Draft BIOPESTICIDE REGISTRATION ACTION DOCUMENT

PLANT-INCORPORATED PROTECTANTS

Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 Proteins and the Genetic Material (Vector PVGMIR13196) 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
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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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 ........18

A. Mammalian Toxicity and Allergenicity Assessment- Cry1A.105 ...................................................18

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) ............................................................23

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
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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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.
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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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. 
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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 plantincorporated 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.
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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.
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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 nextgeneration 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 
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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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 μg 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 μg Cry2Ab2/ml 

diet. Analysis of the glycosylation status showed that the MON 87751-produced Cry2Ab2 protein was 

not glycosylated.
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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 μg/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 μg/g dry weight (dwt) in seed. The mean CrylA.105 

protein level in MON 87751 was 11 μg/g fresh weight (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 μg/g fwt in pollen/anther. 

Summary results are provided below in Tables 1 and 2. 
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Table 1. Summary of Cry1A.105 Protein Levels in Soybean Tissues from MON 87751 Produced in 

United States Field Trials During 2012

Tissue Type1 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.
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Table 2. Summary of Cry2Ab2 Protein Levels in Soybean Tissues from MON 87751 Produced in 

United States Field Trials During 2012

Tissue Type1 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 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.
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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 
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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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 

49332003
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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STUDY TITLE STUDY SUMMARY MRID NO. 

generations.

CLASSIFICATION: ACCEPTABLE 

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 μg/g dwt in seed. The mean 

Cry1A.105 protein level in MON 87751 was 11 μg/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 μg/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 

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 

49332005
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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15

STUDY TITLE STUDY SUMMARY MRID NO. 

and Functional 

Properties of the 

Plant-Produced 

and Escherichia 

coli- Produced 

Cry1A.105 

Protein.

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 Nterminal 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 μg 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 
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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16

STUDY TITLE STUDY SUMMARY MRID NO. 

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

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 

49332006
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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STUDY TITLE STUDY SUMMARY MRID NO. 

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

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 coliproduced 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 μg Cry1A.105/ml diet and 

0.0040 μg 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
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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STUDY TITLE STUDY SUMMARY MRID NO. 

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 coliproduced 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 μg Cry2Ab2/ml diet and 

0.079 μg 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. 
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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19

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 
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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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 nonoccupational 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.
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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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: 
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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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.
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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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 
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

24

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, 
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

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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.
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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 fulllength 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 trypsinresistant 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 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 

493302010
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in Simulated 

Gastric and 

Simulated 

Intestinal Fluids. 

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

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 

49332011
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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

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
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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 thuringiensisprotected 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 thuringiensisprotected 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.
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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 
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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 cost1. 

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 nontarget 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)2. 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 

 

1 Non-target invertebrate hazard tests often are conducted at exposure concentrations several times higher than the maximum 

concentrations expected to occur under realistic exposure scenarios. This has customarily allowed an endpoint of 50% 

mortality to be used as a trigger for additional higher-tier testing. Lower levels of mortality under these conditions of extreme 

exposure suggest that population effects are likely to be negligible given realistic exposure scenarios. Thus, it follows that the 

observed proportion of responding individuals can be compared to a 50% effect to determine if the observed proportion is 

significantly lower than 50%. For example, using a binomial approach, a sample size of 30 individuals is sufficient to allow a 

treatment effect of 30% to be differentiated from a 50% effect with 95% confidence using a one-sided Z test. A one-sided test 

is appropriate because only effects of less than 50% indicate that further experiments are not needed to evaluate risk. 

2 OSCPP Testing Guidelines, Series 850 and 885 website: 

http://www.epa.gov/OSCPPfrs/publications/OSCPP_Harmonized/885Microbial_Pesticide_Test_Guidelines/Series
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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)3. 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.4 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 exposure5. 

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. 

 

3 The 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 or other factors. High dose testing also may not be necessary 

where many species are tested or tests are very sensitive, although the test concentration used must exceed 1X EEC.

4

It is notable that that the 10 X EEC MHD testing approach is not equivalent to what is commonly known as “testing at a 

10X SAFETY FACTOR” where any adverse effect is considered significant. Tier I screen testing is not ‘safety factor testing’. 

In a “10X safety factor” test any adverse effect noted is a “level of concern”, whereas in the EPA environmental risk 

assessment scenario any adverse effect is viewed as a concern only at 1X the field exposure. 

5 The 1X EEC test dose is based on plant tissue content and is considered a high worst case dose (sometimes referred to as 

HEEC). This 1X EEC is still much greater than any amount which any given non-target organism may be ingesting in the 

field because most non-target organisms do not ingest plant tissue.
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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 6(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

 

6 The established peer and EPA Science Board reviewed guidance on screening test levels of concern is 50% mortality at 5X 

environmental concentration. The appropriate endpoints in high dose limit/screening testing are based on mortality of the 

treated, as compared to the untreated (control) non-target organisms. A single group of 30 test animals may be tested at the 

maximum hazard dose.
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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 PIPproducing 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-analysis7of 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 nontarget 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 

 

7

This research was funded by Environmental Protection Agency grant CR-83214Bt 7-01. The Bt crop non-target effects 

database can be found on the National Center for Ecological Analysis and Synthesis (NCEAS). Website. 

(http://delphi.nceas.ucsb.edu/btcrops/).
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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 
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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 
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

37

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
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

38

Data 

Requirement 

OSCPP

Guideline

Test Substance Results Summary and Classification MRID 

No. 

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 

46951416

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

49332002
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

39

Data 

Requirement 

OSCPP

Guideline

Test Substance Results Summary and Classification MRID 

No. 

calculated a risk quotient using the 

expression data from MON 87751 soybean 

and the study previously submitted for MON 

89034.

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 μg 

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 μg/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 μg/g fw pollen and >120 ug/g for 

Cry2Ab2, respectively. The MOE was >15.

Classification: Acceptable for MON 87751.

See above. 

46951422

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

49332002
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

40

Data 

Requirement 

OSCPP

Guideline

Test Substance Results Summary and Classification MRID 

No. 

Honeybee 

testing, 

Honeybee 

larvae,

Apis mellifera

885.4380

margin

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 μg 

Cry1A.105 protein/mL.

Classification: Acceptable for MON 89034

The EEC and NOEC for Aphis mellifera were 

0.022ug and >12 μg 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 

μg Cry1A.105 protein/mL for 19 days. The 

NOAEC was determined to be at least 550 μg 

Cry1A.105 protein/mL.

Classification: Acceptable for MON 89034

The EEC and NOEC for Aphis mellifera were 

11 ug/g fw pollen and >550 μg/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
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

41

Data 

Requirement 

OSCPP

Guideline

Test Substance Results Summary and Classification MRID 

No. 

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 

46951426

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

49332002
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

42

Data 

Requirement 

OSCPP

Guideline

Test Substance Results Summary and Classification MRID 

No. 

MOE was >12.

Classification: Acceptable for MON 87751 

See above.

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 

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

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

49332002

Earthworm 

toxicity, 

Eisenia fetida

850.6200 Purified Cry1A.105 (Lot 

No. 20-100073)

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. 

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

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

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: 

46951428
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

43

Data 

Requirement 

OSCPP

Guideline

Test Substance Results Summary and Classification MRID 

No. 

Acceptable for MON 87751

Environmental 

Risk 

Assessment for 

the Cry1A.105 

and Cry2Ab2 

Proteins as 

expressed in 

MON 87751 

Soybean

Nonguideline

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 
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

44

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
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

45

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/cm2/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 
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

46

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 
Bacillus Thuringiensis Cry1A.105 and Cry2Ab2 in Mon 87751 Soybean

Biopesticide Registration Action Document

47

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.
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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 thuringiensisDerived 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 
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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
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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.
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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 31st each year.

3) Upon request, Monsanto must provide EPA by January 31st 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.