, 2007). These specific receptors have not been found in vertebrate taxa, therefore the Cry proteins are not expected to adversely affect mammals additionally, no adverse effects have been reported in mammals, birds, amphibians or reptiles (OECD, 2007; Schnepf, et al., 1998). Data have been submitted demonstrating the lack of mammalian toxicity at high levels of exposure to purified Cry1A.105 protein. These data demonstrate the safety of the protein at levels well above maximum possible exposure levels that are reasonably anticipated in crops. Furthermore, amino acid sequence comparisons of the Cry1A.105 protein to known toxic proteins showed no similarities.Monsanto Company SY258-14E1-1 Page 29 of 31

The Cry1A.105 protein does not share significant amino acid sequence similarity to known protein allergens. The Cry1A.105 protein is not derived from a source organism known to produce allergenic proteins. Additionally, despite decades of widespread use of B. thuringiensis insecticides on food crops, there have been no confirmed reports of oral allergies to these preparations, and laboratory animal studies submitted to the Agency have not shown any potential for allergic reactions to B. thuringiensis or its components. No significant amino acid sequence similarities between Cry1A.105 and known or putative allergenic protein sequences were identified. Furthermore, the Cry1A.105 protein lose functional activity upon heating and is rapidly digested in simulated gastric fluids. Based on this evidence, Cry1A.105 protein is considered unlikely to be food allergen. The genetic material encoding the Cry1A.105 protein and the regulatory regions controlling expression of the cry1A.105 gene are nucleic acids (DNA and RNA). DNA and RNA occur in all forms of plant and animal life and there is no documented instance of nucleic acids being associated with toxic effects when consumed as a component of food. DNA and RNA are in the category of substances generally recognized as safe (GRAS). EPA exempts nucleic acids that are part of a plant-incorporated protectant from the requirement of a tolerance (40 CFR 174.507). Data characterizing the genetic material necessary for the production of Cry1A.105 in corn and soybean were provided to the Agency. No mammalian toxicity is anticipated fromdietary exposure to the genetic material necessary for production of the subject plantincorporated protectant. Collectively these data support a conclusion that Cry1A.105 protein will be nontoxic to humans and is unlikely to be a food allergen. There is reasonable certainty that no harm will result from mammalian (including human) exposure to the Cry1A.105 protein. D. Aggregate Exposure 1. Dietary exposure. Cry1A.105 is a plant-incorporated protectant in soybean, thus dietary exposure is deemed to be the most relevant route for assessing human risk.
i. Food. Monsanto has considered available information on the aggregate exposure levels to consumers and major identifiable groups of consumers to the protein residue and related substances. Oral exposure may occur at very low levels from ingestion of processed corn andsoybean products. However, a lack of mammalian toxicity and the rapid digestibility of the plantincorporated protectants have been demonstrated.

ii. Drinking Water Exposure. Oral exposure from ingestion of drinking water is unlikely because the proteins are present at low levels within the plant and previously submitted studies demonstrate that the Cry1A.105 protein is rapidly degraded in soil and, therefore, is not present in drinking water.

2. Non-dietary exposure

. Exposure via skin or inhalation is not likely because the plantincorporated protectants are contained within plant cells, which essentially eliminates these exposure routes or reduces them to negligible levels. The use sites for the Cry1A.105 protein areMonsanto Company SY258-14E1-1 Page 30 of 31

agricultural for control of insects. Exposure to workers and bystanders resulting from plant pesticides will be negligible and unlikely to add measurably to any exposure resulting from microbial or other Bacillus thuringiensis formulations. Exposure to infants and children via residential or lawn use is not expected. E. Cumulative Effects There is no indication of mammalian toxicity potential for Cry1A.105 protein. Because there is no indication of mammalian toxicity to the plant-incorporated protectants and the Cry1 class proteins is specific for lepidopteran insects (Crickmore, et al. 1998; de Maagd, et al. 2001; Romeis, et al. 2006) it is reasonable to conclude that there will be no cumulative effects for this protein. F. Safety Determination

1. U.S. population. Sufficient data have been submitted to assess the health risk of Cry1A.105 protein and the genetic material necessary for its production in all food and feed commodities. The absence of toxicity in high dose acute oral studies, the lack of sequence homology with known protein toxins, rapid digestion in a simulated gastric fluids, and minimal allergenicity potential provide a reasonable certainty of no harm for the U.S. general population potentially exposed to the Cry1A.105 protein.

2. Infants and children

. Non-dietary exposure to infants and children is not anticipated due to the patterns of use for these plant-incorporated protectants. The previously submitted data provide no evidence of adverse threshold effects for the Cry1A.105 protein that would warrant application of an additional safety factor for the protection of infants and children. Furthermore, the provisions for consumption patterns, special susceptibility, and cumulative effects do not apply. G. Effects on the Immune and Endocrine Systems The available safety data for Cry1A.105 protein show no adverse effects in mammals at high levels of exposure; thus, no effects on mammalian immune or endocrine systems are predicted. The lack of Cry1A.105 homology to known toxins and allergens and their rapid degradation in simulated gastric fluids suggests minimal risk for adverse effects on the immune system. Furthermore, the Cry1A.105 protein is derived from B. thuringiensis and B. thuringiensis is known not to exert an influence on the endocrine system. H. Existing Tolerances On July 16, 2008, U.S. EPA established an exemption from the requirement of a tolerance for residues of the plant-incorporated protectant Cry1A.105 (40 CFR 174.502) in corn or on the food and feed commodities of corn. The subject of Monsanto’s request is to extend this exemption from the requirement of a tolerance for the PIP, Bacillus thuringiensis Cry1A.105protein and genetic material necessary for its production to all plants and when used as plantincorporated protectant in all food and feed commodities.Monsanto Company SY258-14E1-1 Page 31 of 31

I. International Tolerances No Codex maximum residue levels exist for B. thuringiensis Cry1A.105 protein in plants.