






                Draft BIOPESTICIDE REGISTRATION ACTION DOCUMENT
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                        PLANT-INCORPORATED PROTECTANTS
                                       

Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 Proteins and the Genetic Material (Vector PV-GMIR13196) Necessary For Their Production in MON 87751 (OECD Unique ID. MON-87751-7) Soybean [PC Code 006614 and PC Code 006527]







                        
                        
                        U.S. Environmental Protection Agency
                        Office of Pesticide Programs
                        Biopesticides and Pollution Prevention Division
                        
                        
                        
                        March 5, 2015

Table of Contents
I. Overview	4
   A. Executive Summary	4
   B. Use Profile	6
   C. Regulatory History	6
II. Science Assessment	7
   A. Product Characterization	7
   B. Transformation System	7
   C. Characterization of the DNA Inserted in the Plant and its Inheritance and Stability	7
   D. Protein Characterization and Equivalence	7
   E. Protein Expression	9
   F. Analytical Method	12
   G. Overall Safety Conclusion	12
   H. Supporting Data	12
III. Human Health Assessment of Cry1A.105 and Cry2Ab2 as Expressed in Mon 87751 Soybean	19
   A. Mammalian Toxicity and Allergenicity Assessment- Cry1A.105	19
   B. Mammalian Toxicity and Allergenicity Assessment Cry2Ab2	21
   C.	Determination of Safety for U.S. Population, Infants and Children Toxicity and Allergenicity Conclusions (Cry1A.105 and Cry2Ab2)	23
   D. Overall Safety and Conclusion (Cry1A.105 and Cry2Ab2)	24
   E. Other Consideration (Cry1A.105 and Cry2Ab2)	24
IV. References	29
V. Environmental Risk Assessment for Mon 87751	30
   A. Overall Conclusions	30
   B. Nontarget Organism Tiered Testing and Risk Assessment Process for PIPs	30
   C.	Nontarget Organism Risk Assessment for MON 87751 Soybeans	35
VI.	Risk Assessment Conclusions for MON 87751 Soybeans	43
   A. Effects to Nontarget Wildlife, Invertebrates, and Plants	43
   B. Horizontal Transfer of Transgenes from Bt Crops to Soil Organisms	44
   C.	Gene Flow and Weediness Potential	44
   D.	Impacts on Endangered Species	45
   E.	Conclusions	46
VII. Insect Resistance Management	46
VIII. References	48
IX. Terms and Conditions of Registration	51
X. Regulatory Position for Mon 87751 (Cry1A.105 and Cry2Ab2) Soybean	51


                        
BIOPESTICIDE REGISTRATION ACTION TEAM Office of Pesticide Programs:

Biopesticides and Pollution Prevention Division


Product Characterization and Human Health

Judy Facey, Ph.D.
John Kough, Ph.D. 

Environmental Fate and Effects

In-Soon You, Ph.D.
Shannon Borges, M.S.

Insect Resistance Management

Alan Reynolds, M.S. Jeannette Martinez, Ph.D.

Registration Support

Kenneth Haymes, Ph.D.
Alan Reynolds, M.S.


I. Overview

A. Executive Summary

The United States Environmental Protection Agency (U.S. EPA) proposes to register two new Bacillus thuringiensis (Bt) soybean plant-incorporated protectant (PIP) active ingredients as described in this Biopesticide Registration Action Document (BRAD). 

Monsanto Company (hereafter, "Monsanto") has submitted an application for a FIFRA Section 3 Seed Increase Registration of Event MON 87751 (MON 87751), a new Bt soybean (Glycine max (L.) Merr.), which produces Bt-derived insecticidal proteins Cry1A.105 and Cry2Ab2 as Plant Incorporated Protectants (PIPs). A PIP is defined as a pesticidal substance that is intended to be produced and used in a living plant, and the genetic material necessary for production of such a pesticidal substance (40 CFR Part 174.3).  Two new active ingredients (a.i.s), Cry1A.105 and Cry2Ab2 insect control proteins expressed in MON 87751 soybean, provide protection from feeding damage caused by key lepidopteran soybean pests, including the bean shoot moth (Crocidosema aporema), sunflower looper (Rachiplusia nu) and fall armyworm (Spodoptera frugiferda). Cry1A.105 and Cry2Ab2 proteins are also active against other lepidopteran soybean pests such as the velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens) and corn earworm (Helicoverpa zea).

Product Characterization

MON 87751 was produced by Agrobacterium tumefaciens-mediated transformation of soybean (Glycine max) tissue using the 2T-DNA transformation plasmid vector PV-GMIR13196. The Cry1A.105 and Cry2Ab2 proteins provide protection from feeding damage caused by targeted lepidopteran insect pests.

Mammalian Toxicity and Allergenicity Assessment

Monsanto has developed insect-protected soybean MON 87751 that produces the Cry1A.105 and Cry2Ab2 insecticidal crystal (Cry) proteins (δ- endotoxin) derived from Bacillus thuringiensis (Bt) subsp. kurstaki.  EPA will establish an exemption from the requirement of a tolerance for Cry1A.105 and Cry2Ab2 proteins in soybean when used as a plant-incorporated protectant.

Based on the review and consideration of all the available information, there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to residues of the Cry1A.105 and Cry2Ab2 proteins and the genetic material necessary for their production in soybean. This includes all anticipated dietary exposures and all other exposures for which there is reliable information.  The Agency has arrived at this conclusion because no toxicity to mammals has been observed, and there is no indication of allergenicity potential for the plant- incorporated protectant from available information. 


Environmental Assessment

EPA does not anticipate adverse effects to nontarget organisms, including birds, wild mammals, freshwater and marine/estuarine fish and invertebrates, nontarget insects, honey bees, soil invertebrates, and terrestrial and aquatic plants. EPA also does not anticipate horizontal gene transfer, gene flow, and the development of weediness. Based on these findings and information on habitat requirements of federally-listed threatened and endangered lepidopteran species, the Agency also makes a No Effect determination for direct and indirect effects to threatened and endangered species and their habitats.

Insect Resistance Management

Given the low acreage permitted for MON 87751 (<15,000 total acres per year), it is unlikely that there will be a significant risk of resistance to the main soybean target pests in the United States, soybean looper and velvetbean caterpillar. This conclusion is further supported by the biology of the target insects, which do not overwinter in non-tropical areas and are polyphagous (feeding on a number of wild hosts and cultivated crops). In addition, the registration will have an acreage limitation which will further reduce the risk of resistance.

Public Participation

On October 1, 2009, the EPA announced a policy to provide a more meaningful opportunity for the public to participate in major registration decisions before they occur. According to this policy, the EPA provides a public comment period prior to making a registration decision for the following types of applications: new active ingredients; first food uses; first outdoor uses; first residential uses; or any other registration actions for which the EPA believes there may be significant public interest.

Consistent with the policy of making registration actions more transparent, the proposed plant-incorporated protectant containing the Cry1A.105 and Cry2Ab2 proteins as new active ingredients, will be subject to a comment period. The docket identification number, which is associated with this registration action and can be accessed through http://www.regulations.gov, is EPA-HQ-OPP-2014-0456. The following document is available for comment in EPA-HQ-OPP-2014-0456: (1) draft Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 Proteins and the Genetic Material (Vector PV-GMIR13196) Necessary For Their Production in MON 87751 (OECD Unique ID. MON-87751-7) Soybean [PC Code 006614 and PC Code 006527]. While a final decision on registration is contingent upon review and consideration of public comments, the EPA presently believes that, based upon the risk assessment and the supporting material, it is appropriate to issue this registration. The basis for this preliminary decision can be found in the risk assessment for Mon 87751 Soybean, which is characterized throughout this BRAD.


B. Use Profile
      
      Active Ingredient:	Bacillus thuringiensis Cry1A.105 and Cry2Ab2 insecticidal proteins and the genetic material necessary for its production in soybean
      Trade and Other Name(s):  MON 87751
      
      OPP Chemical Codes (PC Codes): 	006614 and 006615
      Company Name:	Monsanto Company
                  800 North Lindbergh Blvd. 
                  St. Louis, MO 63167
      
      Type of Pesticide:  Plant-incorporated Protectant
      
      Uses: Soybean, Limited to seed increase and a total of 15,000 acres for breeding purposes, agronomic testing, and producing seed in the United States and the Commonwealth of Puerto Rico.
      
      Target Pests for Active Ingredient:  Lepidopteran soybean pests, including the bean shoot moth (Crocidosema aporema), sunflower looper (Rachiplusia nu) and fall armyworm (Spodoptera frugiferda), velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens) and corn earworm (Helicoverpa zea).

C. Regulatory History

On August 28, 2014 (79 FR 63596), EPA published a Notice of Receipt in the Federal Register announcing that Monsanto submitted an application to register two new active ingredients, Bt Cry1A.105 and Cry2Ab2 insecticidal proteins expressed in soybean (Mon 87751 Soybean; EPA File Symbol 524-619) not included in any currently registered pesticide products. 
Concurrently with the FIFRA section 3 application, Monsanto submitted two petitions (PP 4F8275 and PP 4F8276) to establish a permanent exemption from the requirement of a tolerance for residues of Bacillus thuringiensis Cry1A.105 and Cry2Ab2 proteins in soybean. EPA published a Notice of Filing in the Federal Register, initially on October 24, 2014 (79 FR 63596) and again on December 17, 2014 for Cry1A.105 (79 FR 75111) and on January 28, 2015 for Cry2Ab2 (80 FR 4527) and the public was given a 30-day comment period. One public comment was received in response to this publication.

II. Science Assessment
A. Product Characterization
Monsanto has developed insect-protected soybean MON 87751 that produces the Cry1A.105 and Cry2Ab2 insecticidal crystal (Cry) proteins (δ- endotoxin) derived from Bacillus thuringiensis (Bt) subsp. kurstaki. The Cry1A.105 and Cry2Ab2 proteins provide protection from feeding damage caused by targeted lepidopteran insect pests. 

B. Transformation System
MON 87751 was produced by Agrobacterium tumefaciens-mediated transformation of soybean tissue using the 2T-DNA transformation plasmid vector PV-GMIR13196.  PV-GMIR13196 contains two separate T-DNAs that are each delineated by left and right border regions. The first T-DNA, designated as T-DNA I, contains the CrylA.105 coding sequence regulated by the Act2 promoter and RbcS4 terminator; and the Cry2Ab2 coding sequence regulated by the Mt promoter and Ptl terminator. The second T-DNA, designated as T-DNA II, contains the aadA coding sequence regulated by FMV enhancer, EF- Iα promoter and E9 terminator; and the splA coding sequence regulated by the Usp promoter and nos terminator. During transformation, both T-DNAs were inserted into the soybean genome. Subsequently, traditional breeding, segregation, selection and screening were used to isolate those plants that contain the CrylA.105 and Cry2Ab2 expression cassettes (T-DNA I) and do not contain the aadA and splA expression cassettes (T-DNA II), resulting in the production of marker-free, MON 87751.

C. Characterization of the DNA Inserted in the Plant and its Inheritance and Stability
Bioinformatics analysis of next-generation sequence data (comprehensively covering the genome) was used to characterize the insert number of any DNA sequences from the transformation plasmid vector PV-GMIR13196. The data indicated that MON 87751 contains a single integration locus for the DNA insert. Directed sequence data (generated from PCRs targeted at a specific locus) was used to demonstrate that the single DNA insert in MON 87751 contains a single copy of T-DNA 1 from the transformation plasmid vector PV-GMIR13196 and confirmed the integrity of the CrylA.105 and Cry2Ab2 expression cassettes in the T-DNA I insert.  The data also showed that MON 87751 does not contain any sequences from the vector backbone or T-DNA II sequences of PV-GMIR13196. The directed sequencing data also identified the 5' and 3' insert-to-flank junctions in MON 87751 and characterized the insertion site in conventional soybean. Furthermore, bioinformatics analysis of next-generation sequence data (comprehensively covering the genome) was used to evaluate the stability of the DNA insert and demonstrated that the DNA insert in MON 87751 was stably maintained across five generations.

D. Protein Characterization and Equivalence

1.	Cry1A. 105: 
The level of the Cry1A.105 protein in the seed of MON 87751 soybean is relatively low. Therefore, the protein was produced in a bacterial expression system in order to yield a sufficient quantity of the protein for safety testing.  The total protein concentration of the purified MON 87751-produced CrylA.105 protein was measured using the Bio-Rad Protein Assay and was determined to be 0.12 mg/ml.  Apparent molecular weight (MW) and Purity of the MON 87751-produced CrylA.105 protein were determined using SDS-PAGE analysis. The apparent MW was 132.9 kDa and the purity of the MON 87751-produced CrylA.105 protein was 60%.  Identity was confirmed by N-terminal sequence analysis, matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) analysis of trypsin digested CrylA.105 protein, and western blot analysis. The expected N-terminal sequence was observed. Several peptide masses consistent with expected peptide masses for the trypsin digested CrylA.105 protein were observed. The identified peptide masses yielded coverage of 53.5% (632 out of 1181 amino acids) of the expected sequence of the CrylA.105 protein. Anti-Cry1A.105 antibodies recognized the MON 87751-produced CrylA.105. The functional activity of the MON 87751-produced CrylA.105 was determined using a corn earworm diet incorporation assay. The MON 87751  - produced CrylA.105 was shown to be active, with an EC50 of 0.0035 ug CrylA.105/ml diet.  Analysis of the glycosylation status showed that the MON 87751-produced CrylA.105 protein was not glycosylated.

The equivalence of the MON 87751-produced and E. coli-produced CrylA.105 protein was assessed by comparing their apparent MW, immunoreactivity, functional activity, and glycosylation status. Based on these characteristics, the characterized E. coli- produced protein was established to be equivalent to the Cry1A.105 protein isolated from seed of MON 87751 soybean.

      2. Cry2Ab2
The level of the Cry2Ab2 protein in the seed of MON 87751 soybean is relatively low. Therefore, the protein was made in a bacterial expression system in order to produce a sufficient quantity of the protein for safety testing.  The total protein concentration of the purified MON 87751-produced Cry2Ab2 protein was measured using the Bio-Rad Protein Assay and was determined to be 0.40 mg/ml. Apparent molecular weight (MW) and purity of the MON 87751-produced Cry2Ab2 protein were determined using SDS-PAGE analysis. The apparent MW of the MON 87751-produced Cry2Ab2 protein was 61.4 kDa, and the purity of the MON 87751-produced Cry2Ab2 protein was 29%.  Identity was confirmed by N-terminal sequence analysis, matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis of the trypsin digested Cry2Ab2 protein, and western blot analysis. The N-terminal sequence was confirmed using automated Edman degradation chemistry and MALDI-TOF MS analysis. MALDI-TOF MS analysis also produced several peptide masses consistent with expected peptide masses for the trypsin digested Cry2Ab2 protein. The identified peptide masses yielded coverage of 66% (406 out of 619 amino acids) of the expected sequence of the Cry2Ab2 protein. Anti-Cry2Ab2 antibodies detected the MON 87751-produced Cry2Ab2. The functional activity of the MON 87751-produced Cry2Ab2 was determined using a corn earworm diet incorporation assay. The MON 87751-produced Cry2Ab2 protein was shown to be active, with an EC50 of 0.0734 ug Cry2Ab2/ml diet. Analysis of the glycosylation status showed that the MON 87751-produced Cry2Ab2 protein was not glycosylated.

The equivalence of the MON 87751-produced and E. coli-produced Cry2Ab2 protein was assessed by comparing their apparent MW, immunoreactivity, functional activity, and glycosylation status. Based on these characteristics, the characterized E. coli produced protein was established to be equivalent to the Cry2Ab2 protein isolated from seed of MON 87751.

E. Protein Expression
Enzyme-linked Immunosorbent Assay (ELISA) analyses quantified the amount of Cry1A.105 and Cry2Ab2 protein concentrations expressed in Over season leaf (OSL1-4), root, seed, and forage tissue samples collected from MON 87751 Soybean at various growth stages.  The results of the ELISA analysis across field sites showed the majority of the mean Cry1A.105 protein level in MON 87751 as highest in OSL4 at 790 ug/g dwt and lowest in root which was below limit of detection (<LOD). The mean

CrylA.105 protein level in MON 87751 was 2.4 ug/g dry weight (dwt) in seed. The mean CrylA.105 protein level in MON 87751 was 11 ug/g fresh weight (fwt) in pollen/anther.  The mean Cry2Ab2 protein level in MON 87751 across all sites was highest in OSL3 at 32 ug/g dwt and lowest in seed at 4.0 ug/g dwt. The mean Cry2Ab2 protein level in MON 87751 was 7.7 ug/g fwt in pollen/anther. Summary results are provided below in Tables 1 and 2. 
Table 1. Summary of Cry1A.105 Protein Levels in Soybean Tissues from MON 87751 Produced in United States Field Trials During 2012
Tissue Type[1]
                                  Development
                                    Stage2
                                   Mean(SD)
                                     Range
                                (μg/g fwt)[3]
                                   Mean(SD)
                                     Range
                                (μg/g dwt)[4]
                                   LOQ/ LOD
                                (μg/g fwt)[5]

                                       
                                       
                                       
                                       
OSL1
                                   V3  -  V4
                                   130 (50)
                                   580 (250)
                                  1.500/0.406

                                       
                                  61  -  220
                                 260  -  1100
                                       

OSL2
                                   V5  -  V7
                                   120 (54)
                                   590 (270)
                                  1.500/0.406

                                       
                                  13  -  220
                                  68  -  1100
                                       

OSL3
                                   R2  -  R3
                                    79 (45)
                                   400 (220)
                                  1.500/0.406

                                       
                                  8.5  -  160
                                  50  -  780
                                       

OSL4
                                      R6
                                   230 (82)
                                   790 (280)
                                  1.500/0.406

                                       
                                  120  -  480
                                 430  -  1600
                                       

Root
                                      R6
                                 <LOD (N/A)
                                   N/A (N/A)
                                  0.563/0.322

                                       
                                  N/A  -  N/A
                                  N/A  -  N/A
                                       

Forage
                                      R6
                                    62 (21)
                                   230 (91)
                                  1.500/0.524

                                       
                                  31  -  110
                                  110  -  440
                                       

Seed
                                      R8
                                  2.1 (0.46)
                                  2.4 (0.50)
                                  0.900/0.226

                                       
                                  1.5  -  2.9
                                  1.7  -  3.2
                                       

Pollen/Anther
                                      R2
                                   11 (N/A)
                                   N/A (N/A)
                                 1.500/N.D.[6]

                                       
                                  N/A  -  N/A
                                  N/A  -  N/A


1.  OSL= over season leaf
2.  The crop development stage each tissue was collected.
3.  Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of protein per gram (g) of tissue on a fresh weight basis (fwt).  The means, SD, and ranges (minimum and maximum values) were calculated for each tissue across all sites (n=20 except for OSL1 where n=19 due to one sample expressing <LOD and pollen where n=1).  N/A: Not Applicable
4.  Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of protein per gram (g) of tissue on a dry weight basis (dwt).  The dry weight values were calculated by dividing the μg/g fwt by the dry weight conversion factor obtained from moisture analysis data except for pollen tissue.
5.  LOQ = limit of quantitation; LOD = limit of detection.
6.  N.D. = Not determined.  Pollen/anther LOD was not determined due to an insufficient amount of tissue.
Table 2. Summary of Cry2Ab2 Protein Levels in Soybean Tissues from MON 87751 Produced in United States Field Trials During 2012
Tissue Type[1]
                                  Development
                                   Stage[2]
                                   Mean(SD)
                                     Range
                                (μg/g fwt)[3]
                                   Mean(SD)
                                     Range
                                (μg/g dwt)[4]
                                    LOQ/LOD
                                (μg/g fwt)[5]

                                       
                                       
                                       
                                       
OSL1
                                   V3  -  V4
                                  5.4 (0.74)
                                   24 (5.9)
                                  0.625/0.034


                                  4.4  -  6.8
                                   17  -  37
                                       

OSL2
                                   V5  -  V7
                                  5.2 (0.70)
                                   26 (3.1)
                                  0.625/0.034


                                  4.0  -  6.6
                                   20  -  33
                                       

OSL3
                                   R2  -  R3
                                  6.3 (0.80)
                                   32 (5.2)
                                  0.625/0.034


                                  5.2  -  8.0
                                   25  -  43
                                       

OSL4
                                      R6
                                  6.9 (0.79)
                                   24 (2.7)
                                  0.625/0.034


                                  5.5  -  8.5
                                   18  -  29
                                       

Root
                                      R6
                                   4.6 (1.0)
                                   15 (2.7)
                                  1.250/1.241


                                  3.1  -  7.1
                                   11  -  22
                                       

Forage
                                      R6
                                  3.9 (0.60)
                                   14 (2.2)
                                  0.313/0.060


                                  3.0  -  5.1
                                   11  -  18
                                       

Seed
                                      R8
                                  3.6 (0.71)
                                  4.0 (0.77)
                                  0.313/0.094


                                  2.3  -  4.7
                                  2.6  -  5.1
                                       

Pollen/Anther
                                      R2
                                   7.7 (N/A)
                                   N/A (N/A)
                                 0.313/N.D.[6]


                                  N/A  -  N/A
                                  N/A  -  N/A

1.  OSL= over season leaf
2.  The crop development stage each tissue was collected.
3.  Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of protein per gram (g) of tissue on a fresh weight basis (fwt).  The means, SD, and ranges (minimum and maximum values) were calculated for each tissue across all sites (n=20 except for OSL1 where n=19 due to one sample expressing <LOD and pollen/anther where n=1).  N/A: Not Applicable
4.  Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of protein per gram (g) of tissue on a dry weight basis (dwt).  The dry weight values were calculated by dividing the μg/g fwt by the dry weight conversion factor obtained from moisture analysis data except for pollen tissue.
5.  LOQ = limit of quantitation; LOD = limit of detection
6.  N.D. = Not determined. Pollen/anther LOD was not determined due to an insufficient amount of tissue.

F. Analytical Method
ELISA plates were analyzed on a SPECTRAmax Plus 384 (Molecular Devices, Sunnyvale, CA) microplate spectrophotometer, using a dual wavelength detection method.  Protein concentrations were determined by optical absorbance at a wavelength of 450 nm with a simultaneous reference reading of 620 nm.  Data reduction analyses were performed using Molecular Devices SOFTmax PRO GxP version 5.4 software.  Absorbance readings and protein standard concentrations were fitted with a four-parameter logistic curve fit for Cry1A.105 protein.  Absorbance readings and protein standard concentrations were fitted with a five-parameter logistic curve fit for Cry2Ab2 protein.  Following the interpolation from the standard curve, the amount of protein (ng/ml) in the tissue was reported on a "g/g fwt" basis for data that were greater than or equal to the LOQ.  This conversion utilized a sample dilution factor and a tissue-to-buffer ratio.  The protein values in "g/g fwt" were also converted to "g/g dwt" by applying the DWCF, except for expression levels in pollen/anther. 

G. Overall Safety Conclusion
Cry proteins have been found as components of topical Bt microbial pesticides for over 45 years and are widely considered to be non-toxic to humans and other mammalian species (Betz et al., 2000; EPA, 2000; OECD, 2007). The CrylA.105 and Cry2Ab2 proteins produced by MON 89034 are closely related to the Cry proteins that have been used as the active ingredients in existing Bt microbial pesticides and/or biotechnology-derived crops. MON 87751 produces the Cry1A.105 and Cry2Ab2 insecticidal proteins. The Cry1A.105 and Cry2Ab2 proteins are individually expressed in currently registered PIP event MON 89034 maize (EPA Reg. No. 524-575). In addition, the safety data provided for the Cry1A.105 and Cry2Ab2 proteins demonstrate that neither protein exhibits amino acid sequence similarities to know allergenic or toxic proteins, both proteins are degraded in simulated gastric fluid, and both proteins lose functional activity after heating.  

The data submitted and reviewed for the Cry1A.105 and Cry2Ab2 proteins as expressed in MON 87751soybean also support their inclusion in the existing exemptions from the requirement of tolerance for Bacillus thuringiensis Cry1A.105 [40 CFR § 174.502] and Cry2Ab2 [40 CFR § 174.519] proteins, when used as a PIP in food and feed commodities of soybean.

Thus, there is a reasonable certainty that no harm will result from aggregate exposure to the U.S. population, including infants and children, to the Cry1A.105 and Cry2Ab2 proteins and the genetic material necessary for their production in event MON 87751 soybean. This includes all anticipated dietary exposures and all other exposures for which there is reliable information. The Agency has arrived at this conclusion because no toxicity to mammals has been observed, nor any indication of allergenicity potential for a plant-incorporated protectant that has been confirmed as sufficiently equivalent to currently registered plant-incorporated protectants. 

H. Supporting Data
The submitted studies to support the registrant's request for an Experimental Use Pattern and Sec. 3 registration of PIP event MON 87751 soybean expressing Cry1A.105 and Cry2Ab2 proteins are summarized in Table 1. For detailed information of each study, please refer to the individual studies reviewed in the Data Evaluation Reports (DERs) according to its respective assigned MRID number.
    Table 3. Product Characterization Data Submitted for MON 87751 Soybean
                                  STUDY TITLE
                                 STUDY SUMMARY
                                   MRID NO. 
Molecular Characterization of Insect Protected Soybean (MON 87751)

MON 87751 was produced by Agrobacterium tumefaciens-mediated transformation of soybean tissue using the 2T-DNA transformation plasmid vector PV-GMIR13196.  PV-GMIR13196 contains two separate T-DNAs that are each delineated by left and right border regions.  The first T-DNA, designated as T-DNA I, contains the CrylA.105 coding sequence regulated by the Act2 promoter and RbcS4 terminator; and the Cry2Ab2 coding sequence regulated by the Mt promoter and Pt1 terminator.  The second T-DNA, designated as T-DNA II, contains the aadA coding sequence regulated by FMV enhancer, EF-1α promoter and E9 terminator; and the splA coding sequence regulated by the Usp promoter and nos terminator.  During transformation, both T-DNAs were inserted into the soybean genome.  Subsequently, traditional breeding, segregation, selection and screening were used to isolate those plants that contain the CrylA.105 and Cry2Ab2 expression cassettes (T-DNA I) and do not contain the aadA and splA expression cassettes (T-DNA II), resulting in the production of marker-free, MON 87751.
In this study, bioinformatic analysis of next-generation sequence data (comprehensively covering the genome) was used to characterize the insert number of any DNA sequences from the transformation plasmid vector PV-GMIR13196.  The data indicated that MON 87751 contains a single integration locus of DNA insert.  Directed sequence data (generated from PCRs targeted at a specific locus) was used to demonstrate that the single DNA insert in MON 87751 contains a single copy of T-DNA I from the transformation plasmid vector PV-GMIR13196 and confirmed the integrity of the CrylA.105 and Cry2Ab2 expression cassettes in the T-DNA I insert; the data showed that MON 87751 does not contain any sequences from the backbone or T-DNA II sequences of PV-GMIR13196.  The directed sequencing data also identified the 5' and 3' insert-to-flank junctions in MON 87751 and characterized the insertion site in conventional soybean.  Furthermore, bioinformatic analysis of next-generation sequence data (comprehensively covering the genome) was used to evaluate the stability of the DNA insert and demonstrated that the DNA insert in MON 87751 was stably maintained across five generations.

CLASSIFICATION:  ACCEPTABLE 
49332003


Assessment of Cry1A.105 and Cry2Ab2 Protein Levels in Soybean Tissues Collected from MON 87751 Produced in U.S. Field Trails during 2012
The purpose of this study was to determine the levels of Cry1A.105 and Cry2Ab2 proteins in soybean tissues of MON 87751 grown in United States field trials during 2012.  Over season leaf (OSL1-4), root, seed, and forage tissue samples were collected as outlined in Production Plan PLC-2012-0022 and pollen/anther samples were collected as outlined in Production Plan PPN-2012-0041.  

The expression levels of Cry1A.105 and Cry2Ab2 proteins were determined in OSL1-4, root, seed, forage and pollen/anther tissue by validated enzyme-linked immunosorbent assays (ELISAs).  Protein levels for OSL1-4, root, forage, seed and pollen/anther tissues were calculated on a microgram (μg) per gram (g) fresh weight (fwt) basis.  Moisture content was then measured for all tissue types, except pollen/anther, and all protein levels, except for those in pollen/anther, were converted and reported on a dry weight (dwt) basis.

The mean Cry1A.105 protein level in MON 87751 across all sites was highest in OSL4 at 790 μg/g dwt and lowest in root which was below limit of detection (<LOD).  The mean Cry1A.105 protein level in MON 87751 was 2.4 ug/g dwt in seed.  The mean Cry1A.105 protein level in MON 87751 was 11 ug/g fwt in pollen/anther.
The mean Cry2Ab2 protein level in MON 87751 across all sites was highest in OSL3 at 32 μg/g dwt and lowest in seed at 4.0 μg/g dwt.  The mean Cry2Ab2 protein level in MON 87751 was 7.7 ug/g fwt in pollen/anther.

CLASSIFICATION:  ACCEPTABLE
49332004
Protein Characterization: Characterization of the Cry1A.105 Protein Purified from the Soybean Seed of MON 87751 and Comparison of the Physiochemical and Functional Properties of the Plant-Produced and Escherichia coli- Produced Cry1A.105 Protein.
The level of Cry1A.105 protein in the MON 87751 seed is relatively low.  Therefore, the protein was made in a bacterial expression system in order to produce a sufficient quantity of the protein for safety testing.  An Escherichia coli (E. coli) -produced Cry1A.105 protein was engineered to have the same amino acid sequence as the Cry1A.105 protein detected in MON 87751.  Demonstration of physicochemical and functional equivalence between the MON 87751-produced and the E. coli-produced Cry1A.105 proteins justifies the use of the E. coli-produced protein to demonstrate the safety of the MON 87751-produced Cry1A.105 protein.  The purpose of this study was to characterize the Cry1A.105 protein isolated from MON 87751 seed and to assess the equivalence of the MON 87751-produced and the E. coli-produced Cry1A.105 proteins. 
 
MON 87751-produced Cry1A.105 protein was characterized as follows:  The total protein concentration of the purified MON 87751-produced Cry1A.105 protein was measured using the Bio-Rad Protein Assay and was determined to be 0.12 mg/ml.  Apparent molecular weight (MW) and Purity of the MON 87751-produced Cry1A.105 protein were determined using SDS-PAGE analysis.  The apparent MW was 132.9 kDa and the purity of the MON 87751-produced Cry1A.105 protein was 60%.  Identity was confirmed by N-terminal sequence analysis, matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis of trypsin digested Cry1A.105 protein, and western blot analysis.  The expected N-terminal sequence was observed.  Several peptide masses consistent with expected peptide masses for the trypsin digested Cry1A.105 protein were observed.  The identified peptide masses yielded coverage of 53.5% (632 out of 1181 amino acids) of the expected sequence of the Cry1A.105 protein.  Anti-Cry1A.105 antibodies detected the MON 87751-produced Cry1A.105.  The functional activity of the MON 87751-produced Cry1A.105 was determined using a corn earworm diet incorporation assay.  The MON 87751-produced Cry1A.105 was shown to be active, with an EC50 of 0.0035 ug Cry1A.105/ml diet.  Analysis of the glycosylation status showed that the MON 87751-produced Cry1A.105 protein was not glycosylated.  Finally, assessment of the purity and apparent MW of the protein after storage in a -80 °C freezer (49 days) by SDS-PAGE analysis demonstrated that the MON 87751-produced Cry1A.105 protein was stable throughout the experimental phase. 

The equivalence of the MON 87751-produced and E. coli-produced Cry1A.105 proteins was assessed by comparing their apparent MW, immunoreactivity, functional activity, and glycosylation status.  Based on these characteristics, the E. coli-produced protein was established to be equivalent to the Cry1A.105 protein isolated from seed of MON 87751.   

These data provide a detailed characterization of the Cry1A.105 protein isolated from MON 87751 and establish the equivalence of the MON 87751-produced Cry1A.105 protein to the E. coli-produced Cry1A.105 protein
CLASSIFICATION:  ACCEPTABLE
49332005
Protein Characterization:  Characterization of the Cry2Ab2 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 Cry2Ab2 Protein 

The level of the Cry2Ab2 protein in the seed of MON 87751 is relatively low.  Therefore, the protein was made in a bacterial expression system in order to produce a sufficient quantity of the protein for safety testing.  An Escherichia coli (E. coli)-produced Cry2Ab2 protein was engineered to have the same amino acid sequence as the Cry2Ab2 protein detected in MON 87751.  Demonstration of physicochemical and functional equivalence between the MON 87751-produced and the E. coli-produced Cry2Ab2 proteins justifies the use of the E. coli-produced protein to demonstrate the safety of the MON 87751-produced Cry2Ab2 protein.  Hence, the purpose of this study was to characterize the Cry2Ab2 protein isolated from seed of MON 87751 and to assess the equivalence of the MON 87751-produced and the E. coli-produced Cry2Ab2 proteins.  
MON 87751-produced Cry2Ab2 protein was characterized as follows.  The total protein concentration of the purified MON 87751-produced Cry2Ab2 protein was measured using the Bio-Rad Protein Assay and was determined to be 0.40 mg/ml.  Apparent molecular weight (MW) and purity of the MON 87751-produced Cry2Ab2 protein were determined using SDS-PAGE analysis.  The apparent MW of the MON 87751-produced Cry2Ab2 protein was 61.4 kDa, and the purity of the MON 87751-produced Cry2Ab2 protein was 29%.  Identity was confirmed by N-terminal sequence analysis, matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis of the trypsin digested Cry2Ab2 protein, and western blot analysis.  The N-terminal sequence was confirmed using automated Edman degradation chemistry and MALDI-TOF MS analysis.  MALDI-TOF MS analysis produced several peptide masses consistent with expected peptide masses for the trypsin digested Cry2Ab2 protein.  The identified peptide masses yielded coverage of 66% (406 out of 619 amino acids) of the expected sequence of the Cry2Ab2 protein.  Anti-Cry2Ab2 antibodies detected the MON 87751-produced Cry2Ab2.  The functional activity of the MON 87751-produced Cry2Ab2 was determined using a corn earworm diet incorporation assay.  The MON 87751-produced Cry2Ab2 protein was shown to be active, with an EC50 of 0.0734 g Cry2Ab2/ml diet.  Analysis of the glycosylation status showed that the MON 87751-produced Cry2Ab2 protein was not glycosylated.  Finally, assessment of the apparent MW and purity of the protein after storage in a -80 °C freezer (42 days) by SDS-PAGE analysis demonstrated that the MON 87751-produced Cry2Ab2 protein was stable throughout the experimental phase. 
The equivalence of the MON 87751-produced and E. coli-produced Cry2Ab2 proteins was assessed by comparing their apparent MW, immunoreactivity, functional activity, and glycosylation status.  Based on these characteristics, the characterized E. coli-produced protein was established to be equivalent to the Cry2Ab2 protein isolated from seed of MON 87751.   

These data provide a detailed characterization of the Cry2Ab2 protein isolated from MON 87751 and establish the equivalence of the E. coli-produced Cry2Ab2 protein to this plant-derived protein.

CLASSIFICATION:  ACCEPTABLE
49332006
Comparison of the Functional activity of two E. Coli- Produced Cry1A.105 Protein Lots (10000776 and 11349124) Against a susceptible Lepidopteran Species.
The purpose of this study was to compare the biological (insecticidal) activity (EC50 values) of two Escherichia coli-produced Cry1A.105 protein lots (10000776 and 11349124) in a 7-day diet incorporation assay with the corn earworm (CEW), Helicoverpa zea. The EC50 value is defined as the concentration that results in 50% growth inhibition relative to the control and is a reliable and relevant measure of biological activity.

In the 7-day diet incorporation bioassays for both E. coli-produced Cry1A.105 lots (10000776 and 11349124), CEW demonstrated nearly identical concentration-dependent responses in terms of shape and location for growth inhibition. Mean EC50 values for lots 10000776 and 11349124 were 0.0042 ug Cry1A.105/ml diet and 0.0040 ug Cry1A.105/ml diet, respectively, and differ by less than a 5%. Mean EC50 values for both lots fall well within the calculated 95% prediction interval of 0.0030 - 0.0099 μg Cry1A.105/ml diet, which was constructed from historical mean EC50 values for lot number 10000776. Therefore, the two Cry1A.105 lots are concluded to have functionally equivalent biological activity and provide the required information to bridge to the assessments performed with the Cry1A.105 protein in support of MON 89034.

CLASSIFICATION:  ACCEPTABLE
49332007
Comparison of the Functional Activity of two E. coli- Produced Cry2Ab2 Protein Lots (11346423 and 11351673) Against a Susceptible Lepidopteran Species
The purpose of this study was to compare the biological (insecticidal) activity (EC50 values) of two Escherichia coli-produced Cry2Ab2 protein lots (11346423 and 11351673) in a 7-day diet incorporation assay with the corn earworm (CEW), Helicoverpa zea. The EC50 value is defined as the concentration that results in 50% growth inhibition relative to the control and is a reliable and relevant measure of biological activity.
                              
In the 7-day diet incorporation bioassays for both E. coli-produced Cry2Ab2 lots (11346423 and 11351673), CEW demonstrated nearly identical concentration-dependent responses in terms of shape and location for growth inhibition. Mean EC50 values for lots 11346423 and 11351673 were 0.065 ug Cry2Ab2/ml diet and 0.079 ug Cry2Ab2/ml diet, respectively. Mean EC50 values for both lots fall within the calculated 95% prediction interval of 0.043 - 0.25 μg Cry2Ab2/ml diet, which was constructed from historical mean EC50 values for lot number 11346423. Therefore, the two Cry2Ab2 lots are concluded to have functionally equivalent biological activity and provide the required information to bridge to the assessments performed with Cry2Ab2 in support of MON 89034.
CLASSIFICATION:  ACCEPTABLE
49332008

III. Human Health Assessment of Cry1A.105 and Cry2Ab2 as Expressed in Mon 87751 Soybean 
EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide residues. First, EPA determines the toxicity of pesticides. Second, EPA examines exposure to the pesticide through food, drinking water, and through other exposures that occur as a result of pesticide use in residential settings. 

A. Mammalian Toxicity and Allergenicity Assessment- Cry1A.105

   1. Toxicological Profile
Consistent with section 408(b) (2) (D) of the FFDCA, EPA has reviewed the available scientific data and other relevant information in support of this action and considered its validity, completeness and reliability and the relationship of this information to human risk. EPA has also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children.

   2. Mammalian Toxicity Assessment 
Monsanto has submitted acute oral toxicity data demonstrating the lack of mammalian toxicity at high levels of exposure to the pure Cry1A.105 protein. These data demonstrate the safety of the product at a level well above maximum possible exposure levels that are reasonably anticipated in the crop. 

An acute oral toxicity study in mice (MRID No. 46694603) indicated that Cry1A.105 is non-toxic to humans.  Cry1A.105 produced from microbial culture was dosed by gavage as two doses separated by 4 hours (+-20 minutes) to 10 females and 10 males (2072 mg/kg/ body weight). Two control groups were also included in the study: a bovine serum albumin protein control, and a vehicle control.  One male in the test protein group was moribund and sacrificed on day 1 due to a mechanical dosing error; this death was not attributed to the test material.  All other mice survived the study.  There were no significant differences in body weight or body weight change among the three groups during the study, and no treatment-related gross pathological findings were observed. The oral LD50 for males, females, and combined mice was greater than 2072 mg/kg.  

When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad, Roy D., et al., "Toxicological Considerations for Protein Components of Biological Pesticide Products," Regulatory Toxicology and Pharmacology 15, 3-9 (1992)). Therefore, since no acute effects were shown to be caused by Cry1A.105, even at relatively high dose levels, the Cry1A.105 protein is not considered toxic. Further, amino acid sequence comparisons showed no similarities between the Cry1A.105 and known toxic proteins in protein databases that would raise a safety concern.

   3. Allergenicity Assessment 

Since Cry1A.105 is a protein, allergenic potential was also considered. Currently, no definitive tests for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight-of-evidence approach where the following factors are considered: source of the trait; amino acid sequence comparison with known allergens; and biochemical properties of the protein, including in-vitro digestibility in simulated gastric fluid (SGF) and glycosylation. This approach is consistent with the approach outlined in the Annex to the Codex Alimentarius "Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Plants." The allergenicity assessment for Cry1A.105 follows:

    1.    Source of the trait.  Bacillus thuringiensis is not considered to be a source of allergenic proteins. 
    2.    Amino acid sequence.  A comparison of the amino acid sequence of Cry1A.105 with known allergens showed no significant overall sequence similarity or identity at the level of eight contiguous amino acid residues. 
    3.    Digestibility.  The Cry1A.105 protein was rapidly digested in less than 30 seconds in simulated mammalian gastric fluid containing pepsin. 
    4.    Glycosylation.  The Cry1A.105 protein expressed in soybean was shown not to be glycosylated.
    5.    Conclusion.  Considering all of the available information, EPA has concluded that the potential for Cry1A.105 to be a food allergen is minimal.

   4. Aggregate Exposures 

In examining aggregate exposure, EPA considers available information concerning exposures from the pesticide residue in food and all other non- occupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses). 

The Agency has considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of consumers) to the pesticide chemical residue and to other related substances. First, with respect to other related substances, the Cry1A.105 protein is a chimeric Bacillus thuringiensis Cry1A protein with overall amino acid sequence identity of 93.6%, 90.0%, and 76.7% to the Cry1Ac, Cry1Ab, and Cry1F proteins, respectively. All are registered PIPs that were previously assessed as having a lack of mammalian toxicity at high levels of exposure.  Exemptions from the requirement of a tolerance already have been established for Cry1Ab, Cry1Ac and Cry1F [see 40 CFR § 174.511, effective Apr. 25, 2007, 40 CFR § 174.510, effective Apr. 25, 2007, and 40 CFR § 174.504, effective Apr. 25, 2007, respectively]. Second, and specific to the Cry1A.105 protein, these considerations include dietary exposure under the tolerance exemption and all other tolerances or exemptions in effect for the plant-incorporated protectant chemical residue and exposure from non-occupational sources. Exposure via the skin or inhalation is not likely since the plant-incorporated protectant is contained within plant cells, which essentially eliminates these exposure routes or reduces these exposure routes to negligible.  In addition, even if exposure can occur through inhalation, the potential for Cry1A.105 to be an allergen is low, as discussed previously.  Although the allergenicity assessment focused on the Cry1A.105 protein's potential to be a food allergen, the data also indicated a low potential for Cry1A.105 to be an inhalation allergen. Exposure via residential or lawn use to infants and children is also not expected because the use sites for the Cry1A.105 protein are all agricultural for control of insects. Oral toxicity testing done at a dose of 2072 mg/kg showed no adverse effects. Furthermore, the expected dietary exposure from soybean is several orders of magnitude lower than the amounts of Cry1A.105 protein shown to have no toxicity. Therefore, even if negligible aggregate exposure should occur, the Agency concludes that such exposure would present no harm due to the lack of mammalian toxicity and the rapid digestibility demonstrated for the Cry1A.105 protein. 

   5. Cumulative Effects 

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, or revoke a tolerance, the Agency consider "available information" concerning the cumulative effects of a particular pesticide's residues and "other substances that have a common mechanism of toxicity."

Since Cry1A.105 is not considered toxic, EPA has not found Cry1A.105 to share a common mechanism of toxicity with any other substances, and Cry1A.105 does not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, EPA has assumed that Cry1A.105 does not have a common mechanism of toxicity with other substances. Following from this, therefore, EPA concludes that there are no cumulative effects associated with Cry1A.105 that need be considered.  For information regarding EPA's efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see EPA's website at http://www.epa.gov/pesticides/cumulative.

B. Mammalian Toxicity and Allergenicity Assessment Cry2Ab2

   1. Toxicological Profile 

Consistent with section 408(b) (2) (D) of the FFDCA, EPA has reviewed the available scientific data and other relevant information in support of this action and considered its validity, completeness and reliability and the relationship of this information to human risk. EPA has also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children.

   2. Mammalian Toxicity Assessment

Monsanto has submitted acute oral toxicity data demonstrating the lack of mammalian toxicity at high levels of exposure to the pure Cry2Ab2 protein. These data demonstrate the safety of the product at a level well above maximum possible exposure levels that are reasonably anticipated in the crop. 

An acute oral toxicity study in mice (MRID 44966602) indicated that Cry2Ab2 is non-toxic to humans. Three groups of ten male and ten female mice were dosed by oral gavage with 30, 300, or 1000 mg/kg bodyweight of microbially produced Cry2Ab2 protein. Two negative control groups were also included in the study: bovine serum albumin protein control, and a vehicle control (purified water). There were no significant differences between the test and control groups; therefore, the Cry2Ab2 protein does not appear to cause any significant adverse effects at an exposure level of up to 1000 mg/kg bodyweight. 

When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels. Therefore, given that no acute effects were shown to be caused by Cry2Ab2, even at relatively high dose levels, the Cry2Ab2 protein is not considered toxic. Further, amino acid sequence comparisons showed no similarities between the Cry2Ab2 protein and known toxic proteins in protein databases that would raise a safety concern.

   3. Allergenicity Assessment

Since Cry2Ab2 is a protein, allergenic potential was also considered. Currently, no definitive tests for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight-of- evidence approach where the following factors are considered: source of the trait; amino acid sequence comparison with known allergens; and biochemical properties of the protein, including in vitro digestibility in simulated gastric fluid (SGF) and glycosylation. This approach is consistent with the approach outlined in the Annex to the Codex Alimentarius "Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Plants." The allergenicity assessment for Cry2Ab2 follows: 

    1.    Source of the trait.  Bacillus thuringiensis is not considered to be a source of allergenic proteins. 
    2.    Amino acid sequence.  A comparison of the amino acid sequence of Cry2Ab2 with known allergens showed no significant overall sequence similarity or identity at the level of eight contiguous amino acid residues. 
    3.    Digestibility.  The Cry2Ab2 protein was rapidly digested in 15 seconds in simulated mammalian gastric fluid containing pepsin. 
    4.    Glycosylation.  The Cry2AB2 protein expressed in soybean was shown not to be glycosylated.
    5.    Conclusion.  Considering all of the available information, EPA has concluded that the potential for Cry2Ab2 to be a food allergen is minimal.

   4. Aggregate Exposures

Pursuant to FFDCA section 408(b)(2)(D)(vi), EPA considers available information concerning aggregate exposures from the pesticide residue in food and all other non-occupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses). The Agency considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of consumers) to the pesticide chemical residue and to other related substances. These considerations include dietary exposure under the tolerance exemption and all other tolerances or exemptions in effect for the PIP's chemical residue, and exposure from non-occupational sources. Exposure via the skin or inhalation is not likely since the plant incorporated protectant is contained within plant cells, which essentially eliminates these exposure routes or reduces exposure by these routes to negligible. In addition, even if exposure can occur through inhalation, the potential for Cry2Ab2 protein to be an allergen is low, as previously discussed. Although the allergenicity assessment focused on Cry2Ab2 protein's potential to be a food allergen, the data also indicated a low potential for Cry2Ab2 to be an inhalation allergen. Exposure to infants and children via residential or lawn use is also not expected because the use sites for the Cry2Ab2 protein is agricultural. Oral exposure, at very low levels, may occur from ingestion of corn, cotton and soybean products and, theoretically, drinking water. However, oral toxicity testing in laboratory mammals showed no adverse effects.

   5. Cumulative Effects

Pursuant to FFDCA section 408(b)(2)(D)(v), EPA has considered available information on the cumulative effects of such residues and other substances that have a common mechanism of toxicity. These considerations included the cumulative effects on infants and children of such residues and other substances with a common mechanism of toxicity. Because there is no indication of mammalian toxicity from the plant incorporated protectant, we conclude that there are no cumulative effects for the Cry2Ab2 protein.

C.	Determination of Safety for U.S. Population, Infants and Children Toxicity and Allergenicity Conclusions (Cry1A.105 and Cry2Ab2)
The data submitted and cited regarding potential health effects for the Cry1A.105 and Cry2Ab2 proteins include the characterization of the expressed Cry1A.105 and Cry2Ab2 proteins in soybean, as well as the previous submitted acute oral toxicity, heat stability, and in vitro digestibility of the proteins. The results of these studies were used to evaluate human risk, and the validity, completeness, and reliability of the available data from the studies were also considered. 

As discussed above, the previous submitted acute oral toxicity data supports the prediction that the Cry1A.105 and Cry2Ab2 proteins would be nontoxic to humans.  Moreover, Cry1A.105 and Cry2Ab2 showed no sequence similarity to any known toxin.  Because of this lack of demonstrated mammalian toxicity, no protein residue chemistry data for Cry1A.105 and Cry2Ab2 proteins were required for a human health effects assessment.  Even so, preliminary expression level analysis showed the Cry1A.105 and Cry2Ab2 proteins are present at relatively low levels. Dietary exposure is expected to be correspondingly low. 

Since Cry1A.105 and Cry2Ab2 are proteins, its potential allergenicity is also considered as part of the toxicity assessment. Data considered as part of the allergenicity assessment include that the Cry1A.105 and Cry2Ab2 proteins came from Bacillus thuringiensis which is not a known allergenic source, showed no sequence similarity to known allergens, was readily degraded by pepsin, and was not glycosylated when expressed in the plant. Therefore, there is a reasonable certainty that Cry1A.105 and Cry2Ab2 proteins will not be an allergen. 

Considered together, the lack of mammalian toxicity at high levels of exposure to the Cry1A.105 and Cry2Ab2 proteins and the minimal potential for the proteins to be a food allergen demonstrate the safety of the products at levels well above possible maximum exposure levels anticipated in the crop.

Finally, and specifically in regards to infants and children, FFDCA section 408(b)(2)(C) provides that EPA shall assess the available information about consumption patterns among infants and children, special susceptibility of infants and children to pesticide chemical residues, and the cumulative effects on infants and children of the residues and other substances with a common mechanism of toxicity.  In addition, FFDCA section 408(b)(2)(C) provides that EPA shall apply an additional tenfold margin of safety for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the data base unless EPA determines that a different margin of safety will be safe for infants and children. 

D. Overall Safety and Conclusion (Cry1A.105 and Cry2Ab2)

Based on the review and consideration of all the available information, as discussed in more detail above, there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to residues of the Cry1A.105 and Cry2Ab2 proteins and the genetic material necessary for their production in soybean. This includes all anticipated dietary exposures and all other exposures for which there is reliable information.  The Agency has arrived at this conclusion because no toxicity to mammals has been observed, and there is no indication of allergenicity potential for the plant- incorporated protectant from available information. 

E. Other Consideration (Cry1A.105 and Cry2Ab2)

   1.	Endocrine Disruptors

As required under FFDCA section 408(p), EPA has developed the Endocrine Disruptor Screening Program (EDSP) to determine whether certain substances (including pesticide active and other ingredients) may have an effect in humans or wildlife similar to an effect produced by a "naturally occurring estrogen, or other such endocrine effects as the Administrator may designate."  The EDSP employs a two-tiered approach to making the statutorily required determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal systems.  Chemicals that go through Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP where EPA will determine which, if any, of the Tier 2 tests are necessary based on the available data. Tier 2 testing is designed to identify any adverse endocrine related effects caused by the substance, and establish a dose-response relationship between the dose and the E, A, or T effect.

Between October 2009 and February 2010, EPA issued test orders/data call-ins for the first group of 67 chemicals, which contains 58 pesticide active ingredients and 9 inert ingredients.  This list of chemicals was selected based on the potential for human exposure through pathways such as food and water, residential activity, and certain post-application agricultural scenarios.  This list should not be construed as a list of known or likely endocrine disruptors.

The proposed PIP, Cry1A.105 and Cry2Ab2 proteins from MON 87751 (as expressed in soybean) is not among the group of 58 pesticide active ingredients on the initial list to be screened under the EDSP.  Under FFDCA sec. 408(p) the Agency must screen all pesticide chemicals.  Accordingly, EPA anticipates issuing future EDSP test orders/data call-ins for all pesticide active ingredients. 
For further information on the status of the EDSP, the policies and procedures, the list of 67 chemicals, the test guidelines and the Tier 1 screening battery, please visit our website:  http://www.epa.gov/endo/.

   2.	Codex Maximum Residue Level 

In making its tolerance decisions, EPA seeks to harmonize U.S. tolerances with international standards whenever possible, consistent with U.S. food safety standards and agricultural practices. In this context, EPA considers the international maximum residue limits (MRLs) established by the Codex Alimentarius Commission (Codex), as required by FFDCA section 408(b)(4). The Codex Alimentarius is a joint U.N. Food and Agriculture Organization/World Health Organization food standards program, and it is recognized as an international food safety standards-setting organization in trade agreements to which the United States is a party. EPA may establish a tolerance that is different from a Codex MRL; however, FFDCA section 408(b)(4) requires that EPA explain the reasons for departing from the Codex level.

The Codex has not established a MRL for Bacillus thuringiensis Cry1A.105 and Cry2Ab2 proteins in soybean.


Table 4. Human Health Data Submitted for MON 87751 Soybean
                                       
Assessment of the in vitro Digestibility of Cry1A.105 Protein in Simulated Gastric and Simulated Intestinal Fluids

In this study, the in vitro digestibility of the cry1a.105 protein was assessed in simulated gastric fluid (sgf) containing the proteolytic enzyme, pepsin, and in simulated intestinal fluid (sif) containing pancreatin, a mixture of enzymes.  The digestibility of the cry1a.105 protein was also assessed in a sequential digestion of the protein, i.e. Brief digestion in sgf followed by digestion in sif.  Digestibility of the cry1a.105 protein was assessed using sodium dodecyl sulfate-polyacrylamide gel (sds-page) and/or western blot analysis.  The extent of digestion of the cry1a.105 protein was evaluated by visual analysis of scanned colloidal brilliant blue g stained polyacrylamide gels or by visual analysis of scanned developed western blot x-ray films.
Greater than 98.7% of the full-length cry1a.105 protein was digested in sgf within 0.5 min when analyzed using colloidal brilliant blue g stained sds-page, and at least 98.4% of the full-length cry1a.105 protein was digested within 0.5 min when analyzed by western blot using a cry1a.105-specific antibody.  Colloidal brilliant blue g stained sds-page analysis showed that a transient peptide fragment of ~60 kda was present at 0.5 min but gone by 2 min and a transiently-stable fragment under 5 kda was observed for 20 min but was gone by 30 min in the sgf digestion.  When the cry1a.105 protein was subjected to a sequential digestion, the transiently-stable fragments observed at early time points in the sgf analysis were degraded within 0.5 min upon exposure to sif.  Greater than 98.4% of the full-length cry1a.105 protein was digested in sif within 5 min when analyzed by western blot using a cry1a.105-specific antibody.  As expected, a trypsin-resistant core (~55 kda) was observed throughout the course of the sif digestion. 
The results show that full-length cry1a.105 protein is rapidly digested in sgf and sif.  Rapid digestion of the full-length cry1a.105 protein in sgf, and of all visible cry1a.105 protein fragments in sequential digestion indicates that it is highly unlikely that the cry1a.105 protein will pose any safety concern to human or animal health.

CLASSIFICATION:  ACCEPTABLE
49332009
Assessment of the in vitro Digestibility of Cry2Ab2 protein in Simulated Gastric and Simulated Intestinal Fluids. 
In this study, the in vitro digestibility of the Cry2Ab2 protein was assessed in simulated gastric fluid (SGF) containing the proteolytic enzyme pepsin, and in simulated intestinal fluid (SIF) containing pancreatin, a mixture of enzymes.  Digestibility of the Cry2Ab2 protein was assessed using sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and/or western blot analysis.  The extent of digestion of the Cry2Ab2 protein was evaluated by visual analysis of scanned Colloidal Brilliant Blue G stained polyacrylamide gels or by visual analysis of scanned developed western blot X-ray films.
Greater than 99.4% of the full-length Cry2Ab2 protein was digested in SGF within 0.5 min when analyzed using Colloidal Brilliant Blue G stained SDS-PAGE and at least 98.4% of the full-length Cry2Ab2 protein was digested within 0.5 min when analyzed by western blot using a Cry2Ab2-specific antibody. Colloidal Brilliant Blue G stained SDS-PAGE analysis showed that peptide fragments of ~4-5 kDa were observed in the 0.5 min time points in SGF, but were gone by 2 min.
Greater than 96.8% of the full-length Cry2Ab2 protein was digested in SIF within 5 min when analyzed by western blot using a Cry2Ab2-specific antibody.  As expected, a trypsin-resistant core (~50 kDa) was observed throughout the course of the SIF digestion.  Other fragments ranging in size from ~57 kDa to just under 10 kDa were transiently present during SIF digestion, but all were gone by 4 hr.
These results show that the full-length Cry2Ab2 is rapidly digested in SGF and SIF.  Rapid and complete digestion of the Cry2Ab2 protein in SGF indicates that the Cry2Ab2 protein is highly unlikely to pose any safety concern to human or animal health.

CLASSIFICATION:  ACCEPTABLE
493302010
Effect of Heat treatment on the Functional Activity of Escherichia coli (E. coli)- produced MON 87751 Cry1A.105 Protein
The purified E. coli-produced MON 87751 Cry1A.105 protein (referred to elsewhere in this document as Cry1A.105 protein) was heated to 25, 37, 55, 75, and 95 °C for either 15 or 30 minutes. Heated samples and an unheated control sample of the protein (maintained on wet ice) were analyzed by an insect bioassay to assess the impact of temperature on the functional activity of the Cry1A.105 protein.  Additionally, all samples were analyzed by SDS-PAGE to assess Cry1A.105 protein intactness following the heat treatment.
Results of this study demonstrated that the Cry1A.105 protein is functionally active following heat treatment at 25 ºC, 37 ºC and 55 ºC for 15 or 30 min.  Cry1A.105 protein heated to 75 ºC and 95 ºC demonstrated no activity at the highest dose level tested in the insect bioassay.  SDS-PAGE analysis demonstrated that treatment at 25, 37, 55, 75 and 95 ºC for 15 minutes or 25, 37, 55 and 75 ºC for 30 minutes did not significantly change the band intensity of the Cry1A.105 protein. When treated at 95 ºC for 30 minutes, a slight reduction in band intensity of the Cry1A.105 protein was observed. These data demonstrate that the Cry1A.105 protein behaves with a predictable tendency towards loss of functional activity at elevated temperatures.

CLASSIFICATION:  ACCEPTABLE
49332011
Effect of Heat treatment on the Functional Activity of Escherichia coli (E. coli)- produced MON 87751 Cry2Ab2 Protein
The purified Escherichia coli (E. coli)-produced MON 87751 Cry2Ab2 protein (referred to elsewhere in this document as Cry2Ab2 protein) was heated to 25, 37, 55, 75, or 95 °C for either 15 or 30 minutes.  Heated samples and an unheated control sample of the protein (maintained on wet ice) were analyzed by an insect bioassay to assess the impact of temperature on the functional activity of the Cry2Ab2 protein.  Additionally, all samples were analyzed by SDS-PAGE to assess Cry2Ab2 protein intactness following the heat treatment.
Results of this study demonstrated that the Cry2Ab2 protein is functionally active following heat treatment at 25 ºC and 37 ºC for 15 or 30 minutes.  When the Cry2Ab2 protein was heated at a temperature of 55 ºC for 15 minutes, a 96% reduction of functional activity and a 99% reduction at 55 ºC for 30 minutes was observed relative to the control substance.  Furthermore, when the Cry2Ab2 protein was heated at a temperature 75 and 95 ºC for 15 or 30 minutes, a >99% loss of functional activity was observed relative to the control. SDS-PAGE analysis demonstrated that treatment at 25, 37, or 55 ºC for 15 or 30 minutes did not change the band intensity of the Cry2Ab2 protein relative to control substance.  When treated at 75 ºC for 15 minutes a reduction in band intensity of the Cry2Ab2 protein was observed.  The reduction in the band intensity became more pronounced for the Cry2Ab2 protein heated at 75 ºC for 30 minutes and 95 ºC for 15 minutes, and the band intensity was reduced further to only slightly higher than a 10% reference standard when heated at 95 ºC for 30 minutes.  These data demonstrate that Cry2Ab2 behaves with a predictable tendency towards loss of functional activity at elevated temperatures.

CLASSIFICATION:  ACCEPTABLE
49332012


IV. 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: Uses 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.

Gill, S.S., E.A. Cowles and P.V. Pietrantonio.  1992.  The mode of action of Bacillus thuringiensis- protected plants to control insect pests.  Regulatory Toxicology and Pharmacology 32: 156-173.

OECD. 2007. OECD Environment, Health and Safety Publications.  Series on Harmonization of Regulatory Oversight in Biotecnology. No. 42. Consensus Document on Safety Information on Transgenic Plants Expressing Bacillus thuringiensis- Derived Insect Control Proteins.  Environment Directorate.  Organization for Economic Cooperation and Development. Paris 2007.  Enj/jm/mono (2007)14.

Richter, S. and G.K. Lamppa. 1998. A chloroplast processing enzyme functions as the general stromal processing peptidase.  Proceedings of the National Academy of Sciences of the United States of America 95:7463-7468.

Sjoblad, Roy D., et al., 1992. Toxicological Consideration for Protein Components of Biological Pesticide Products. Regulatory Toxicology and Pharmacology 15, 3-9, 1992.

US 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)]. US Environmental Protection Agency, Office of Pesticide Programs, Biopesticides and Pollution Prevention Division, Washington, D.C.
U.S. EPA. 2000. FIFRA Scientific Advisory Panel on Sets of scientific issues being considered by the Environmental Protection Agency regarding: Bt plant-insecticides risk and benefit assessments, October 18-20, 2010. 
Widner, W.R. and H.R. Whiteley. 1989.  Two highly related insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki possess different host range specificities.  Journal of Bacteriology 171: 965-974.

V. Environmental Risk Assessment for Mon 87751

A. Overall Conclusions

Monsanto has applied for a FIFRA Section 3 seed increase registration to support seed production of MON 87751 for further development of combined-trait soybean products. EPA has also completed an assessment of the environmental risks of the proposed FIFRA Section 3 seed increase registration of MON 87751, and has examined the effects of the soybean PIP consisting of insecticidal proteins Cry1A.105 and Cry2Ab2 and the genetic material (PV-GMIR13196) necessary for their production in MON 87751 soybean on avian and mammalian wildlife, aquatic animals, nontarget insects (including honey bees), and nontarget plants.  Gene flow, development of weediness, horizontal gene transfer, and fate in the environment have also been examined, as well as effects on federally listed threatened and endangered species.  

Monsanto submitted no new guideline testing data for the ecological risk assessment of MON 87751 PIP expressed in soybean for a Section 3 seed increase registration, but requested that the data developed for a previously registered PIP (event MON 89034 in corn, EPA Reg. No. 524-575), containing functionally equivalent active ingredients be used to support the registration of MON 87751 in soybean. Based on the previous assessment conducted by the Agency for MON 89034 (US EPA, 2010a) and in comparison of the levels of the insecticidal proteins expressed in MON 87751 to those of the proteins expressed in MON 89034 in corn, no significant increase in risk of nontarget organisms to the insecticidal proteins expressed by event MON 87751 in soybean is expected to occur. For the proposed seed increase registration of MON 87751, adverse effects to nontarget organisms, including birds, wild mammals, aquatic animals, nontarget insects, honey bees, soil invertebrates, and nontarget plants, are not likely to occur.  Horizontal gene transfer, gene flow, and the development of weediness are also not likely to occur.  Based on these findings and information on habitat requirements of federally-listed threatened and endangered lepidopteran species, the Agency also makes a No Effect determination for direct and indirect effects to federally listed threatened and endangered species and their habitats.

B. Nontarget Organism Tiered Testing and Risk Assessment Process for PIPs

The paragraphs below describe the process and rationale developed by BPPD for evaluating hazard of PIPs to nontarget organisms.  This process has been described in several of BPPD's documents and is presented again here as background information.

To minimize data requirements and avoid unnecessary tests, risk assessments are structured such that risk is determined first from estimates of hazard under "worst-case" exposure conditions.  A lack of adverse effects under these conditions would provide enough confidence that there is no risk and no further data would be needed.  Hence, such screening tests conducted early in an investigation tend to be broad in scope but relatively simple in design, and can be used to demonstrate acceptable risk under most conceivable conditions.  When screening studies suggest potentially unacceptable risk, additional studies are designed to assess risk under more realistic field exposure conditions.  These later tests are more complex than earlier screening studies. Use of this "tiered" testing framework saves time and resources by organizing the studies in a cohesive and coherent manner and eliminating unnecessary lines of investigation.  Lower tier, high dose screening studies also allow tighter control over experimental variables and exposure conditions, resulting in a greater ability to produce statistically reliable results at relatively low cost.  

Tiered tests are designed to first represent unrealistic worst case scenarios and would progress to real world field scenarios if the earlier tiered tests fail to indicate adequate certainty of acceptable risk.  Screening (Tier I) non-target organism hazard tests are conducted at exposure concentrations several times higher than the highest concentrations expected to occur under realistic field exposure scenarios.  This has allowed an endpoint of 50% mortality to be used as a trigger for additional higher-tier testing.  Less than 50% mortality under these conditions of extreme exposure suggest that population effects are likely to be negligible given realistic field exposure scenarios. 

The EPA uses a tiered (Tiers I-IV) testing system to assess the toxicity of a PIP to representative non-target organisms that could be exposed to the PIP in the field environment. Tier I high dose studies reflect a screening approach to testing designed to maximize any toxic effects of the PIP on the tested non-target organism.  The screening tests evaluate single species in a laboratory setting with mortality as the end point.  Tiers II  -  IV generally encompass definitive hazard level determinations, longer term greenhouse or field testing, and are implemented when unacceptable effects are seen at the Tier I screening level.

Testing methods which utilize the tiered approach were last published by the EPA as Harmonized OSCPP Testing Guidelines, Series 850 and 885 (EPA 712-C-96-280, February 1996). These guidelines, as defined in 40 CFR 152.2150, apply to microbes and microbial toxins when used as pesticides, including those that are naturally occurring, and those that are strain-improved, either by natural selection or by deliberate genetic manipulation.  Therefore, PIPs consisting of microbial toxins (e.g., Cry proteins) and the genes encoding the toxins are also covered by these testing guidelines. 

The Tier I screening maximum hazard dose (MHD) approach to environmental hazard assessment is based on some factor (whenever possible >10) times the maximum amount of active ingredient expected to be available to terrestrial and aquatic non-target organisms in the environment (estimated environmental concentration or EEC). Tier I tests serve to identify potential hazards and are conducted in the laboratory at high dose levels which increase the statistical power to test the hypotheses.  Elevated test doses, therefore, add certainty to the assessment, and such tests can be well standardized. The Guidelines call for initial screening testing of a single group or several groups of test animals at the maximum hazard dose level. The Guidelines call for testing of one treatment group of at least 30 animals or three groups of 10 test animals at the screening test concentration, including proper controls, to be observed for approximately 30 days. In cases where an insect species cannot be cultured for 30 days, testing be continued until negative control mortality rises above 20 percent. 

Failing the Tier I (e.g., 10 X EEC) screening at the MHD dose does not necessarily indicate the presence of an unacceptable risk in the field but it triggers the need for additional testing. A less than 50% mortality effect at the MHD is taken to indicate minimal risk.  However, greater than 50% mortality does not necessarily indicate the existence of unacceptable risk in the field, but it does trigger the need to collect additional dose-response information and a refinement of the exposure estimation before deciding if the risk is acceptable or unacceptable. Where potential hazards are detected in Tier I testing (i.e. mortality is greater than  50%), additional information at lower test doses is required which can serve to confirm whether any effect might still be detected at more realistic field [1X EEC] concentrations and routes of exposure.  

When screening tests indicate a need for additional data, the OSCPP Harmonized Guidelines call for testing at incrementally lower doses in order to establish a definitive LD50 and to quantify the hazard.  In the definitive testing, the number of doses and test organisms evaluated must be sufficient to determine an LD50 value and, when necessary, the Lowest Observed Effect Concentration (LOEC), No Observed Adverse Effect Level (NOAEL) , or reproductive and behavioral effects such as feeding inhibition,  weight loss, etc.  In the final analysis, a risk assessment is made by comparing the LOAEC (Low Observed Adverse Effect Concentration) to the EEC; when the EEC is lower than the LOAEC, a no risk conclusion is made. These tests offer greater environmental realism, but they may have lower statistical power. Appropriate statistical methods, and appropriate statistical power, must be employed to evaluate the data from the definitive tests. Higher levels of replication, the number of test species, and/or repetition are needed to enhance statistical power in these circumstances. 

Data that shows less than 50 % mortality at the maximum hazard dosage level  -  (i.e. LC50, ED50, or LD50 >10 X EEC) is sufficient to evaluate adverse effects, making lower field exposure dose definitive testing unnecessary. It is also notable that the recommended >10X EEC maximum hazard dose level is a highly conservative factor.  The published EPA Level of Concern [LOC] is 50% mortality at 5X EEC  (US EPA, 1998). 

   1. Validation

The tiered hazard assessment approach was developed for the EPA by the American Institute of Biological Sciences (AIBS) and confirmed in 1996 as an acceptable method of environmental hazard assessment by a FIFRA Scientific Advisory Panel (SAP) on microbial pesticides and microbial toxins. The December 9, 1999 SAP agreed that the Tiered approach was suitable for use with PIPs; however, this panel recommended that, for PIPs with proteinaceous insecticidal properties (e.g., Bt-derived PIPs), additional testing of beneficial invertebrates closely related to target species and/or likely to be present in genetically engineered (GE) crop fields should be conducted. Testing of Bt Cry proteins on species not closely related to the target insect pest was not recommended, although it is still performed to fulfill the published EPA non-target species data requirements.  In October 2000, another SAP also recommended that field testing should be used to evaluate population-level effects on non-target organisms. The August 2002 SAP, and some public comments, generally agreed with this approach, with the additional recommendation that indicator organisms should be selected on the basis of potential for field exposure to the subject protein (US EPA, 2000, 2001, 2002, and 2004). 

   2. Chronic studies

Since delayed adverse effects and/or accumulation of toxins through the food chain are not expected to result from exposure to proteins, protein toxins are not routinely tested for chronic effects on non-target organisms.  However, the 30 day test duration requirement does amount to subchronic testing when performed at field exposure test doses. Proteins also do not bioaccumulate. The biological nature of proteins makes them readily susceptible to metabolic, microbial, and abiotic degradation once they are ingested or excreted into the environment.  Although there are reports that some proteins (e.g.,Bt Cry proteins) from both plants and micro-organisms bind to some substances in soil, thus increasing the duration of their persistence in soil (Stotzky, 2000, Saxena et al., 2002a and 2002b),  no adverse effects have been reported in this increase in exposure (OECD, 2007).

   3. Conclusion

The tiered approach to test guidelines ensures, to the extent possible, that the Agency requires the minimum amount of data needed to make science-based regulatory decisions. The EPA believes that maximum hazard dose Tier I screening testing presents a reasonable approach for evaluating hazards related to the use of biopesticides and for identifying negative results with a high degree of confidence. The Agency expects that Tier 1 testing for short-term hazard assessment will be sufficient for most studies submitted in support of PIP registrations. However, if long range adverse effects must be ascertained, then higher-tier longer-term field testing will be required.  As noted above, the October 2000 SAP and the National Academy of Sciences (NAS, 2000) recommended testing of non-target organisms directly in the field. This approach, with an emphasis on testing invertebrates found in corn fields, was also recommended by the August 2002 SAP and was supported by several public comments. Based on these recommendations, the Agency has required field studies on long term invertebrate population/community and Bt Cry protein persistence in soils as a condition of registration due to the lack of baseline data on the potential for long-term environmental effects from the cultivation of PIP-producing plants.

Since the commercialization of Bt PIP-producing plants, the number of field studies published in scientific literature in combination with the post-registration field studies submitted to the Agency has accumulated to a level where empirical conclusions can be made.  As a result, the issue of long range effects of cultivation of these PIP plants containing Bt Cry proteins on the invertebrate community structure in Bt crop fields has since been adequately addressed.  Specifically, a meta-analysis of the data collected from 42 field studies indicated that non-target invertebrates are generally more abundant in Bt cotton and Bt corn fields than in non-Bt crop fields managed with insecticides (Marvier et al., 2007).  In addition, a comprehensive review of short and long term field studies on the effects of invertebrate populations in Bt corn and cotton fields indicated that no unreasonable adverse effects are taking place as a result of wide scale Bt crop cultivation (Sanvido et al., 2007).  Another review of field tests published concluded that the large-scale studies in commercial Bt cotton have not revealed any unexpected non-target effects except subtle shifts in the arthropod community caused by the effective control of the target pests (Romeis et al., 2006).  Slight reductions in some invertebrate predator populations are an inevitable result of all pest management practices, which result in reductions in the abundance of the pests as prey.  

The Agency is generally in agreement with the conclusions of the above studies and collectively, these results provide sufficient data to support that Bt crops registered to date have not caused long term environmental effects on a population level to organisms not targeted by Bt-derived Cry proteins.  These available field studies further support the tiered testing approach in that the absence of hazard in the Tier I toxicity studies is predictive of an absence of ecological harm in the field.  These results are also consistent with earlier studies of Bt strains used as microbial insecticides, which have shown no significant detrimental impacts on populations of nontarget organisms that were studied. Based on these information, the tiered testing approach serves as an appropriate method for assessing environmental risk of Bt crops while ensuring, to the extent possible, that the Agency requires the minimum amount of data needed to make science-based regulatory decisions. 

C.	Nontarget Organism Risk Assessment for MON 87751 Soybeans

   1. Environmental Exposure Assessment

Two separate FIFRA SAP reports (US EPA, 2000 & 2002) recommended that non-target testing of Bt Cry proteins should focus on invertebrate species exposed to the crop being registered.  Following SAP recommendations, the EPA determined that non-target organisms with the greatest exposure potential to Bt Cry proteins in transgenic crop fields are beneficial insects, especially those that feed on pollen and nectar, and soil invertebrates.  While EPA's risk assessments of Bt PIP crops have focused primarily on these taxa, it recognizes that exposure to other nontarget organisms can occur and has continued to require testing on representative species.

The EPA risk assessment is focused on adverse effects at the field exposure rates, which for PIPs are typically based on protein expression levels within the plant. Although it is recommended that non-target testing be conducted at a test dose 10X the EEC whenever possible, the test dose margin can be less than 10X where uncertainty in the system is low or where high concentrations of test material are not possible to achieve due to test organism feeding habits.  The Agency may also allow for testing at lower doses in cases where many species are tested or tests are very sensitive, although the concentration used must exceed 1X EEC. 

The exposure assessment is based mainly on the exposure which was determined from the expression data using the MON 87751 soybean containing Cry1A.105 and Cry2Ab2 proteins (MRID No. 49332002).  Further details can also be found in Table 5 of this document.

   2. Ecological Effects Data 
As discussed above, in the absence of PIP-specific risk assessment data requirements, EPA requires applicants for PIP registrations to meet the 40 CFR Part 158 data requirements for microbial pesticides. These requirements include testing on birds, mammals, nontarget insects, honey bee, plants, and aquatic species, and information has been submitted to address these requirements. 
No new guideline testing data using the MON 87751 soybean plant parts or soybean plant residues containing both the insecticidal proteins (Cry1A.105 and Cry2Ab2) were submitted. Instead, the applicant has requested to bridge to the Agency database of previously reviewed toxicity studies of select nontarget organisms that supported the currently registered MON 89034 in corn (EPA Reg. No. 524-575).  

The October 2000 SAP recommended that while actual plant material is the preferred test material, bacteria derived protein is also a valid test substance, particularly in scenarios where test animals do not normally consume soybean plant tissue and where large amounts of Cry protein are needed for maximum hazard dose testing. For the MON 89034 corn registration, test substances used in the submitted studies included bacterially produced purified Cry1A.105 and Cry2Ab2 proteins (see Table 5).  Comparative analyses were performed to verify the equivalence of the bacterially produced and purified Cry1A.105 and Cry2Ab2 and the Cry1A.105 and Cry2Ab2 proteins produced in MON 89034 corn (see Table 3).  These analyses showed that the two bacterially produced insecticidal proteins were equivalent to the corresponding proteins produced by the MON 89034 corn (US EPA, 2010a).

The toxicity of Cry1A.105 and Cry2Ab2 proteins has been previously evaluated on several species of invertebrates including: lady beetle (Coleomegilla maculata), honey bee, adult and larvae (Apis mellifera), collembola (Folsomia candida), minute pirate bug (Orius insidiosus), parasitic wasp (Ichneumon promissorius) and earthworm (Eisenia fetida). Two dietary toxicity studies were submitted in support for the currently registered MON 89034 corn, which included broiler chicken (Gallus domesticus) and a freshwater invertebrate (Daphnia magna). The individual results for the cited studies and a newly submitted environmental risk assessment prepared by the applicant in support for the Section 3 seed increase registration of MON 87751 are summarized in Table 5.  The evaluation of these studies are described in more detail in the environmental risk assessment for Cry1A.105 and Cry2Ab2 expressed in MON 89034 corn (US EPA, 2010a).

EPA conducted an environmental risk assessment based mainly on the following lines of evidence to support the seed increase registration of MON 87751 soybean: a) Determination of protein equivalency of Cry1A.105 and Cry2Ab2 proteins produced in MON 87751 soybean to the previously characterized corn, cotton or microbe-derived Cry1A.105 and Cry2Ab2 protein test substances; and b) Finding of no significant increase in exposure to nontarget organisms based on calculated margins of exposure for protein concentrations in  MON 87751 soybean plant tissue matrices relative to the dose concentrations used in the nontarget organisms laboratory toxicity tests conducted on Cry1A.105 and Cry2Ab2 for MON 89034. 

   a.	Finding of No Increase in Exposure to Nontarget Organisms

The Agency previously assessed MON 89034, a PIP corn containing insecticidal genes cry1A.105 and cry2Ab2, which were initially derived from PV-ZMIR245, for its ecological effects of the insecticidal proteins Cry1A.105 and Cry2Ab2, singly or in combination, on nontarget organisms. A summary of the assessment is described in Table 5 (see below) which was in turn derived from the Biopesticide Registration Action Document (BRAD) entitled "Bacillus thuringiensis Cry1A.105 and Cry2Ab2 Insecticidal Proteins and the Genetic Material Necessary for Their Production in Corn" (US EPA, 2010a).  Finding of no significant increase in exposure to nontarget organisms is based on calculated margins of exposure (MOEs which are based on the ratio of the NOEC to EEC) for protein concentrations in MON 87751 soybean plant tissue matrices (per MRID No. 49332002) relative to the dose concentrations used in the non-target organisms laboratory toxicity tests conducted on Cry1A.105 and Cry2Ab2 for MON 89034.

Table 5.  Summary of the assessment of the environmental effects data based on previously submitted data for plant- or microbe-produced Cry1A.105 and/or Cry2Ab2 (e.g., the MON 89034 corn produced insecticidal proteins) in support of data requirements published in 40 CFR PART 158.2150. 
The summary was prepared based on a previous assessment carried out by the Agency (US EPA, 2010a), and also on an environmental risk assessment prepared by the registrant (MRID No 49332002) in support of the registration of event MON 87751 in soybean.
                               Data Requirement 
                                     OSCPP
                                   Guideline
                                Test Substance
                      Results Summary and Classification
                                   MRID No. 
Avian dietary testing, 
broiler chicken, Gallus domesticus 

885.4050*
MON 89034 corn grain**   
A 42-day dietary study showed no adverse effects to broiler chickens.
Classification:  Acceptable 
 46951412
Avian acute oral
850.2100
MON 89034 corn grain
An 8-day dietary study showed that the LC50 for MON 89034 is >500,000 ppm in the diet of Northern bobwhite.  
Classification:  Acceptable for MON 89034.
Acceptable for MON 87751, conditional on acceptance of protein equivalence data
 46951427
Avian inhalation testing
885.4100

N/A
Not Required
N/A
Wild mammal testing
885.4150

N/A
Acceptable waiver rationale
N/A
Freshwater fish testing, 
885.4200

N/A
Acceptable waiver rationale
N/A
Freshwater invertebrate testing, 
Daphnia  magna 
885.4240
 
MON 89034 corn pollen
A 48-hour static renewal limit bioassay resulted in 17% mortality compared with 0% mortality in the control groups when exposed to 120 mg/liter. The acute EC50 was estimated to be >120 mg/liter and NOEC was 100 mg/liter. However, The Guideline for 885.4240 calls for a 7-14 day study, not 48 hour acute study.
Classification: Unacceptable for MON 89034---------------------------------------------------------
A new study was submitted for Mon 89034. The study was acceptable and satisfies the condition of registration for additional aquatic invertebrate toxicity testing.
Classification:  Acceptable for MON 89034
Acceptable for MON 87751
 46951417
 
 
 
 
 
 ------------
 47838801
Estuarine and marine animal testing 
885.4280

N/A
Acceptable waiver rationale
 N/A
Non-target plant testing
885.4300

N/A
Acceptable waiver rationale
 N/A
Non-target insect testing, collembolan Folsomia candida
885.4340
MON 89034 corn leaf tissue (80ug
Cry1A.105 and 70 ug Cry2Ab2/g diet 



--------------------------------------------------------------------------------

Lyophilized MON 89034 corn leaf tissue for NOEC determinations, but  
MON 87751 soybean root for EEC determinations.
Collembola were fed a diet containing 50% Brewer's yeast and 50% lyophilized leaf tissue for 28 days.  No statistically significant effects on survival or reproduction were found among the test and negative control groups. The NOEC for Folsomia candida is at least 80 ug Cry1A.105 and 70 ug Cry2Ab2 per gram of diet.
--------------------------------------------------------------------------------
Classification:  Acceptable for MON 89034
The EEC and NOEC for Folsomia candida were <0.322 ug Cry1A.105/g fw root and >80 ug/g, respectively. The EEC and NOEC for Folsomia candida were 7.5 ug Cry2Ab2/g dry root wt and >70 ug/g, respectively. (It is noted that the above NOECs were calculated as 50% of the maximum Cry1A.105 and Cry2Ab2 expressed in the MON 89034 corn leaves.) Margins of Exposure (MOEs***) were >248 for Cry1A.105 and >9.3 for Cry2Ab2. 
Acceptable for MON 87751. Note that this was not a new study, but the registrant calculated a risk quotient using the expression data from MON 87751 soybean and the study previously submitted for MON 89034.
 46951416
 
 
 
 
 
 
 ------------
 49332002
 
Non-target insect testing, ladybird beetle, Coleomegilla maculata 
885.4340

Purified Cry1A.105 protein (Lot No. 20-100073)


-----------------------
MON 87751 soybean pollen (fresh)


No statistically significant differences in survival, development to adult, or mean adult weight were observed in Coleomegilla maculata larvae when exposed to a diet containing 240 ug Cry1A.105 protein/g of diet for 14 days.  
Classification:  Acceptable for MON 89034
----------------------------------------------------------
The EEC and NOEC for Coleomegilla maculata were stated to be 11 ug/g fw pollen and >240 ug/g for Cry1A.105, respectively.  The MOE was 21.
Classification:  Acceptable for MON 87751. See above.   
 46951421
 
 
 
 
 ------------49332002
Non-target insect testing, ladybird beetle, Coleomegilla maculate
885.4340
Purified Cry2Ab2 protein (Lot No. 20-100071)


--------------------------------------------------------------------------------

MON 87751 soybean pollen (fresh)
No statistically significant differences in survival, or development to adult were observed between the test and control groups when Coleomegilla maculata larvae were exposed to a diet containing 120  Cry2Ab2.105 protein/g of diet for 14 days. 
Classification:  Acceptable for MON 89034
----------------------------------------------------------
The EEC and NOEC for Coleomegilla maculata were   7.7 ug/g fw pollen and >120 ug/g for Cry2Ab2, respectively.  The MOE was >15.
Classification:  Acceptable for MON 87751. See above. 
 46951422
 
 
 
 
 ------------
 49332002
Honeybee testing, Honeybee larvae,
Apis mellifera
885.4380margin

Purified Cry1A.105 (Lot No. 20-100073)




--------------------------------------------------------------------------------

MON 87751 soybean pollen (fresh)

Two-to-three day old honeybee larvae in brood frames were administered a single 10 ul dose of Cry1A.105 protein brood cell (equivalent to 12 ug total protein/cell.  On day 18 after dosing, mean survival of the test group was 95%.  .  The NOAEC was determined to be at least 12 ug Cry1A.105 protein/mL.
--------------------------------------------------------------------------------
Classification:  Acceptable for MON 89034
The EEC and NOEC for Aphis mellifera were 0.022ug and >12 ug Cry1A.105 /larva fw pollen, respectively.  The MOE was >545.
Classification: Acceptable for MON 87751. See above.
 46951420
 
 
 
 
 
 
 ------------
 49332002
 
Honeybee testing, Honeybee larvae,
Apis mellifera
885.4380
MON 87751 soybean pollen (fresh)
The EEC and NOEC for Aphis mellifera were 0.0154 Cry2Ab2 ug/fw pollen and >0.60 ug/larva, respectively.  The MOE was >38.
Classification: Acceptable for MON 877511.   See above.
 49332002
Honeybee testing, Honeybee adult,
Apis mellifera
885.4380

Purified Cry1A.105 (Lot No. 20-100073)




--------------------------------------------------------------------------------

MON 87751 soybean pollen (fresh)
87751No statistically significant differences in mortality were observed between the test group and negative controls when adult honey bees were fed a 30% sucrose solution containing 550 ug Cry1A.105 protein/mL for 19 days.  The NOAEC was determined to be at least 550 ug Cry1A.105 protein/mL.
--------------------------------------------------------------------------------
Classification:  Acceptable for MON 89034
The EEC and NOEC for Aphis mellifera were 11 ug/g fw pollen and >550 ug/ml for Cry1A.105, respectively.  The MOE was >50.   
Classification: Acceptable for MON 87751.  See above. 
 4695140
 
 
 
 
 
 
 ------------
 49332002
 
Honeybee testing, Honeybee adult,
Apis mellifera 
885.4380
MON  87751 soybean pollen (fw)
The EEC and NOEC for Aphis mellifera were 7.7 ug/g fw pollen and >68 Cry2Ab2 ug ml, respectively. The MOE was 8.8.  
Classification: Acceptable for MON 87751. See above.
 49332002
Non-target insect testing, minute pirate/insidious flower bug Orius insidiosus
885.4340
Purified Cry2Ab2 protein (Lot No. 20-100071)

--------------------------------------------------------------------------------

MON 87751 soybean pollen (fresh)
Orius nymphs were fed a diet containing 100 ug Cry2Ab2 protein/diet for 14 days.  No adverse effects were observed.
--------------------------------------------------------------------------------
Classification:  Acceptable for MON 89034
--------------------------------------------------------------------------------

The EEC and NOEC for Orius insidiosus were 7.7 Cry2Ab2 ug and >100 ug/g fw pollen, respectively. 
The MOE was >12. 
Classification: Acceptable for MON 87751. See above.
 46951424
 
 
 
 ------------
 49332002
 
Non-target insect testing, minute pirate/insidious flower bug Orius insidiosus
885.4340
Purified Cry1A.105protein (Lot No. 20-100073)






--------------------------------------------------------------------------------

MON 87751 soybean pollen (fresh)
Orius nymphs were fed a diet containing 30 to 240 ug Cry1A.105/g diet for for 14 days. In an initial maximum dose test (240 ug) the survival rate was 47% compared to 88% in the control groups.  In the three subsequent dose-response tests, the mean survival rate of the 240 ug group was 55% compared to 91% and 89% in the control groups.  No statistically significant effects on survival or development were observed at concentrations less than or equal to 120 ug Cry Cry1A.105/g diet.
--------------------------------------------------------------------------------
Classification:  Acceptable for MON 89034
The EEC and NOEC for Orius insidiosus were 11 Cry1A.105 ug and 120 ug/g fw pollen, respectively. 
The MOE was 10.9.
Classification: Acceptable for MON 87751 
 46951423
 
 
 
 
 
 
 
 
 ------------
 49332002
 
Non-target insect testing, parasite wasp, Ichneumon promissorius
885.4340
Purified Cry2Ab2 protein (Lot No. 20-100071)


-----------------------
MON 87751 soybean pollen (fresh)

Adult female wasps were fed a sucrose solution containing 100 ug Cry2Ab2 protein/ml for 21 days. Mortality in the Cry2Ab2 group was 3% and the LC50 was determined to be >100 ug/L 
Classification:  Acceptable for MON 89034
----------------------------------------------------------
The EEC and NOEC for Ichneumon promissorius were 7.7ug/g fw pollen and >100ug/g. 11 for Cry2Ab2, respectively. The MOE was >12.
Classification: Acceptable for MON 87751   See above.
 46951426
 
 
 
 ------------
 49332002
 
 
Non-target insect testing, parasite wasp, Ichneumon promissorius
885.4340
Purified Cry1A.105protein (Lot No. 20-100073)
--------------------------------------------------------------------------------

MON 87751 soybean pollen (fesh)
Adult female wasps were fed a sucrose solution containing 240 ug Cry1A.105 protein/ml for 21 days. Mortality in the Cry2Ab2 group was 7% and the LC50 was determined to be >240 ug/L 
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Classification:  Acceptable for MON 89034
The EEC and NOEC for Ichneumon promissorius were 11 Cry1A.105 ug/g fw pollen and >240 ug/ml, respectively. The MOE was >21.
Classification: Acceptable for MON 87751.  See above.
 46951425
 
 
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 49332002
 
Earthworm toxicity, 
Eisenia fetida
850.6200
Purified Cry1A.105 (Lot No. 20-100073)



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MON 87751 soybean root (fresh)
Adult earthworms were exposed to artificial soil containing 178 mg Cry1A.105protein/kg dry soil for 14 days.  The 14-day LC50 for earthworms was determined to be >178 mg Cry1A.105 protein/kg dry soil and the NOAEC was determined to be >=178 mg Cry1A.105 protein/kg dry soil. 
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Classification:  Acceptable for MON 89034: Acceptable for MON 87751 
The EEC and NOEC for Eisenia fetidas were <0322 ug/g fw root and >178 mg/kg dry soil for Cry1A.105, respectively. The MOE was >552.
Classification: Acceptable for MON 87751.  See above. 
 46951418
 
 
 
 
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 49332002

Earthworm toxicity, 
Eisenia fetida
850.6200
MON 87751 soybean root
(fresh)
The EEC and NOEC for Esenia fetida were 22 ug/g dw root and >330 mg/kg dry soil for Cry2Ab2, respectively. The MOE was >15.
Classification: Acceptable for MON 87751
 
Aerobic soil degradation 
885.5200
Purified Cry1A.105 (Lot No. 20-100073) and Cry2Ab2 protein (Lot No. 20-100071)
Results of this degradation study indicate that  Cry1A.105 and Cry2Ab2 proteins do not persist in soil beyond about three weeks  
Classification:  Acceptable for 89034 PIP corn:  
Acceptable for MON 87751 
 46951428
 
 
 
Environmental Risk Assessment for the Cry1A.105 and Cry2Ab2 Proteins as expressed in MON 87751 Soybean
Non- guideline
N/A
The applicant submitted voluntarily an environmental risk assessment (ERA) for the registration of the current submission, MON 87751 soybean containing cry1A.105 and cry 2Ab2 genes encoding Cry1A.105 and Cry2Ab2 insecticidal proteins. Monsanto requested data bridging to the previously conducted testing on the effects of Cry1A.105 and Cry2Ab2 proteins produced in MON 89034 corn( or other organisms) on various nontarget organisms. In the ERA, Cry1A.105 and Cry2Ab2 protein levels in select soybean tissues were presented in order to calculate expected environmental concentrations (EECs) from MON 87751.  EECs, no observed effect concentrations (NOECs) for nontarget organism studies and margins of exposure (MOEs) for Cry1A.105 and Cry2Ab2 proteins were summarized.
Classification: Supplemental for risk assessment purposes 
 49332002
 
Note: Earthworm and honey bee studies for Cry2Ab2 protein were submitted and reviewed with previously registered products (US EPA, 2005).  The interaction study between Cry1A.105 and Cry2Ab2 was reviewed for the MON 89034 Experimental Use Permit (US EPA, 2007)
*OSCPP Microbial test guidelines
**Cry1A.105 and Cry2Bb2 are the active ingredients in MON 89034
*** Margins of Exposure (MOEs) were calculated based on the ratio of the NOEC to EEC.  The MOE was determined based on the expression level of the Cry1A.105 and CryAb2 proteins in the tissue of MON 87751 deemed relevant to nontarget insect exposure. 
Classification for MON 87751 is acceptable for protein equivalence data

   VI. Risk Assessment Conclusions for MON 87751 Soybeans 

A. Effects to Nontarget Wildlife, Invertebrates, and Plants

The active ingredients of the MON 87751 soybean are Cry1A.105 and Cry2Ab2 insecticidal proteins which are very similar to the Cry1A.105 and Cry2Ab2 proteins produced in the registered MON 89034  corn. It is stated that the Cry1A.105 and Cry2Ab2 proteins in the MON 87751 share greater than 99% and 98% identity, respectively, with the Cry1A.105 and Cry2Ab2 proteins in MON89034 in their deduced amino acid sequences. The Cry1A.105 and Cry2Ab2 proteins produced in MON 87751 are determined to be functionally equivalent to those produced in MON 89034 (MRID No. 49332002).  
Monsanto submitted no new testing data for the FIFRA Section 3 seed increase registration for MON 87751  soybean, but referenced the data/information submitted previously for the MON 88034 registration (see Table 5 above and also refer to the BRAD for MON 89034, US EPA, 2010a).

The Agency's previous environmental risk assessment of the MON 89034 corn (US EPA, 2010a) is briefly described as follows:  Maximum hazard dose (MHD) testing on representative nontarget organisms was performed in support of the registration of the MON 89034 corn producing Cry1A.105 and Cry2Ab2 proteins.  Avian oral toxicity testing data were submitted. The toxicity of the insecticidal Cry1A.105 and/or Cry2Ab2 proteins was evaluated on several species of invertebrates, including the ladybird beetle, minute pirate bug, parasitic wasp, Daphnia, honey bee, Collembola, and earthworm. Developmental observations were also made in the ladybird beetle, minute pirate bug and honey bee studies. Possible reproductive effects were also examined.  Earthworm studies were provided to the Agency to evaluate the potential effects of the Cry1A.105 and/or Cry2Ab2 proteins on soil decomposer species. 

As shown in Table 5 above, the test substance used for the studies provided to the Agency in support of the registration of the MON 89034  corn included bacterially-produced, purified Cry 1A.105 and Cry2Ab2 proteins, MON 89034 corn leaf tissue, pollen, and grain. Even though actual plant material is the preferred test material, bacterially derived protein is also a valid test substance, especially when test animals do not normally consume specific plant tissue and large amounts of Cry protein are needed for maximum hazard dose testing (US EPA, 2000).  

B. Horizontal Transfer of Transgenes from Bt Crops to Soil Organisms 

The EPA has evaluated the potential for horizontal gene transfer (HGT) from Bt crops to soil organisms and has considered possible risk implications if such a transfer were to occur. Genes that have been engineered into Bt crops are mostly found in, or have their origin in, soil-inhabiting bacteria. Soil is also the habitat of anthrax, tetanus and botulinum toxin-producing bacteria. Transfer of these genes and/or toxins to other microorganisms or plants has not been detected. Furthermore, several published experiments, that were conducted to assess the likelihood of HGT, have been unable to detect gene transfer under typical environmental conditions.  Horizontal gene transfer to soil organisms has only been detected with very promiscuous microbes under laboratory conditions designed to favor transfer. As a result of these findings and the fact that the Bt toxins engineered into MON 87751 were derived from soil-inhabiting bacteria, the EPA has concluded that HGT of transgenes found in Cry1A.105 and Cry2Ab2 producing soybean is not expected.

   C.       Gene Flow and Weediness Potential 

Movement of transgenes from crop plants into weeds is a significant concern, due to uncertainty regarding the effect that a new pest resistance gene may have on plant populations in the wild. Under FIFRA, the Agency has reviewed the potential for gene capture and expression of Cry proteins in commercial Bt soybeans by wild or weedy relatives of cotton in the United States, its possessions or territories. 

The potential for gene flow and the weediness potential of MON 87751 soybean is expected to be low since no known wild or weedy relatives exist in the North America with which soybean (Glycine max (L.) Merr.) can form viable hybrids in nature (OECD, 2000), and because soybean is not weedy in character or invasive, BPPD determined that there is no significant risk of gene capture and expression of any Bt endotoxin by wild or weedy relatives of soybean in the U.S., its possessions or territories (US EPA, 2007).

   A. Impacts on Endangered Species 

Because of the selectivity of Cry1A.105 and Cry2Ab2 insecticidal proteins for lepidopteran species and lack of evidence of effects on other nontarget species, the Agency has investigated concerns for federally-listed threatened and endangered insect species in the order Lepidoptera.  Because soybean pollen is not expected to move beyond the planted soybean field and its immediate margins, any exposure to lepidopterans would be expected to occur within those areas (US EPA, 2010b & 2014). 

Exposure could occur via direct consumption of MON 87751 soybean plants or consumption of MON 87751 pollen that falls on non-soybean plants within the soybean field and its immediate margins.  However, exposure to significant amounts of Cry1A.105 and Cry2Ab2 via pollen consumption is not likely.  Little pollen is expected to be released from soybean flowers, since soybean plants are primarily self-pollinated and anthers usually dehisce and release pollen before flowers open.  Airborne pollen concentrations have been measured at very low levels within soybean fields (mean of 0.18 grains/cm[2]/day) (Yoshimura et al., 2006).  Based on previous analyses for Bt soybeans (e.g., see USEPA 2010b and 2014), EPA concludes that exposure resulting from pollen falling on potential non-soybean food plants in the field and immediate margins is not sufficient to cause effects in federally listed lepidopterans.  Therefore, any significant exposure would have to occur through consumption of the MON 87751 soybean plants on the field.
 
A search of EPA's LOCATES database indicates that four species of federally listed lepidopterans are present in U.S. counties in which soybeans are grown.  These are the Karner blue butterfly (Lycaeides melissa samuelis), St. Francis's Satyr Butterfly (Neonympha mitchellii fransisci), Mitchell's Satyr Butterfly (Neonympha mitchellii mitchellii), and Uncompahgre Fritillary Butterfly (Boloria acrocnema).

The potential effects of Bt PIPs in soybean on the Karner blue butterfly, Mitchell's satyr butterfly, and St. Francis's satyr butterfly were previously analyzed in  the endangered species assessment section in the BRAD for event MON 87701 soybean expressing Cry1Ac insecticidal protein (US EPA, 2010b) and event DAS-81419-2 soybean expressing Cry1Ac and Cry1F insecticidal proteins (US EPA, 2014). Based on their biology and habitat requirements, EPA determined that these species would not be exposed to the Cry proteins expressed in the MON 87701 or DAS-81419-2 soybean plants. Since MON 87751 soybeans would be expected to be grown in the same locations as soybeans expressing previously registered Bt PIPs, these conclusions would also apply to the Cry1A.105 and Cry2Ab2 proteins expressed in MON 87751 soybeans. 

According to the LOCATES database, the Uncompahgre Fritillary Butterfly (Boloria acrocnema) is present in one county (Delores) in Colorado in which soybeans are also grown (US FWS, 2009).   However, all known populations of this species are associated with large patches of snow willow above 12,400 feet which provide food and cover. The species has been located only on northeast-facing slopes, which are the coolest and wettest microhabitat available.  Snow willow serves as the larval food, while the adult butterflies take nectar from a wide range of alpine plants.  Movement of the Uncompahgre Fritillary Butterfly is limited to their habitat patches.  Based on its limited habitat in high altitudes, effects to this species as a result of the proposed seed increase registration of MON 87751 soybeans are not expected, since this butterfly species will not be exposed. 

Based on the above analysis, the Agency determines that there will be no direct effect to federally listed lepidopteran species as a result of the cultivation of MON 87751 soybeans as proposed.  Obligate relationships between insectivorous listed species with lepidopterans that are expected to be found in soybean fields, especially pest species that feed on MON 87751 plants, are not currently known.  Since the Cry1A.105 and Cry2Ab2 in MON 87751 soybeans target only lepidopteran insects, loss of the pest insects as a result of MON 87751 are expected to be offset by the presence of other insects that could act as food sources for listed species, including beneficial insects that are known not to be affected by Cry1A.105 and Cry2Ab2.  Effects on species other than insects have also been determined to be  unlikely because of the specificity of Cry1A.105 and Cry2Ab2.  Therefore, no effects to listed species due to indirect effects or effects on habitat are anticipated to occur.

E.	Conclusions 

The environmental risk assessment indicates that, based on information submitted to support this proposed registration, will not result in unreasonable adverse environmental effects.  The Agency additionally concludes registering MON 87751 soybeans will have no direct or indirect effect on endangered  and/or threatened species listed by the FWS and National Marine Fisheries Service or their habitats.

VII. Insect Resistance Management

An insect resistance management (IRM) assessment was not conducted specifically for MON 87751 soybean. However, BPPD did conduct an IRM review (BPPD 2010) for MON 87701 soybean, which expresses Cry1Ac protein. MON 87751 expresses both Cry1A.105 and Cry2Ab2 proteins. Like MON 87751, MON 87701 is a "breeding only" registration and was limited to 15,000 acres per year in the U.S. Both products target lepidopteran soybean pests, including soybean looper and velvetbean caterpillar.

BPPD's review for MON 87701 concluded that there is not likely to be a significant risk of resistance in the U.S. to the lepidopteran target pests. This conclusion was supported by the approved low acreage (maximum 15,000 per year, with no more than 1,000 in any individual county) and the biology of the target pests, which do not overwinter in non-tropical areas and are polyphagous (feeding on a number of non-soybean wild hosts and cultivated crops). BPPD also evaluated the potential impact of Bt soybean on the natural refuge strategy in place for Bt cotton PIPs in the southeastern U.S. The review noted that the total annual acreage for MON 87701 is a small fraction of the of the total soybean acres in each state and should not significantly reduce the amount of natural refuge available for Bt cotton.

The IRM review conclusions for MON 87701 are also applicable to MON 87751 soybean. Both will be grown on comparable acreage and target the same lepidopteran pests. In addition, MON 87751 is a pyramided product that expresses two Bt proteins (Cry1A.105 and Cry2Ab2), while MON 87701 expresses a single toxin (Cry1Ac). Pyramided PIPs are inherently more durable than single toxin varieties (Roush 1998); therefore, it is expected that the risk of resistance will be lower for MON 87751 than for MON 87701.

As part of the IRM strategy for MON 87751 soybean, a resistance monitoring program will be required. Similar to the approach in place for MON 87701, the monitoring plan can be based on investigations of unexpected pest damage in MON 87751 fields. An associated remedial action plan should be applied in the event of confirmed resistance (similar to the remedial action plans used for Bt corn PIPs).

It should be noted that BPPD's IRM assessment is applicable only to an acreage-limited, breeding registration. A separate evaluation will be necessary if a full commercial product (i.e., without acreage restrictions) is proposed for registration. Data needed to support such a review would include additional information about pest biology, dose, simulation modeling, and resistance monitoring. Further information to evaluate the impact on natural refuge for Bt cotton will also be necessary.


VIII. References
BPPD, 2010. Review of Insect Resistance Management Considerations for Bt Soybean Event MON 87701. A. Reynolds memorandum to M. Mendelsohn, June 1, 2010.

Marvier, M., McCreedy, C., Regetz, J. & Kareiva, P. (2007). A meta-analysis of effects of Bt cotton and maize on nontarget invertebrates. Science 316: 1475 - 1477.

Monsanto. 2014. Environmental Risk Assessment for the Cry1A.105 and Cry 2Ab2 Proteins as Expressed in MON 87751 Soybean. MRID No. 49332002.

National Academy of Science (NAS). 2000. Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation is available from the National Academy Press, 2101 Constitution Avenue, N.W., Lockbox 285, Washington, DC 20055; (800) 624-6242 or (202) 334-3313 (in the Washington metropolitan area); http://www.nap.edu.

OECD. 2000. Consensus Document on the Biology of Glycine max (L.) Merr. (Soybean). ENV/JM/MONO(2000)9.

OECD. 2007. Consensus Document on Safety Information on Transgenic Plants Bacillus thuringiensis- Derived Insect Control Protein. ENV/JM/MONO(2007)14.

Romeis, J., Meissle, M. and Bigler, F.  2006.  Transgenic Crops expressing Bacillus thuringiensis toxins and biological control. Nature Biotechnology 24:  63-71. 

Roush, R.T., 1998. Two toxin strategies for management of insecticidal transgenic crops: will pyramiding succeeed where pesticide mixtures have not? Phil. Trans. R. Soc. Lond. 353: 1777-1786.

Sanvido, O., Romeis, J., Bigler, F.  2007. Ecological Impacts of Genetically Modified Crops: Ten Years of Field Research and Commercial Cultivation. Adv Biochem Engin/Biotechnol 107: 235 - 278.

Saxena, D.S. Flores, and G. Stotzky. 2002a.  Bt toxin released in root exudates from 12 transgenic corn hybrids representing  three transformation events. Soil Biol. Biochem. 34:133-137.

Saxena, D.S. Flores, and G. Stotzky. 2002b. Vertical movement in soil of insecticidal Cry1Ab protein from  Bacillus thuringiensis. Soil Biol. Biochem. 34:111-120.

Stotzky, G. 2000. Persistence and biological activity in soil of insecticidal proteins from Bacillus  thuringiensis and of bacterial DNA on clays and humic acids. J. Environ. Qual. 29:691-705.

United States Environmental Protection Agency (US EPA). "Guidelines for Ecological Risk Assessment."  EPA 630/R-95-002F. Washington, DC, USA. [Federal Register, May 14, 1998. 63(93): 26846-26924.]

US EPA.  2000. SAP report No 99-06. Sets of scientific issues being considered by the Environmental Protection Agency regarding: Section I - Characterization and Nontarget Organism Data Requirements for Protein Plant Pesticides. Dated February 4, 2000. Available at the EPA website: http://www.epa.gov/scipoly/sap/1999/index.htm#december

US EPA. 2001. SAP Report No. 2000-07. Sets of scientific issues being considered by the Environmental Protection Agency regarding: Bt plant-pesticides risk and benefit assessments. Dated March 12, 2001. Web site: http://www.epa.gov/scipoly/sap/2000/october/octoberfinal.pdf

US EPA.  2002. SAP Report No. 2002-05. A set of scientific issues being considered by the Environmental Protection Agency regarding: Soybean rootworm plant-incorporated protectant nontarget insect and insect resistance management issues. Dated November 6, 2002.  http://www.epa.gov/scipoly/sap/2002/august/august2002final.pdf

US EPA.  2004. SAP Report No.2004-05.  Product characterization, human health risk, ecological risk, and insect resistance management for Bt cotton products. Dated August 19, 2004.  http://www.epa.gov/scipoly/sap/meetings/2004/june/final1a.pdf

US EPA.  2005.  Biopesticides Registration Action Document: Bacillus thuringiensis Cry2Ab2 Protein and its Genetic Material Necessary for its Production in Cotton.  Washington, DC. 
    http://www.epa.gov/pesticides/biopesticides/ingredients/tech_docs/brad_006487.pdf

US EPA.  2007. Ecological risk assessment for Monsanto's MON 87701 Bt soybean EUP. Memorandum from T. Milofsky to M. Hunter.

US EPA.  2010a. Biopesticides Registration Action Document: Bacillus thuringiensis Cry1A.105and Cry2Ab2 Insecticidal Proteins and the Genetic Material Necessary for Their Production in Corn.  Washington, DC. 
   http://www.epa.gov/pesticides/biopesticides/ingredients/tech_docs/brad_006514-

US EPA.  2010b.  Biopesticides Registration Action Document: Bacillus thuringiensis Cry1Ac Protein and the Genetic Material Necessary for its Production in Event MON 87701 Soybean. US Environmental protection Agency, Washington, D.C.
 	http://www.epa.gov/pesticides/biopesticides/pips/bt-cry1ac-protein.pdf


US EPA.  2014.  Biopesticides Registration Action Document (BRAD) Plant-Incorporated protectants: Bacillus thuringiensis Cry1Ac protein and the Genetic Material Necessary for its Production (PC Code 006527) and Bacillus thuringiensis Cry1F  Protein and the Genetic Material Necessary for its Production (PC Code 006528) as Expressed in Event  DAS-814 Soybean. Washington, DC.
   http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPP-2013-0703-0003

U.S. Fish and Wildlife Service (US FWS).   2009.  Uncompahgre Fritillary Butterfly. Grand Junction, CO.  
http://ecos.fws.gov/speciesProfile/profile/speciesProfile?spcode=I01Q

Yoshimura, Y., K. Matsuo, and K. Yasuda.  2006.  Gene flow from GM glyphosate-tolerant to conventional soybeans under field conditions in Japan.  Environmental Biosafety Research 5:169-173.

IX. Terms and Conditions of Registration

 1) The subject registration is limited to a combined yearly total of 15,000 acres for breeding purposes, agronomic testing, and producing seed in the United States and the Commonwealth of Puerto Rico.

 2) Monsanto must submit reports on IRM monitoring and remedial action plans to EPA annually by August 31[st] each year.

 3) Upon request, Monsanto must provide EPA by January 31[st] each year annual reports on the acreage where MON 87751 has been grown by States and territory.

 4) Harvested seed are not allowed for sale as commercial seed in the U.S. under the conditions of this registration, but any non-treated seed containing MON 87751 may be sold as grain.

X. Regulatory Position for Mon 87751 (Cry1A.105 and Cry2Ab2) Soybean

Pursuant to FIFRA section 3(c)(5), EPA may unconditionally register a pesticide if EPA determines that, when used in accordance with widespread and commonly recognized practice, it will not generally result in unreasonable adverse effects to the environment. Monsanto has submitted or cited data sufficient for EPA to determine that an unconditional breeding registration of Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 Proteins and the Genetic Material (Vector PV-GMIR13196) Necessary For Their Production in MON 87751 (OECD Unique ID. MON-87751-7) Soybean under FIFRA 3(c)(5) will not result in unreasonable adverse effects to the environment. Monsanto submitted and/or cited satisfactory data pertaining to the proposed use. The human health effects data and nontarget organism effects data are considered sufficient for the limited acreage, and geographic limitations of the unconditional registration. These data demonstrate that no foreseeable human health hazards or ecological effects are likely to arise from the use of the product and that the risk of resistance developing to Cry1A.105 and Cry2Ab2 protein, during this limited registration is not expected to be significant.

The registration is limited to seed increase and to a total of 15,000 acres per year, with no more than 1,000 acres per county per year.

