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 
Monsanto 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 

Submitter Date 

Debbie Mahadeo, B.S. 

Monsanto Company 

Regulatory Affairs Manager 
Monsanto 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 26
Monsanto 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

and feed sources. Furthermore, due to the lack of mammalian toxicity for the δ-endotoxins

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 46694604
Monsanto 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

feed sources. Furthermore, due to the lack of mammalian toxicity for the δ-endotoxins tested at

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 46951401
Monsanto 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 in

Corn Grain from MON 89034 Following Heat Treatment. Monsanto Technical Report MSL19899. MRID 46694607
Monsanto 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 47127505
Monsanto 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 for

residues 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 CFR

174.502 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 47127505
Monsanto 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 referenced

in 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 for

residues 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. Both
Monsanto 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 from

dietary 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 and

soybean 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 are
Monsanto 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.105

protein 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.