BIOPESTICIDE REGISTRATION ACTION DOCUMENT

Bacillus thuringiensis Cry1Ac Protein and the Genetic Material (Vector PV-GMIR9) Necessary 

for Its Production in MON 87701 (OECD Unique Identifier: MON 877Ø1-2) Soybean

[PC Code 006532] 

U.S. Environmental Protection Agency 

 Office of Pesticide Programs 

 Biopesticides and Pollution Prevention Division
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

Biopesticide Registration Action Document 

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TABLE of CONTENTS 

I. OVERVIEW............................................................................................................................................................ 3

A. EXECUTIVE SUMMARY .................................................................................................................................... 3

B. USE PROFILE........................................................................................................................................................ 4

C. REGULATORY HISTORY .................................................................................................................................. 5

II. SCIENCE ASSESSMENT......................................................................................................................................... 6

A. PRODUCT CHARACTERIZATION

B. HUMAN HEALTH ASSESSMENT

D. ENVIRONMENTAL ASSESSMENT FOR MON 89034..................................................................................... 15

E. INSECT RESISTANCE MANAGEMENT (IRM)............................................................................................. 33

III. TERMS AND CONDITIONS OF REGISTRATION........................................................................................... 38

IV. REGULATORY POSITION FOR CRY1AC SOYBEAN.................................................................................... 38
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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BIOPESTICIDE REGISTRATION ACTION TEAM

Office of Pesticide Programs:

Biopesticides and Pollution Prevention Division

Product Characterization and Human Health 

John Kough, Ph.D. 

Chris Wozniak, Ph.D. 

Environmental Fate and Effects 

Zigfridas Vaituzis, Ph.D. 

Shannon Borges, M.S. 

Insect Resistance Management 

Alan Reynolds, M.S. 

Jeannette Martinez, M.S. 

Registration Support 

Mike Mendelsohn 

I. OVERVIEW 

 A. EXECUTIVE SUMMARY 

Monsanto Company (hearafter, “Monsanto”) has submitted an application to register the soybean 

product MON 87701, which expresses the Bacillus thuringiensis (Bt) subsp. kurstaki insecticidal 

protein Cry1Ac as a Plant Incorporated Protectant (PIP). Cry1Ac provides protection from feeding 

damage by target lepidopteran pests, including the velvetbean caterpillar (Anticarsia gemmatalis), 

soybean looper (Pseudoplusia includens), soybean axil borer (Epinotia aporema), and sunflower 

looper (Rachiplusia nu). Monsanto has applied for a FIFRA Section 3 registration for breeding and 

seed increase activities to support commercial production of MON 87701 in tropical and subtropical 

regions outside the U.S. and its territories. 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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Product Characterization

MON 87701, which was developed by Agrobacterium-mediated transformation of soybean (Glycine 

max) using the 2T-DNA plasmid vector PV- GMIR9, produces one Bacillus thuringiensis protein, 

Cry1Ac. This protein is intended to provide protection from feeding damage caused by a number of 

lepidopteran pests, such as the soybean looper, beet armyworm, green cloverworm and velvetbean 

caterpillar. 

Mammalian Toxicity and Allergenicity Assessment

Cry1Ac protein is a δ-endotoxin from B. thuringiensis that has been used extensively in both 

microbial and plant-incorporated protectants as a means of insect pest management. An existing 

exemption from the requirement of a tolerance for Cry1Ac (CFR 40 Section 174.510; 72 FR 20435, 

April 25, 1997) in food and feed commodities precluded the need for a separate tolerance action in 

conjunction with this review of product characterization and human health assessment. 

Environmental Assessment

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, are not anticipated. Horizontal gene transfer, gene flow, and the 

development of weediness are also not anticipated. 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 87701 (<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). 

The use of MON 87701 on a limited basis (as described by Monsanto in their submissions) should 

not impact the natural refuge strategy in place for Bt cotton PIPs that express Cry1Ac. The total 

proposed acreage for MON 87701 would represent a small fraction of total soybean acres in each 

state and should not significantly reduce the amount of natural refuge. Further, MON 87701 will 

not be planted in states (i.e., Mississippi Delta states) with lower natural refuge. 

The Agency is requiring adoption of a resistance monitoring plan, remedial action strategy, and 

annual sales reporting for MON 87701. 

B. USE PROFILE
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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Active Ingredient Name: Bacillus thuringiensis Cry1Ac Protein and the Genetic Material 

(Vector PV-GMIR9) Necessary for Its Production in MON 

87701 (OECD Unique Identifier: MON 877Ø1-2) Soybean

Trade and Other Name(s): MON 87701 

OPP Chemical Codes: 006532 

Basic Manufacturer: Monsanto Company 

800 North Lindbergh Blvd. 

St. Louis, MO 63167 

Type of Pesticide: Plant-incorporated Protectant 

 

Uses: Soybean. Limited to seed increase and to a total of 15,000 acres per year in the 

Atlantic Coast States within the Continental United States with no more than 1,000 acres per 

county per year. 

Target Pests for Active Ingredient: Velvetbean caterpillar (Anticarsia gemmatalis), 

soybean looper (Pseudoplusia includens), soybean axil borer (Epinotia apoerema), and 

sunflower looper (Rachiplusia nu) 

C. REGULATORY HISTORY 

Monsanto previously submitted EUP requests for event MON 87701 in order to conduct field 

evaluations and gather data. Cry1Ac protein is a δ-endotoxin from B. thuringiensis and has been 

used extensively in both microbial and plant-incorporated protectants as a means of insect pest 

management. An existing exemption from the requirement of a tolerance for Cry1Ac (CFR 40 

Section 174.510; 72 FR 20435, April 25, 1997) in food and feed commodities precludes the need 

for a separate tolerance action in conjunction with this review of product characterization and 

human health assessment. 

On April 14, 2010, EPA announced its receipt of this application pursuant to FIFRA secion 3(c)(4) 

in the Federal Register and in regulations.gov under docket number EPA-HQ-OPP-2010-0023. No 

comments were received in response to this notice. 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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II. SCIENCE ASSESSMENT

 

A. PRODUCT CHARACTERIZATION

MON 87701 was developed by Agrobacterium-mediated transformation of soybean using the 2TDNA plasmid vector PV- GMIR9 and produces Bacillus thuringiensis protein δ-endotoxin, Cry1Ac. 

This protein is intended to provide protection from feeding damage caused by a number of 

lepidopteran pests. 

Transformation System and Genetic Elements: 

PV- GMIR9 is a binary vector containing two separate transfer DNAs (2T-DNAs). The first TDNA (T-DNA I) contains the cry1Ac gene under the direction of the RbcS4 (small subunit 

RuBISCO from Arabidopsis thaliana) promoter and termination sequences from T-7Sά (3’ region 

of the Sphas gene of Glycine max encoding the 7Sά seed storage protein conglycinin). The 

chloroplast transit peptide coding sequence from Arabidopsis thaliana from RbcS4 small subunit 

1A was placed at the 5’ end of the gene (N-terminus) to direct accumulation of the insecticidal 

protein to the chloroplast. In the Cry1Ac protein produced in MON 87701, 4 additional amino acids 

are present at the N-terminus of the protein as a result of the use of this targeting sequence. 

The second T-DNA, designated as T-DNA II, contains the cp4-epsps gene under the direction of 

the Figwort Mosaic Virus 35S promoter. During transformation, both T-DNAs were inserted into 

the soybean genome independently. The cp4-epsps gene was used as the selectable marker 

(tolerance to glyphosate) to select transformed cells and plants. After the transformed cells and 

subsequent regenerated plants were identified, the cp4-epsps selectable marker gene was no longer 

needed. Therefore, traditional breeding and segregation were employed to isolate plants that contain 

only the cry1Ac expression cassette (T-DNA I), producing marker-free MON 87701 plants lacking 

the cp4-epsps selectable marker gene. 

Analysis of the insert junction of the single copy T-DNA-I into MON 87701 genomic DNA 

indicated that the entire sequence, as present in T-DNA-I from PV- GMIR9, is intact in the final 

transformant. Sequencing of the insertion site indicated a loss of 32 bp as well as an addition of 14 

bp at the junction with soybean genomic DNA. This has been previously noted in the literature 

regarding Agrobacterium-mediated integration events (Gorbunova and Levy, 1997; Salomon and 

Puchta, 1998). 

Protein Characterization: 

Protein characterization data demonstrate that the plant-produced Cry1Ac has biochemical and 

functional activities that are similar to those of the E. coli-produced protein that was used in several 

toxicity studies. The following techniques were used to characterize and compare the plantproduced and the E. coli-produced proteins: sodium dodecyl sulfate polyacrylamide gel 

electrophoresis (SDS-PAGE), western blot analysis, densitometry, matrix-assisted laser 

desorption/ionization time-of-flight (MALDI-TOF) / mass spectrometry, glycosylation analysis, Nterminal amino acid sequencing, and insecticidal activity bioassays. Glycoslyation analysis 

indicated that the protein is not glycoslyated. These analyses demonstrated the structural and 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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functional similarity between the plant-produced and the E. coli-produced Cry1Ac protein and 

justified the use of E. coli-produced protein in toxicity studies. 

Soybean MON 87701 derived Cry1Ac protein contains an additional 4 amino acids derived from 

the chloroplast transit peptide (CTP1) sequence at the N-terminus, as deduced by N-terminal 

sequencing. While this addition differs from the protein that was produced in E. coli for the oral 

toxicity study and the insect bioassays with corn earworm, it should be noted that the Cry1Ac 

protein is processed at both the N- and C- termini during maturation of the protein into its active 

form (tryptic core) in the insect gut. The CTP1 residual 4 amino acid residues would be cleaved off 

by protease action as part of that maturation process in situ. The equivalency of the protein used as a 

test substance and that as produced in planta were reviewed in this risk assessment and found to be 

acceptable. 

Analytical Detection Methods: 

An exemption from the requirement of a food tolerance is already in existence (since 1997) for this 

protein and an appropriate analytical detection method already reviewed and in place for Cry1Ac. 

Hence, there is no separate review of an analytical method in this risk assessment summary. 

Protein Expression: 

Expression level data were provided for Cry1Ac in different plant tissues and at different growth 

stages. The data were produced using ELISA methods for this protein. Summary results are 

provided below in Table 1. Table 2 provides summaries of the product characterization studies and 

data provided. 

TABLE 1. Cry1Ac protein levels in MON 87701 tissues 

Tissue type1

Mean (SD)2,4

(μg/g fwt) 

Range5

(μg/g fwt) 

Mean (SD)3,4

(μg/g dwt) 

Range5

(μg/g dwt) 

OSL-1 30 (8.5) 12-40 220 (70) 110-350 

OSL-2 38 (16) 18-80 260 (100) 130-500 

OSL-3 34 (17) 14-77 240 (110) 94-480 

OSL-4 53 (36) 15-110 340 (290) 78-960 

Root <LOD <LOD NA6 NA6

Forage 9.0 (8.8) 2.5-32 34 (36) 8.2-140 

Seed 4.2 (0.73) 3.1-5.0 4.7 (0.79) 3.4-5.7 

Pollen 2.3 (0.58) 1.8-3.1 NA NA 

Data from pp. 17 and 18 of 20, MRID 47841703 

1Tissues were collected at the following growth stages: OSL-1: V3-V4; OSL-2: V6-V8; OSL-3: V10-V12; OSL-4: V14-

V16; Root: R6; Forage: R6; Seed: R8, harvested at or dried to 10-15% moisture; Pollen: R2. 

2Protein levels expressed as μg of protein/g of tissue on a fresh weight basis. 

3Protein levels expressed as μg/g on a dry weight basis, calculated by dividing the fresh weight by the dry weight. 

conversion factors obtained from moisture analysis data. Moisture analysis was not performed for pollen. 

4The mean and standard deviation were calculated across sites (n=15, except OSL-1 n=13 and pollen n=4). 

5Minimum and maximum values were determined for each tissue type across sites. 

6Protein levels <LOD on a fresh weight basis were not converted to dry weight values. 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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Table 2. Product Characterization Data Submitted 

Study Type/Title Summary MRID #

Human Health and 

Environmental 

Assessment of the 

Plant-Incorporated 

Protectant Bacillus 

thuringiensis

Cry1Ac Protein 

Produced in InsectProtected Soybean 

MON 87701 

MON 87701 is an insect-protected soybean that produces the 

Cry1Ac insecticidal protein derived from Bacillus thuringiensis

subsp. kurstaki. Cry1Ac provides protection from feeding 

damage by target lepidopteran pests. Characterization studies 

have confirmed that the genetic modification in MON 87701 

contains a single insert with the intended sequence, and that the 

insert is stable over multiple generations. The mean Cry1Ac 

content in MON 87701 tissues (dry weight basis) was 

determined to be 4.7 μg/g in harvested seed, 34 μg/g in forage, 

220 to 340 μg/g in leaves, 2.3 μg/g in pollen/anthers (fresh 

weight basis), and below the limit of detection in roots. MON 

87701 was effective against major lepidopteran pests of soybean 

in screenhouse and open field trials at multiple locations in the 

US and Argentina. The Cry1Ac protein has a history of safe use 

in cotton and corn products. No structurally relevant similarity 

exists between Cry1Ac and any known toxic or biologically active 

proteins or known allergens that would be harmful to human or 

animal health. MON 87701 is not expected to produce adverse 

effects in non-target organisms, including threatened or endangered 

species, based on tests with mice, two avian species, soil 

decomposers, and beneficial insects. Tests on aquatic species were 

not conducted since there is no meaningful, ecologically relevant 

exposure to aquatic organisms from soybean. A laboratory soil 

degradation study showed that 72% to 96% of the Cry1Ac in 

MON 87701 biomass incorporated into soil degraded within six 

months, indicating Cry1Ac is unlikely to persist or accumulate 

in the environment. 

Classification: ACCEPTABLE

47841701 

Assessment of the 

Cry1Ac Protein 

Levels in Soybean 

Tissues Collected 

from MON 87701 

Produced in U.S. 

Field Trials During 

2007

MON 87701 is an insect-protected soybean that produces the 

Cry1Ac insecticidal protein derived from Bacillus thuringiensis

subsp. kurstaki. Cry1Ac provides protection from feeding 

damage by the target lepidopteran pests. An enzyme linked 

immunosorbent assay method was used to determine the level 

of Cry1Ac protein expressed in MON 87701 tissues from 

plants grown in field trials conducted in Alabama, Arkansas, 

Georgia, Illinois, and North Carolina in 2007. The mean 

Cry1Ac levels across all sites were 4.7 μg/g dry weight in 

harvested seed, 34 μg/g dry weight in forage, and below the 

47841703 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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Study Type/Title Summary MRID #

limit of detection (LOD) in roots. The mean Cry1Ac level in 

leaves harvested during difference stages of the growing season 

ranged from 220-340 μg/g dry weight. The mean Cry1Ac level 

in pollen from the single site sampled was 2.3 μg/g fresh 

weight.

Classification: ACCEPTABLE

Characterization of 

the Cry1Ac protein 

purified from the 

harvested seed of 

MON 87701 

soybean and 

comparison of the 

physicochemical 

and functional 

properties of the 

MON 87701-

produced and E 

coli-produced 

Cry1Ac protein

The information and data presented are in accord with the 

conclusion of the study author that MON-87701-derived and E. 

coli-derived Cry1Ac are equivalent proteins. Relative 

molecular mass measurements derived through SDS-PAGE and 

fragment analysis through MALDI-TOF / MS clearly indicate 

that the proteins derived from in planta expression in MON 

87701 and from expression in E. coli are of the same mass. Nterminal sequencing of the first 7 amino acids and their identity 

to the Cry1Ac sequence aided in verifying that the protein of 

interest was consistent with what is known of the insecticidal 

protein, along with the ambiguous cysteine residue present in 

accord with the use of the CTP1 peptide at the terminus. 

Reactivity to antiserum directed against Cry1Ac also indicates 

the sequence similarities between the two proteins and any 

products of catalysis as noted on the blots. 

An absence of glycosylation in both microbial and plant 

produced forms of Cry1Ac is as expected for this protein and 

further suggests the homology between the proteins from the 

two sources. The EC50 values for the two proteins, when tested 

against the corn earworm, identify the insecticidal activity of 

the two proteins as so similar in value as to be the same. The 

weight of evidence from the variety of testing parameters 

indicates that the two sources of the Cry1ac test substance 

protein are identical and interchangeable for the testing 

performed. 

Classification: ACCEPTABLE

47841704 

Functional 

Equivalence of E. 

coli-Produced Full 

Length Cry1Ac 

Protein and the 

Cry1Ac Tryptic 

Core Protein

The functional activity of the 131.7 kDa full-length E. coliproduced Cry1Ac protein (protoxin) was compared to that of 

the 57.8 kDa E. coli-produced Cry1Ac tryptic core (toxin). The 

EC50 values from 18 previous dietary bioassays of corn 

earworm (Helicoverpa zea) exposed to the Cry1Ac tryptic core 

were compared to the EC50 values from 12 previous studies 

exposing corn earworm to full length Cry1Ac. To correct for 

differences in the molecular mass, the EC50 values (μg/mL) for 

each set of bioassays were converted to molar concentrations, 

47841706 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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Study Type/Title Summary MRID #

and the means were compared statistically. There was no 

statistically significant difference (p = 0.39) in the mean EC50

values for the tryptic core and full length Cry1Ac protein 

expressed on a molar basis, indicating that both forms are 

functionally equivalent in potency against susceptible 

Lepidoptera. Therefore, results of safety and efficacy studies using 

the tryptic core can be used to evaluate the safety and insecticidal 

activity of the full length protein. 

Classification: ACCEPTABLE

Molecular Analysis 

of Insect-Protected 

Soybean MON 

87701 – Amended 

Report for 

MSL0022176 

(Amendment 3)

MON 87701 contains one copy of the insert at a single integration locus and all expression elements were present in the TDNA I; MON 87701 does not contain detectable plasmid 

backbone or selectable marker sequences; and the integrity of 

the inserted cry1Ac expression cassette within the inserted 

sequences confirmed and identified the 5' and 3' insert togenomic DNA junctions. The insertion site in conventional 

soybean confirmed the flanking genomic DNA is native to the 

soybean genome and was maintained through five generations 

of breeding. 

Classification: ACCEPTABLE

47873801 

B. HUMAN HEALTH ASSESSMENT OF CRY1AC AS EXPRESSED IN MON 87701 

Toxicological Profile 

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 health risk 

The human health studies submitted for Cry1Ac are summarized in Table 3 below. 

Table 3. Summary of Cry1Ac Human Health Data 

Study Type/Title Summary MRID #

Immunodetection of 

Cry1Ac protein in 

MON 87701 ground 

seed following heat 

treatment.

Heating of MON 87701 ground seed in a manner that simulates 

baking results in the loss of immunodetectable Cry1Ac protein 

below the LOD, indicating a decrease ≥ 94% of the Cry 1Ac 

relative to the protein in unheated MON 87701 samples. 

Classification: ACCEPTABLE 

47841705 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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Study Type/Title Summary MRID #

An Acute Study of 

Cry1AcProtein 

Administered by the 

Oral (Gavage) 

Route to Mice; 

Human Healthand 

Environmental 

Assessment 

In an acute oral toxicity study, twenty male and twenty female 

fasted, approximately 8-10 week old CD-1 mice were given an 

oral dose of control Bovine serum albumin (BSA) or Cry1Ac 

Protein at doses of 1280 or 1290 mg protein/kg body weight, 

respectively. In an additional study phase, twenty male fasted, 

approximately 6 week old CD-1 mice were given an oral dose 

of control BSA or Cry1Ac Protein at dose of 1620 or 1460 mg 

protein/kg body weight, respectively. The control and test 

material were administered as two doses separated by 

approximately 4 hours. The animals were observed for 14 

days. The Oral LD50 for female mice was > 1290 mg / kg BW 

and for male mice it was > 1460 mg / kg BW. Based on the 

results of this study, Cry1Ac Protein is in EPA Toxicity 

Category III. All animals survived the study. No test materialrelated clinical signs, body weight effects, and food 

consumption effects were noted during the study. No test 

material-related gross abnormalities were noted in any animal 

at necropsy. 

Classification: ACCEPTABLE

47841707 

Assessment of the 

in vitro digestibility 

of the Cry1Ac 

protein in simulated 

gastric and 

simulated intestinal 

fluids

The data presented in this evaluation of protein digestibility in 

simulated gastric and simulated intestinal fluids clearly indicate 

a trend toward digestion of the Cry1Ac protein in a rapid 

manner. Within the limits of detection of the colloidal 

Coomassie Brilliant Blue protein binding dye (between 10 - 20 

ŋg) following SDS-PAGE, the Cry1Ac protein was degraded 

(i.e., not visible) within 30 sec of treatment with SGF. 

Treatment with trypsin (SIF) results in a peptide fragment 

representing the tryptic core of Cry1Ac. This tryptic core 

fragment is typical of δ-endotoxins from B. thuringiensis. With 

sequential treatment of SGF and SIF, Cry1Ac was rapidly 

degraded suggesting that under conditions typical of a 

monogastric mammalian stomach, the Cry1Ac protein would 

not enter the small intestine in intact form, but rather as small, 

peptide fragments. Similar results were obtained using Western 

blotting techniques with chemiluminescent detection; Cry1Ac 

protein was degraded within 30 seconds of SGF treatment with 

a limit of detection of approximately 1 ŋg. A small stained band 

of approximately 4 kDa was noted on the SDS-PAGE gel 

following SGF digestion. This band was examined by Nterminal sequencing and determined to consist of two peptides 

from Cry1Ac. Following treatment with SIF for 5 min, this 

band of two peptides was no longer detected. The study results 

47841708 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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Study Type/Title Summary MRID #

show that full-length Cry1Ac protein is rapidly digested in the 

gastrointestinal digestive system. 

Classification: ACCEPTABLE 

Bioinformatics 

evaluation of the 

Cry1Ac protein 

present in MON 

87701 soybean 

utilizing the AD8, 

TOXIN6, and 

PROTEIN 

databases

No significant sequence homology between any sequential 

MON 87701 Cry1Ac protein sequences and known allergens, 

toxins, or reactive proteins was found as based upon FASTA 

alignment procedures and an 8 amino acid sliding window 

comparison. 

Classification: ACCEPTABLE 

47841709 

HUMAN HEALTH ASSESSMENT Cry1Ac in MON 87701

. 

Mammalian Toxicity and Allergenicity Assessment

Monsanto has submitted acute oral toxicity data demonstrating the lack of mammalian toxicity at 

high levels of exposure (ingestion) to the pure Cry1Ac protein. These data demonstrate the safety 

of the product at a level well above maximum possible consumption levels that are reasonably 

anticipated as expressed in the crop. Basing this conclusion on acute oral toxicity data without 

requiring further toxicity testing and residue data is similar to the Agency position regarding 

toxicity testing and the requirement of residue data for the microbial Bacillus thuringiensis 

products from which this plant-incorporated protectant was derived (See 40 CFR Sec. 

158.740(b)(2)(i)). For microbial products, further toxicity testing (Tiers II & III) and residue data 

are triggered by significant adverse acute effects in studies, such as the acute oral toxicity study, to 

verify the observed adverse effects and clarify the source of these effects. 

An acute oral toxicity study in mice (MRID 47841707) indicated that Cry1Ac is non-toxic to 

humans and other mammals. Three groups of ten male and ten female mice were dosed by oral 

gavage with 1290 or 1460 mg/kg bodyweight of microbially-produced Cry1Ac protein. A negative 

control group was also included in the study: bovine serum albumin protein control. There were no 

significant differences between the test and control groups; therefore, the Cry1Ac protein does not 

appear to cause any significant adverse effects at an exposure level of > 1460 mg/kg bodyweight. 

When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels 

(Sjoblad et al., 1992). Therefore, since no acute effects were shown to be caused by Cry1Ac, even 

at relatively high dose levels, the Cry1Ac protein is not considered toxic. Further, amino acid 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

Biopesticide Registration Action Document 

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sequence comparisons showed no similarities that would raise a safety concern between the Cry1Ac 

protein and known toxic proteins in protein databases. 

Since Cry1Ac 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-ofevidence 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) followed by simulated intestinal fluid (SIF), 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 Cry1Ac 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 Cry1Ac with known 

allergens showed no significant overall sequence similarity or identity at the level of eight 

contiguous amino acid residues. 

3. Digestibility. The Cry1Ac protein was digested within 30 seconds in simulated gastric fluid 

containing pepsin. Small peptides remaining following gastric simulated digestion were 

completely degraded to amino acid residues in SIF upon contact. 

4. Glycosylation. Cry1Ac expressed in soybean was shown not to be glycosylated. 

5. Conclusion. Considering all of the available information, EPA has concluded that the 

potential for Cry1Ac to be a food allergen is minimal. 

Although Cry1Ac was only shown not to be glycosylated in corn, it is unlikely to be glycosylated in 

any other crops because in order for a protein to be glycoslyated, it must contain specific 

recognition sites for the enzymes involved in glycosylation, and the mechanisms of protein 

glycosylation are similar in different plants (Lerouge et al., 1998).

Overall Safety Conclusions 

The continued use of Cry1Ac protein in plant expression systems (plant-incorporated 

protectants) and in microbial biopesticides is fully supported by the information presented in this 

registration submission relative to human and animal health concerns. The lack of mammalian 

toxicity and allergenicity effects following thorough examination of pertinent information, as 

well as the efficacy noted in the insect bioassays, indicates that the specificity of the δ-endotoxin 

Cry1Ac as an insect management mechanism is safe as proposed for use in soybean intended for 

cultivation and human and animal consumption. 

Endocrine Disruptors 

As required under FFDCA section 408(p), the Agency 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 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

Biopesticide Registration Action Document 

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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 the 

Agency 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 quantitative relationship between the dose and the E, A, or T effect. 

Between October 2009 and February 2010, the Agency 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 Cry1Ac protein is not among the group of 58 pesticide active ingredients on the initial list to 

be screened under the EDSP. Under FFDCA section 408(p), the Agency must screen all pesticide 

chemicals. Accordingly, the Agency anticipates issuing future EDSP 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/. 

REFERENCES: 

Gorbunova, V. and A.A Levy, (1997) Non-homologous DNA end joining 

in plant cells is associated with deletions and filler DNA insertions. 

Nucleic Acids Res., 25:4650–4657. 

Lerouge, P. Cabanes-Macheteau, M., Rayon, C., Fichette-Lainé, A-C., Gomord, V., and Faye, 

L., (1998) N-Glycoprotein biosynthesis in plants: recent developments and future trends. Plant 

Molecular Biology 38: 31-48. 

Salomon, S. and H. Puchta (1998) Capture of genomic and T-DNA sequences during 

double-strand break repair in somatic plant cells. The EMBO Journal, 17:6086–6095. 

Sjoblad, Roy D., et al., (1992) Toxicological Considerations for Protein Components of 

Biological Pesticide Products. Regulatory Toxicology and Pharmacology 15:3-9. 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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C. ENVIRONMENTAL ASSESSMENT for MON 87701 

Overall Conclusions 

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, are not anticipated. Horizontal gene transfer, gene flow, and the 

development of weediness are also not anticipated. 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. 

1. 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 is described in several of BPPD’s 

documents and is presented again here as background information. 

To minimize data requirements and avoid unnecessary tests, risk assessments are structured such 

that risk is determined first from estimates of hazard under “worst-case” exposure conditions. A 

lack of adverse effects under these conditions would provide enough confidence that there is no 

risk and no further data would be needed. Hence, such screening tests conducted early in an 

investigation tend to be broad in scope but relatively simple in design, and can be used to 

demonstrate acceptable risk under most conceivable conditions. When screening studies suggest 

potentially unacceptable risk additional studies are designed to assess risk under more realistic 

field exposure conditions. These later tests are more complex than earlier screening studies. Use 

of this “tiered” testing framework saves valuable time and resources by organizing the studies in 

a cohesive and coherent manner and eliminating unnecessary lines of investigation. Lower tier, 

high dose screening studies also allow tighter control over experimental variables and exposure 

conditions, resulting in a greater ability to produce statistically reliable results at relatively low 

cost.1

 

 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. 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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Tiered tests are designed to first represent unrealistic worst case scenarios and ONLY progress to 

real world field scenarios if the earlier tiered tests fail to indicate adequate certainty of acceptable 

risk. Screening (Tier I) non-target organism hazard tests are conducted at exposure 

concentrations several times higher than the highest concentrations expected to occur under 

realistic field exposure scenarios. This has allowed an endpoint of 50% mortality to be used as a 

trigger for additional higher-tier testing. Less than 50% mortality under these conditions of 

extreme exposure suggest that population effects are likely to be negligible given realistic field 

exposure scenarios. 

The EPA uses a tiered (Tiers I-IV) testing system to assess the toxicity of a PIP to representative 

non-target organisms that could be exposed to the toxin in the field environment. Tier I high dose 

studies reflect a screening approach to testing designed to maximize any toxic effects of the test 

substance on the test (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 OPPTS Testing Guidelines, Series 850 and 885 (EPA 712-C-96-280, February 

1996).2

 These guidelines, as defined in 40 CFR 152.20, apply to microbes and microbial toxins 

when used as pesticides, including those that are naturally occurring, and those that are strainimproved, either by natural selection or by deliberate genetic manipulation. Therefore, PIPs 

containing microbial toxins are also covered by these testing guidelines. 

The Tier I screening maximum hazard dose (MHD) approach to environmental hazard 

assessment is based on some factor (whenever possible >10) times the maximum amount of 

active ingredient expected to be available to terrestrial and aquatic non-target organisms in the 

environment (EEC).3

 Tier I tests serve to identify potential hazards and are conducted in the 

laboratory at high dose levels that 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. 

The Guidelines further state that the duration of all Tier I tests should be approximately 30 days. 

 2

 OPPTS Testing Guidelines, Series 850 and 885 website: 

http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/885Microbial_Pesticide_

Test_Guidelines/Series

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. 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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Some test species, notably non-target insects, may be difficult to culture and the suggested test 

duration has been adjusted accordingly. Control and treated insects should be observed for at 

least 30 days, or in cases where an insect species cannot be cultured for 30 days, until negative 

control mortality rises above 20 percent. 

Failing the Tier I (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. Greater than 50% 

mortality, however, does not necessarily indicate the existence of unacceptable risk in the field, 

but it does trigger the need to collect additional dose-response information and a refinement of 

the exposure estimation before deciding if the risk is acceptable or unacceptable. Where potential 

hazards are detected in Tier I testing (i.e. mortality is greater than 50%), additional information 

at lower test doses is required which can serve to confirm whether any effect might still be 

detected at more realistic field [1X EEC] concentrations and routes of exposure.5 

When screening tests indicate a need for additional data, the OPPTS 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 to the EEC; when the EEC is lower than the 

LOAEC, a no risk conclusion is made. These tests offer greater environmental realism, but they 

may have lower statistical power. Appropriate statistical methods, and appropriate statistical 

power, must be employed to evaluate the data from the definitive tests. Higher levels of 

replication, the number of test species, and/or repetition are needed to enhance statistical power 

in these circumstances. 

Data that show less than 50 % mortality at the maximum hazard dosage level – (i.e. LC50, ED50, 

or LD50 >10 X EEC) are sufficient to evaluate adverse effects, making lower field exposure dose 

definitive testing unnecessary. It is also notable that the recommended >10X EEC maximum 

 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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hazard dose level is a highly conservative factor. The published EPA Level of Concern [LOC] is 

50% mortality at 5X EEC (US EPA, 1998).6 

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 Plant-Incorporated Protectants (PIPs); however, this panel recommended that, 

for PIPs with insecticidal properties, additional testing of beneficial invertebrates closely related to 

target species and/or likely to be present in GM 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, 2001a, 

2002, and 2004). 

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. But, 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 (Cry proteins) bind to soil particles, it has also 

been shown that these proteins are degraded rapidly by soil microbial flora upon elution from 

soil particles. 

Conclusion: The tiered approach to test guidelines ensures, to the greatest extent possible, that 

the Agency requires the minimum amount of data needed to make scientifically sound regulatory 

decisions. EPA believes that maximum hazard dose Tier I screening testing presents a reasonable 

approach for evaluating hazards related to the use of biological pesticides 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. But, 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 non-target organisms directly in the 

 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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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 Cry protein accumulation in soils as a condition of registration due to 

the lack of baseline data on the potential for long-term environmental effects from the cultivation 

of PIP-producing plants. 

Since the commercialization of Bt crops, 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 Cry proteins on the invertebrate community structure in Bt

crop fields has since been adequately addressed. Specifically, a meta-analysis7 of the data 

collected from 42 field studies indicated that non-target invertebrates are generally more 

abundant in Bt cotton and Bt maize fields than in non-transgenic 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 to date concluded that the largescale studies in commercial Bt cotton have not revealed any unexpected non-target effects other 

than 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. 

Overall, the Agency is in agreement with the conclusions of these studies and collectively, these 

results provide extensive data to support that Bt crops have not caused long term environmental 

effects on a population level to organisms not targeted by Bt proteins. Based on these 

considerations, regulatory testing of the specialist predators and parasitoids of target pests may 

eventually be considered unnecessary. 

2. Nontarget Organism Risk Assessment for Cry1Ac as Expressed in MON 87701 

Soybeans 

 

a. Exposure Assessment

Two separate SAP reports (October 2000 and August 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 

 

7 This research was funded by Environmental Protection Agency grant CR-832147-01. 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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greatest exposure potential to 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, BPPD recognizes that 

exposure to other nontarget organisms can occur and has required testing on representative 

species. 

The EPA risk assessment is centered only 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. BPPD 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 the EEC. For the 

purposes of the non-target organism studies submitted in support of MON 87701 soybeans, the 

test material dose levels were based on an estimated concentration of Cry1Ac protein expressed 

in the tissue(s) to which the assessed taxa are likely to be exposed. For nontarget insects and 

honeybees, tests were performed at concentrations of 19X to 322X the expression level in MON 

87701 pollen. For soil invertebrates, Monsanto tested at 15X to 18X the estimated soil 

concentration based on the expected amount resulting from plant incorporation in the soil. For 

birds, tests were performed using raw soybeans. Details of these dosing amounts are discussed 

further in the summary of the toxicity studies presented below. 

b. 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 toxins. These requirements include testing on birds, mammals, nontarget insects, honey 

bee, plants, and aquatic species, and information has been submitted to address these requirements. 

Limit dose testing and dietary studies were performed on representative organisms from several 

taxa in support of the MON 87701 Soybean Section 3 registration. As stated above, BPPD’s risk 

assessments focus heavily on beneficial nontarget invertebrates, since they are most closely related 

to organisms susceptible to the insecticidal action of Bt toxins. The Cry1Ac protein is meant to 

target species within the order Lepidoptera (moths and butterflies). Bt toxins are known typically 

to have a limited host range; however, to address any unforeseen change in activity spectrum as a 

result of laboratory protein synthesis and to fulfill the published registration data requirements 

EPA requires that test species used for non-target insect evaluations should include several 

invertebrate species that are not related to the target pests, as well as tests on birds and other 

vertebrates as appropriate. Earthworm studies are also recommended. Data to evaluate the toxicity 

of Cry1Ac on several species of invertebrates were submitted, including the lady beetle, honey 

bee, and earthworm. 

Since exposure may also occur to other nontarget organisms, EPA has received data to comply 

with the Agency’s published non-target data requirements on other nontarget organisms. A 
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study to determine soil degradation of Cry1Ac has also been submitted, as well as a description 

of the MON 87701 soybean Cry1Ac in soil. The individual results for nontarget organism and 

soil degradation testing for Cry1Ac are summarized in Table 1. The studies are described in 

more detail within the risk assessment conclusions below, and full reviews of each study for each 

event can be found in the individual Data Evaluation Records. 

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. In support of the MON 87701 soybean 

registration, test substances used in the submitted studies included bacterially produced purified 

Cry1Ac protein. Comparative analyses have been performed to verify the equivalence of the 

bacterially produced and purified Cry1Ac and the Cry1Ac protein produced in MON 87701 

soybean plants (MRID 47841701). These studies show that the bacteria produced protein is 

equivalent to those produced by MON 87701 soybean plants. 

Table 4. Summary of environmental effects studies for Cry1Ac as expressed in MON 

87701 Soybean 

Data 

Requirement 

OPPTS 

Guideline

Test Substance Results Summary and Classification MRID 

No. 

Avian dietary 

testing, 

broiler chicken, 

Gallus domesticus 

885.4050 MON 87701 

Soybean meal 

A 42-day dietary study showed no adverse affects to 

broiler chickens when fed a diet composed of 

approximately 30% MON 87701 soybean meal 

containing Cry1Ac. The presence of active Cry 

protein in the test material was not confirmed. 

Classification: Unacceptable 

47841712 

Avian dietary 

testing, 

Northern 

bobwhite, Colinus 

virginianus 

885.4050 Raw ground MON 

87701 soybean 

An 8-day dietary study showed no adverse affects to 

Northern bobwhite fed a diet composed of 20% raw 

ground MON 87701 soybean. This exposure level is 

consistent with previously accepted studies for other 

PIPs. 

Classification: Acceptable 

47841711 

Non-target insect 

testing, ladybird 

beetle, 

Coleomegilla 

maculata 

885.4340 Microbiallyproduced Cry1Ac No adverse effects on survival, development to adult, 

or mean adult weight were observed in Coleomegilla 

maculata larvae exposed to 60 μg Cry1Ac protein/g 

of diet for 20 days. The LC50 was determined to be 

>60 μg Cry1Ac/g of diet and the NOAEC is ≥ 60 μg 

Cry1Ac/g of diet. 

Classification: Acceptable

47841715 

Non-target insect 

testing, parasitic 

wasp, Pediobius 

foveolatus 

885.4340 Microbiallyproduced Cry1Ac No adverse effects on survival were observed in adult 

Pediobius foveolatus fed 250 μg Cry1Ac protein/g of 

diet in a 21-day laboratory bioassay. The nominal 

LC50 was determined to be >250 μg Cry1Ac/g diet 

and the NOAEC is ≥250 μg Cry1Ac/g diet. 

Classification: Acceptable

47841716 

Honeybee testing, 

Honeybee larvae, 

Apis mellifera

885.4380 Microbiallyproduced Cry1Ac No adverse effects on survival were observed in 

honey bee larvae treated in natal cells with 410 

μg/mL (4.1 μg/cell) solution of Cry1Ac protein. The 

LC50 was determined to be > 410 μg Cry1Ac/mL or 

>4.1 μg/cell. 

47841713 
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Data 

Requirement 

OPPTS 

Guideline

Test Substance Results Summary and Classification MRID 

No. 

Classification: Acceptable

Honeybee testing, 

Honeybee adult, 

Apis mellifera

885.4380 Microbiallyproduced Cry1Ac No adverse effects on survival attributable to the test 

material were observed in honey bee adults fed 175 

μg Cry1Ac protein/mL for 10 days. The LC50 was 

determined to be > 175μg Cry1Ac/mL and the 

NOAEC was determined to be ≥175 μg Cry1Ac/mL. 

Classification: Acceptable

47841714 

Earthworm 

toxicity, 

Eisenia foetida

850.6200 Microbiallyproduced Cry1Ac No adverse effect on survival or burrowing time were 

observed in adult earthworms exposed to 250 mg 

Cry1Ac protein/kg dry soil for 14 days. The LC50

was determined to be >250 mg Cry1Ac protein/kg 

dry soil and the NOAEC was determined to be ≥250 

mg Cry1Ac protein/kg dry soil. 

Classification: Acceptable 

47841717 

Aerobic soil 

degradation 

885.5200 Lypohilized MON 

87701 soybean 

plants 

Based on the measured bioactivity in corn earworm 

(Helicoverpa zea), the DT50 for Cry1Ac in three 

different soils was ranged from 0.4 –13 days. 

Classification: Acceptable

47841718 

3. Risk Assessment Conclusions for MON 87701 Soybeans 

 a. Effects to Nontarget Wildlife, Invertebrates, and Plants 

 1. Avian Wildlife

The primary routes of exposure of birds to Cry1Ac expressed in MON 87701 soybean plants are 

expected to occur through consumption of MON 87701 plant material (e.g., seeds) and 

invertebrates that feed on MON 87701 soybean plant material. Exposure via consumption of 

soybean seeds may be limited, however, by compounds that interfere with digestion and nutrient 

uptake, such as trypsin inhibitors. Other routes of exposure (e.g., inhalation) are not expected to 

be significant, since the Cry1Ac protein is contained primarily within the plant. 

The registrant has submitted two toxicity studies for use in determining risks to birds. In a 42-

day feeding study, broiler chickens (MRID 47841712) exposed in a diet containing 

approximately 30% processed soybean meal showed no adverse effects. We note, however, that 

soybean meal processing involves heat, which could inactivate the Cry1Ac protein. Given that 

confirmation of the presence of the Cry protein in the diet was not performed, it is unknown 

whether the test animals were exposed to Cry1Ac, so this study may not be reliable for risk 

assessment. Confirmation of the presence of the Cry1Ac in the diet would resolve this 

uncertainty. An 8-day feeding study with Northern bobwhite (MRID 47841711) is available, in 

which the birds were fed a diet of 20% raw ground soybeans. As mentioned above, consumption 

of raw soybeans by birds can have detrimental effects; however, the inclusion of raw soybean in 

the diet at this level without significant effects was supported by an additional non-guideline 

study (MRID 47896601). This study shows that effects are not expected when birds consume a 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

Biopesticide Registration Action Document 

23

diet containing 20% MON 87701 soybeans. Additionally, no adverse effects were observed in a 

study of birds exposed to Cry1Ac expressed in Monsanto’s Bollgard cotton (USEPA 2001b) 

tested an exposure of 10% cottonseed in the diet. The level of exposure to the birds in these 

studies is consistent with the Agency’s previous assumptions of EECs of PIPs for birds (e.g., 

USEPA 2001b, USEPA 2005a, USEPA 2005b, USEPA 2008), and based on those assumptions 

adverse effects to birds are not anticipated. A refined estimate of exposure to birds in the field 

for soybeans has not been determined (e.g., based on their potential avoidance of soybean seeds 

and/or consumption of other foods, such as invertebrates). Therefore, a feeding test at a higher 

concentration of Cry1Ac or a limit dose test at the MHD would increase the certainty of risk 

conclusions. 

Cry1Ac has been registered as a PIP in several crops with no identified concerns for birds, and it 

has a history of use with no reported incidents involving birds in the field, including use in a crop 

that is typically ingested by birds (corn). Therefore, based on this history, current toxicity data, 

and the BPPD’s assumptions of avian exposure, adverse effects to birds as a result of the 

registration of MON 87701 soybeans are not expected 

2. Wild Mammals

Wild mammals could be exposed to Cry1Ac produced by MON 87701 soybean plans by 

consumption of plant material and invertebrates that feed on MON 87701 soybean plant material. 

As with birds, these are expected to be the primary sources for exposure. 

Data are available with which to determine the risk of Cry1Ac from MON 87701 soybeans to 

wild mammals. The results from the acute oral toxicity studies showed no significant toxicity to 

mice from acute oral testing with the Cry1Ac protein, with a NOAEL that exceeds 1290 mg/kg 

body weight for females and 1460 mg/kg for males (MRID 47841707). These data are 

sufficiently representative of toxicity that would be expected in wild mammals, and based on the 

results of this study risk to wild mammals resulting from exposure to Cry1Ac expressed in MON 

87701 soybean is not expected. 

3. Freshwater Animals

The Agency has previously determined that the exposure of aquatic systems to registered Cry 

proteins produced in their respective crops is minimal. Off-field pollen movement is a possible 

mechanism by which Cry proteins may be transported to aquatic habitats. But, given that 

soybeans are primarily self-pollinated and cultivated soybean plants release pollen prior to 

flower opening (Abud et al. 2007, Caviness 1966), off-site movement of pollen in this case is not 

expected. The Agency has concluded that plant litter from both Bt corn and Bt cotton crops are 

not deposited in amounts high enough to result in adverse effects (USEPA 2001b). Moreover, 

given that soybean produces less above-ground residue under cultivation (Green and Blackmer 

1995), this conclusion is also made for MON 87701 soybeans. Monsanto submitted data waiver 

rationales that are consistent with the Agency’s position on aquatic exposure with Bt PIPs. 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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Based on the expected lack of exposure, risk to freshwater fish as a result of the registration of 

Cry1Ac in MON 87701 soybeans is not expected. While exposure is expected to be very low in 

aquatic habitats, and effects on freshwater invertebrates is not expected, a published laboratory 

study with lepidopteran-active Cry proteins has revealed that the leaf shredding (caddis fly) 

trichopteran, Lepidostoma liba, had 50% lower growth rate when fed Bt corn litter (RosiMarshall, et al. 2007). Two previous field study reports by the same authors did not find adverse 

effects on head stream invertebrates. Because of concerns raised in this study, EPA has been 

requiring registrants of Bt PIPs to submit a 7-10 day freshwater invertebrate toxicity study for 

each PIP to reduce uncertainty regarding this issue. 

 4. Estuarine and marine animals

BPPD typically waives the requirement of studies with estuarine/marine animals for Bt Cry 

proteins because of an expected lack of exposure in these environments. Therefore, data were not 

required for Cry1Ab for the registration of MON 87701 soybeans, and based on expected lack of 

exposure, adverse effects to these species are not anticipated. 

 5. Terrestrial and aquatic plant species

BPPD typically waives nontarget plant testing for Bt Cry proteins, since the active ingredient is 

an insect toxin (Bt δ-endotoxin) that has never shown any toxicity to plants. Therefore, BPPD 

has concluded that adverse effects of Cry1Ac expressed in MON 87701 soybeans to terrestrial 

and aquatic plants are not anticipated. 

 6. Invertebrate species 

BPPD assumes that nontarget insects receive exposure primarily through consumption of MON 

87701 soybean pollen and/or nectar, though they may also consume pest insects that feed on 

soybean plant tissue or occasionally consume other soybean plant tissues themselves. The 

principal route of exposure to soil-dwelling invertebrates, such as collembola, earthworms, and 

rove beetles, is assumed to be consumption of decomposing plant tissue, and also possibly plant 

exudates, in soil during feeding. 

Monsanto submitted studies with which to determine the effects of Cry1Ac expressed in MON 

87701 to nontarget insects and soil invertebrates. Studies were submitted with a lady bird beetle 

and a parasitic wasp species, each of which showed that adverse effects did not occur to the test 

insects at >60 μg/g diet and >250 μg/g diet, respectively. Assuming a maximum expected 

environmental concentration of 3.1 μg/g diet (see MRID 47841701) based on the estimated 

expression level in pollen, adverse effects would not be expected at concentrations up to 19X and 

80X this assumed maximum EEC for lady bird beetle and parasitic wasp, respectively. 

Additionally, a study was submitted with earthworms that showed no effects on mortality or 

burrowing time resulting from exposure to Cry1Ac present in artificial soil at approximately 18X 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

Biopesticide Registration Action Document 

25

maximum EEC. The maximum EEC of the soil was determined based on incorporation of entire 

soybean plants into the top six inches of soil, and is described in MRID 47841717. 

Two studies that tested the effect of Cry1Ac expressed in MON 87701 soybean in honey bee 

were also submitted. One study tested the effects in honey bee larvae at 410 μg Cry1Ac/mL test 

solution or 4.1 μg Cry1Ac/cell (larva). This study tested effects at 132X EEC based on the 

concentration of the test solution. No adverse effects were observed in the larvae in this study, 

and the number emerging to adults in the group exposed to Cry1Ac was not significantly 

different from the controls. The other bee study tested the effects of oral exposure to Cry1Ac in 

adult bees. In this study, adult bees were fed a solution containing 175 μg Cry1Ac/mL test 

solution (56X EEC). The test solution contained a buffer, which was shown to cause a decrease 

in survival. A decrease in survival was also observed in the group exposed to Cry1Ac, but these 

effects were determined to be statistically similar to those in the buffer control group. Therefore, 

the effects observed on adult honey bees in the Cry1Ac group were determined to be due to the 

buffer and not the Cry1Ac. 

The Agency has additional information for nontarget insects on Cry1Ac submitted to support the 

registration of Bollgard cotton. The Cry1Ac protein produced in MON 87701 soybeans is 

identical to the Cry1Ac produced in Bollgard cotton, with the exception of four additional Nterminal amino acids derived from a chloroplast transit peptide targeting sequence that is not 

expected to impact activity or toxicity because of excision of the N-terminus during prototoxin 

activation (MRID 47841701). Studies with the Cry1Ac protein produced in Bollgard 

additionally showed no adverse effects in two Collembola spp. (Folsomia candida and Xenylla 

grisea) and minute pirate bug (Orius albidipennis) (USEPA 2001b). 

Based on the studies submitted for MON 87701, as well as other information for Cry1Ac, risk to 

nontarget insects, soil invertebrates, and honey bees resulting from registration of Cry1Ac in 

MON 87701 soybean is not expected. 

b. Fate of Cry1Ac in Soil 

A study was submitted to show that Cry1Ac expressed in MON 87701 soybean plants degrades 

quickly and does not accumulate in soil (MRID 47841718). Lyophilized MON 87701 soybean 

plants (leaves + stems + roots), containing 63 μg of Cry1Ac/g of soybean biomass were 

incorporated into soils collected in three locations with soybean-growing regions. Soils were 

incubated for 181 days, and samples collected weekly were subjected to susceptible insect (corn 

earworm [Helicoverpa zea]) bioassays and ELISA to determine Cry1Ac content and bioactivity. 

Both the ELISA and the bioassays showed that Cry1Ac degraded in all three soils, with the most 

rapid degradation occurring during the first 16 days. At test end, 72% to 96% of the Cry1Ac had 

degraded regardless of soil texture, pH, clay content, or the method used to analyze the soil 

(ELISA or bioassay), and the DT50 of Cry1Ac in the soils was ranged from 0.4 – 13 days. These 

results indicate that Cry1Ac from MON 87701 breaks down rapidly in soil and is unlikely to 

persist or accumulate in the environment after continuous cultivation. Because currently 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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registered PIPs have not been shown to persist in soil, the Agency has determined that no 

additional long-term field studies are required for MON 87701 soybeans. 

 c. Effects on Soil Microorganisms 

Numerous published studies indicate that exposure to Cry protein produced in Bt PIP crop plants 

does not adversely affect soil microorganisms (Sanvido et al., 2007). Although a minimal 

transient increase and shift in microbial populations may result from the presence of transgenic 

plant tissue in soil, no adverse effects have been attributed to the Cry protein. In addition, the 

soil degradation study discussed above shows that the Cry1Ac protein from MON 87701 plants 

degrades quickly in soil. 

With regard to the impact of genetically engineered crops on soil, it is important to note that 

agricultural practices themselves cause large changes in soil and soil microbial composition. 

Furthermore, factors such variations in seasons and weather, plant growth stage, and plant 

varieties, independent of being genetically engineered, are also responsible for significant shifts 

in soil microbial communities. Most studies with genetically engineered crops to date have 

shown minor or no effects on soil microbes beyond the variation caused by the factors listed 

above. If reports of adverse effects became available, the Agency will take appropriate action to 

mitigate potential risks. 

 d. 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, soilinhabiting 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 87701 were 

derived from soil-inhabiting bacteria, the EPA has concluded that HGT of transgenes found in 

Cry1Ac producing soybean is not expected. 

 e. Gene Flow and Weediness Potential 

Movement of transgenes from crop plants into weeds is a significant concern, due to uncertainty 

regarding the effect that a new pest resistance gene may have on plant populations in the wild. 

Under FIFRA, the Agency has reviewed the potential for gene capture and expression of Cry 

proteins in commercial Bt soybeans by wild or weedy relatives of cotton in the United States, its 

possessions or territories. 
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An Experimental Use Permit (EUP) registration was granted for MON 87701 soybeans in 2007. 

At that time, BPPD examined the potential for gene flow and the weediness potential of MON 

87701 soybean. Since no known wild or weedy relatives exist in the U.S. with which Glycine 

max (L.) Merr. can form viable hybrids in nature, 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 

(BPPD 2007). 

f. Impacts on Endangered Species 

Because of the selectivity of Cry1Ac 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, as discussed above, any 

exposure to lepidopterans would be expected to occur within those areas. 

Exposure could occur via direct consumption of MON 87701 soybean plants or consumption of 

MON 87701 pollen that falls on non-soybean plants within the soybean field and its immediate 

margins. However, exposure to significant amounts of Cry1Ac 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). To examine the potential exposure from 

pollen falling on food plants, BPPD performed a similar analysis as that used in Hellmich et al. 

(2001), which was also employed in the Bt crops reassessment BRAD (USEPA 2001b) and the 

assessment of nontarget lepidopteran risk for WideStrike cotton (USEPA 2005a). Based on the 

most sensitive EC50 value reported for CEW (used in confirmatory tests) in nontarget organism 

studies submitted for MON 87701 soybean (0.0022 μg Cry1Ac/mL diet), an expression level of 

3.1 μg Cry1Ac/g for pollen, and assumptions of leaf consumption and pollen weight as given in 

Hellmich et al. (2001), BPPD determined that an estimated number of pollen grains resulting in 

the EC50 is 3.51 grains/day. Based on the assumed daily food consumption, 18.49 grains 

pollen/cm2

/day must be present on potential food plants to result in an amount equal to the EC50

for the sensitive species. This amount is 100X the mean levels of pollen measured in soybean 

fields. The Hellmich et al. analysis was performed for corn. Corn pollen is recognized as being 

relatively heavy; however, since corn is wind pollinated, soybean pollen is likely to be heavier. 

However, even if a soybean pollen grain weighs 5x that of corn pollen, the estimated number of 

pollen grains resulting in the EC50 would still be 20x the amount of pollen measured in soybean 

fields. These are expected to be conservative estimates, since higher pollen weights for corn are 

reported (e.g., 2.0 million grains/gram [Mascarenhas et al. 1984], 3.5 million grains/gram [RosiMarshall et al. 2007]). Additionally, based on the mean amount of pollen measured in soybean 

fields, and assuming a 14 day flowering period for a soybean field, 2.52 grains/cm2 would be 

expected on potential food plants at the end of the flowering period if they are entirely 
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undisturbed and no other organisms consume the pollen, and the amount needed to result in the 

equivalent EC50 would not be attained. Based on this analysis, BPPD 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 listed lepidopterans. Therefore, any significant 

exposure would have to occur through consumption of the MON 87701 soybean plants on the 

field. 

A search of EPA’s LOCATES database indicates that three species of 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), and 

Mitchell’s Satyr Butterfly (Neonympha mitchellii mitchellii). 

The potential effects of Bt PIPs in corn on the Karner blue butterfly was extensively analyzed in 

BPPD’s Bt Crops Reassessment (USEPA 2001b). This species requires a specific wild lupine 

species (Lupinis perrenis) as a site of oviposition and larval food source, and the concern for 

exposure in the case of Bt corn resulted from the possibility of pollen falling on this required 

plant species. As shown in the analysis above, soybean pollen is not expected to be deposited on 

plants in or around soybean fields in amounts sufficient to cause effects in sensitive 

lepidopterans. Additionally, EPA has previously reviewed information on the proximity of 

Karner blue habitats, and none are known to exist immediately adjacent to agricultural fields 

(“adjacent to” was defined as 0-3 m for corn fields, which is reasonably applied to soybean 

fields) (USEPA 2001b). Therefore, effects to the Karner blue butterfly via consumption of 

pollen on its food/host plant are not expected. Additionally, because the Karner blue butterfly 

has an obligate relationship with a specific host plant, it will not be exposed to Cry1Ac via direct 

consumption of MON 87701 soybeans. Therefore, effects to this species as a result of the 

proposed registration of MON 87701 soybeans are not expected. 

Mitchell’s satyr butterfly has a strict reliance on fen habitats, which are characterized as stable, 

highly alkaline, and nutrient poor wetland habitats. Fen habitats can occur near agricultural 

areas; however this species is considered to be a strict habitat specialist (USFWS 1998, USFWS 

2009). Szymanski et al. (2004) describes habitat for this species as “mosaics of open shrubby 

and forested communities and associated ecotones.” They also note that all active habitats for 

this species are sedge-dominated, and contain scattered deciduous or coniferous shrubs, and 

observed that individuals stay within their habitat patch. Edge habitat and scattered shrubs 

appear to be crucial habitat components for these species, and vegetation structure is as 

important as the presence of suitable host plants (Lee 2000, Shuey 1997). The species has a 

strong preference for habitats with dense stands of sedges (USFWS 1998), and meadows and 

fens without shrubs are considered to be unsuitable habitat (Shuey 1997). In a study by Barton 

and Bach (2005) captures of adults at their study sites showed clear preference for edge and 

scattered shrub cover and avoidance by this species of open areas and habitats other than suitable 

fen habitats, including agricultural fields. Host plants have been determined to be almost 

certainly sedges, and Carex stricta is considered likely to be their primary host plant (USFWS 

1998, USFWS 2009), although they have been observed ovipositing on other small (<5 cm) 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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herbaceous plants (Lee 2000, Szymanski et al. 2004), reported to be located under dense stands 

of sedge (Szymanski et al. 2004). Sources cited within Barton and Bach (2005) observed 

ovipositioning within 1 m of the nearest tree or shrub. The species is also considered to be 

sedentary and a weak flyer (USFWS 1998, USFWS 2009, Szymanski et al. 2004, Lee 2000), 

although some populations may be less sedentary than others (Barton and Bach 2005, USFWS 

1998). Corridors containing suitable habitat are considered to be critical for dispersal and 

conservation (Barton and Bach 2005, Szymanski et al. 2004), so an open, agricultural field is not 

likely to be used for this purpose. Adults are generally not observed feeding, and the butterflies 

do not live for more than a few weeks in this life stage. U.S. Fish and Wildlife Service (FWS) 

documents describing habitat for this species do not associate it with agricultural fields (USFWS 

1998, USFWS 2009), though the extent to which such habitats have been surveyed for this 

species is not reported. Mitchell’s satyr butterfly is recognized as a strict specialist of fen 

habitats that cannot be provided by soybean cultivation. Based on the life history described in 

the documents and studies examined, BPPD concludes that the occurrence of Mitchell’s satyr in 

MON 87701 soybean fields is extremely unlikely and its use of MON 87701 soybean plants as 

ovipositioning sites or larval food plants will not occur. Therefore, effects to this species as a 

result of the registration of MON 87701 soybeans are not anticipated. 

The St. Francis’s satyr butterfly is closely related to the Mitchell’s satyr, but occupies bog 

habitats that tend to be more transitory compared to fens. This species is also considered to be a 

strict habitat specialist, and is most closely associated with Carex spp. The St. Francis’s satyr 

has habits similar to those described for Mitchell’s satyr above, in that it relies heavily on early 

successional habitats dominated by sedges, is sedentary, and is a weak flyer. All occupied 

habitats have been created by some kind of disturbance (e.g., beaver activity, fire). Dispersal has 

been recorded rarely over unsuitable habitats. Dispersing individuals show a strong tendency to 

fly along streams, remaining within riparian vegetation, and have been observed almost always 

turning back to the colony once the edge of the wetland is reached. Movements >1 km are 

probably rare (NatureServe 2009). The St. Francis’s satyr butterfly exists in limited number of 

small populations (one metapopulation). It is currently known to occupy only a small area (10-

20 ha) within Department of Defense (DOD) lands in the sandhills region of North Carolina 

(Bartel and Sexton 2009, Haddad et al. 2008, Kuefler et al. 2008, USFWS 1996), which 

substantially restricts its potential for exposure. Since this species is recognized as a strict 

habitat specialist, and because it has not been detected outside of DOD lands, despite surveys of 

surrounding areas, cultivation of MON 87701 soybean plants will not result in effects to the St. 

Francis’s satyr. 

Based on the above analysis, the Agency determines that there will be no direct effect to listed 

lepidopteran species as a result of the cultivation of MON 87701 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 87701 plants, are not currently 

known. Since the Cry1Ac in MON 87701 soybeans targets only lepidopteran insects, loss of the 

pest insects as a result of MON 87701 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 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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be affected by Cry1Ac. Effects on species other than insects have also been determined to be 

very unlikely because of the specificity of Cry1Ac. Therefore, no effects to listed species due to 

indirect effects or effects on habitat are anticipated to occur. 

4. Data Needed to Confirm MON 87701 Soybean Non-Target Hazard Assessment

BPPD has sufficient information to believe that there is no risk from the proposed uses of MON 

87701 soybean to non-target wildlife and terrestrial invertebrates, fish and aquatic invertebrates, 

plants and soil organisms. As indicated above, the Agency has been alerted to possible concerns 

of effects of Bt PIPs on freshwater aquatic invertebrates. As a result, the Agency is requiring 

registrants of Bt PIPs to submit a 7 -10 day freshwater invertebrate toxicity study to reduce 

uncertainty with this issue. Consistent with this determination, this study must be submitted to 

support a full commercial registration of Cry1Ac in MON 87701 soybeans. 

CONCLUSION

The environmental risk assessment indicates that, based on information submitted to support this 

proposed registration, as well as previous assessments for Cry1Ac, cultivation of MON 87701 

soybeans as intended will not result in unreasonable adverse environmental effects. The Agency 

additionally concludes that MON 87701 soybeans will have no direct or indirect effect on 

endangered and/or threatened species listed by the FWS and the National Marine Fisheries 

Service or their habitats. 

References 

Abud, S., P.I.M. de Souza, G.R. Vianna, E. Leonardecz, C.T. Moreira, F.G. Faleiro, J.N. Junior, 

P.M.F.O Monteiro, E.L. Rech, and F.J.L. Aragao. 2007. Gene flow from transgenic to 

nontransgenic soybean plants in the Cerrado region of Brazil. Genetics and Molecular 

Research 6:445-452. 

Bartel, R.A. and J.O. Sexton. 2009. Monitoring habitat dynamics for rare and endangered 

species using satellite images and niche-based models. Ecography 32:888-896. 

Barton, B.J. and C.E. Bach. 2005. Habitat use by the federally endangered Mitchell’s satyr 

butterfly (Neonympha mitchellii mitchellii) in a Michigan prairie fen. American Midland 

Naturalist 153:41-51. 

BPPD. 2007. Ecological risk assessment for Monsanto’s MON 87701 Bt soybean EUP. 

Memorandum from T. Milofsky to M. Hunter. 

Caviness, C.E. 1966. Estimates of natural crosspollination in Jackson soybeans in Arkansas. 

Crop Science 6:211-212. 
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Green. C.J. and A.M. Blackmer. 1995. Residue decomposition effects on nitrogen availability 

to corn following corn or soybean. Soil Science Society of America Journal 59:1065-

1070. 

Haddad, N.M., B. Hudgens, C. Damiani, K. Gross, D. Kuefler, and K. Pollock. 2008. 

Determining optimal population monitoring for rare butterflies. Conservation Biology 

22:929-940. 

Hellmich, R.L., B.D. Siegfried, M.K. Sears, D.E. Stanley-Horn, M.J. Daniels, H.R. Mattila, T. 

Spencer, K.G. Bidne, and L.C. Lewis. 2001. Monarch larvae sensitivity to Bacillus 

thuringiensis-purified proteins and pollen. Proceedings of the National Academy of 

Sciences 98:11925-11930. 

Kuefler, D., N.M. Haddad, S. Hall, B. Hudgens, B. Bartell, E. Hoffman. 2008. Distribution, 

population structure, and habitat use of the endangered Saint Francis satyr butterfly, 

Neonympha mitchellii fransisci. American Midland Naturalist 159:298-320. 

Lee, Y. 2000. Special animal abstract for Neonympha mitchellii mitchellii (Mitchell’s satyr 

butterfly). Michigan Natural Features Inventory, Lansing, MI. 4pp. 

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. 

Mascarenhas, N.T., D. Bashe, A. Eisenberg, R.P. Willing, C.M. Xiao, and J.P. Mascarenhas. 

1984. Messenger RNAs in corn pollen and protein synthesis during germination and 

pollen tube growth. Theoretical and Applied Genetics 68:323-326. 

National Academy of Science (NAS). 2000. Environmental Effects of Transgenic Plants: The 

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NatureServe. 2009. NatureServe Explorer: An online encyclopedia of life [web application]. 

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Romeis, J., Meissle, M. and Bigler, F. 2006. Transgenic Crops expressing Bacillus 

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Rosi-Marshall E. J., J. L. Tank, T. V. Royer, M. R. Whiles, M. Evans-White, C. Chambers, N. A. 

Griffiths, J. Pokelsek, and M. L. Stephen. 2007. Toxins in transgenic crop byproducts 
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may affect headwater stream ecosystems. Proceedings of the National Academy of 

Sciences 104:16204–16208. 

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. 

Shuey, J.A. 1997. Conservation status and natural history of Mitchell’s Satyr, Neonympha 

mitchellii mitchellii French (Insecta: Lepidoptera: Nymphalidae). Natural Areas Journal 

17:153-163. (abstract) 

Szymanski, J., J.A. Shuey, and K. Oberhauser. 2004. Poulation structure of the endangered 

Mitchell’s Satyr, Neonympha mitchellii mitchellii (French): implications for conservation. 

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USEPA. 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. 

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USEPA. 2001a. 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 

USEPA. 2001b. Biopesticides Registration Action Document: Bacillus thuringiensis (Bt) 

Incorporated Plant Protectants. Washington, DC. 

http://www.epa.gov/opp00001/biopesticides/pips/bt_brad.htm

USEPA. 2002. SAP Report No. 2002-05. A set of scientific issues being considered by the 

Environmental Protection Agency regarding: Corn rootworm plant-incorporated 

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USEPA. 2004. SAP Report No.2004-05. Product characterization, human health risk, 

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USEPA. 2005a. Biopesticides Registration Action Document: Bacillus thuringiensis var. 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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aizawai Cry1F and the Genetic Material (From the Insert of Plasmid PGMA281) 

Necessary for its Production in Cotton and Bacillus thuringiensis var. kurstaki Cry1Ac 

and the Genetic Material (From the Insert of Plasmid PMYC3006) Necessary for its 

Production in Cotton. Washington, DC. 

http://www.epa.gov/pesticides/biopesticides/ingredients/tech_docs/brad_006512-

006513.pdf

USEPA. 2005b. 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

USEPA. 2008. Biopesticides Registration Action Document: Bacillus thuringiensis Cry1A.105 

and Cry1Ab2 Insecticidal Proteins and the Genetic Material Necessary for Their 

Production in Corn. Washington, DC. 

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006515.pdf

U.S. Fish and Wildlife Service (USFWS). 1996. Saint Francis’ Satyr Recovery Plan. Atlanta, 

GA. http://ecos.fws.gov/docs/recovery_plan/960423.pdf 

USFWS. 1997. Recovery Plan for Mitchell’s Satyr Butterfly (Neonympha mitchellii mitchellii 

French), Ft. Snelling, MN. 

USFWS. 2009. Mitchell’s Satyr Butterfly Spotlight Species Action Plan. Ft. Snelling, MN. 

http://ecos.fws.gov/docs/action_plans/doc3038.pdf 

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. 

D. INSECT RESISTANCE MANAGEMENT (IRM) 

1. Overall Conclusions 

a) Given the low acreage projected by Monsanto for MON 87701 (<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). 

b) The use of MON 87701 on a limited basis should not impact the natural refuge strategy in 

place for Bt cotton PIPs. The total proposed acreage for MON 87701 would represent a small 
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fraction of total soybean acres in each state and should not significantly reduce the amount of 

natural refuge. Further, MON 87701 will not be planted in states (i.e., Mississippi Delta states) 

with lower natural refuge. 

c) BPPD is requiring adoption of a resistance monitoring plan, remedial action strategy, and 

annual sales reporting for MON 87701. 

2. Insect Resistance Management Assessment 

Based on the materials submitted to support registration, BPPD does not have sufficient 

information to completely assess the risk of resistance to the primary target pests for a full 

commercial registration of MON 87701 soybean. However, Monsanto’s proposal to limit the 

overall acreage and distribution of MON 87701 in the U.S. should be sufficient to mitigate 

potential resistance development. 

a. Target Pests 

Soybean looper (SL, Pseudoplusia includens) and velvetbean caterpillar (VBC, Anticarsia 

gemmatalis) are the primary target pests of MON 87701 in the U.S., but only cursory 

information on pest biology was provided in the submission. These insects have not been 

targeted by other registered Bt crops (e.g. Bt cotton or Bt corn) and have not been assessed as 

part of any other IRM plan. The other two insects targeted by MON 87701, soybean axil borer 

(Epinotia apoerema), and sunflower looper (Rachiplusia nu), have South American distributions 

and do not occur in the U.S. 

Soybean looper occurs throughout the U.S. where soybeans are grown, but overwinters only in 

tropical regions migrating annually from Mexico, Central America, and the Caribbean (Pedigo, 

1999). The insect is multivoltine with two (northern regions) to seven (southern states) 

generations per season. In addition to soybean, SL is known to feed on a wide range of 

vegetables, field crops (including corn and cotton), and weeds (Capinera, 2005). 

Velvetbean caterpillar, similar to SL, is a tropical species that migrates annually northward into 

the U.S. It can range as far north as New England, but overwinters only in the southern end of 

the Florida peninsula (Pedigo, 1999; Barbara, 2008). VBC is also polyphagous and feeds on a 

number of field crops (soybean, alfalfa, cotton, peanut, other legumes) and weeds (Barbara, 

2008). 

b. IRM Risk Considerations 

To assess the risk of resistance for new PIPs, BPPD typically considers (in addition to pest 

biology) information on dose expression and simulation modeling. Regarding the dose of MON 

87701, Monsanto referenced a published article (McRae et al., 2005) that concluded the tested 

soybean cultivar (TIC107 with a cry1A gene) exhibited high dose control against the target pests. 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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A copy of this article was not provided in the submission, though BPPD was able to obtain a 

reprint. This research showed that the tested soybean line appears to meet the criteria for high 

dose using two of the techniques established by the 1998 SAP, including serial dilutions with 

lyophilized plant tissue (technique #1) and field trials to demonstrate high efficacy against 

potential heterozygotes (technique #3). But, BPPD cannot verify the high dose claims from the 

article because it is unclear whether the tested variant (TIC107) and toxin (a Cry1A toxin 

described as being “similar” to Cry1Ac) are equivalent to MON 87701. 

Simulation modeling was not conducted for MON 87701 and a refuge-based resistance 

management strategy was not proposed by Monsanto. Rather, the company has indicated that a 

number of factors should reduce the risk of resistance so that a formalized refuge strategy is 

unnecessary. These factors include the distribution of the target pests (SL and VBC), the 

abundance of natural refuge for these insects, and anticipated limited plantings of MON 87701 

for seed increase purposes. 

BPPD believes that additional information on pest biology, dose, simulation modeling, and cross 

resistance would be necessary to assess the risks for an unlimited commercial registration of 

MON 87701. But, for a limited “seed increase” registration, BPPD agrees with Monsanto’s 

rationale regarding the low risk of resistance for the target pests. 

Biological attributes of SL and VBC reduce the overall likelihood of resistance development. 

Both insects are polyphagous and feed on numerous other wild and cultivated hosts, which 

should provide a source of natural refuge to reduce selection pressure for Cry1Ac resistance. 

Monsanto’s submission also indicated that soybeans have relatively small acreage (ca. 13% of 

U.S. soybean acres) in the southeast, where these two insects are most prevalent. Further, SL 

and VBC overwinter only in tropical areas (e.g., Florida in the U. S.); populations migrate 

annually from the Caribbean, Mexico and Central America northward through the U.S. Because 

of this, it is unlikely that any resistant individuals that might evolve during the growing season 

north of tropical areas would be able to successfully overwinter and propagate the resistant trait 

to future generations. 

Monsanto suggested in their submission that the proposal for MON 87701 is similar to the 

requirements in place for “breeding and seed multiplication activities” with other registered PIPs. 

Seed production is often not compatible with refuges due to the need for purity in seed stock. 

For IRM purposes, the seed activity is geographically limited to reduce the selection pressure for 

resistance. To illustrate, an annual per county limit of 20,000 acres for seed production has been 

imposed for Bt corn PIPs. Also, refuges have not generally been used with PIP Experimental 

Use Permits because of smaller and dispersed acreages. 

Based on the proposed acreage-limited (seed increase) registration and in consideration of the 

biological aspects of the target pests discussed above, BPPD concludes the risk of resistance to 

Cry1Ac should be low for SL and VBC. Therefore, a refuge strategy is not recommended at this 

time for MON 87701. BPPD notes that Monsanto’s submission provided an estimate of the 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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geographic limitations for MON 87701. Projected acreage is not expected to exceed 15,000 

(U.S.) total and 1,000 per county. A follow-up submission from Monsanto (April 8, 2010 letter) 

indicated that a maximum of 15,000 acres would be planted in Atlantic Coast states (Maryland 

through Georgia). Monsanto’s submission also indicated, however, that “MON 87701 

plantings....will be initially limited to breeding and seed multiplication activities...” (emphasis 

added). Should any request be made to expand the scope of MON 87701 to a commercial 

registration (with large or unlimited acreage), BPPD will require a formal IRM assessment to 

better evaluate the resistance risk and develop appropriate mitigation measures. Such a review 

should include appropriate data for dose expression, simulation modeling, and cross resistance. 

c. Natural Refuge Considerations 

In addition to the primary target pests of soybean, BPPD is also concerned about other insect 

pests that are targeted by Bt cotton but may also be exposed to Bt soybean. These insects, 

tobacco budworm (TBW, Heliothis virescens) and cotton bollworm (CBW, Helicoverpa zea), are 

similar to SL and VBC in that they are highly polyphagous and are known to exploit numerous 

crop and wild plant hosts. Cry1Ac is expressed in both MON 87701 and Bt cotton (Bollgard and 

WideStrike varieties). 

The established resistance management plan for Bt cotton involves the use of “natural refuge” in 

which non-PIP hosts (such as weeds and other non-cotton cultivated crops) provide sources of 

susceptible insects to dilute any potential resistance genes arising from transgenic cotton. One of 

the non-cotton crops included in calculations of natural refuge is soybean. Should a significant 

portion of the soybean crop be devoted to a Bt variety, the potential amount of natural refuge 

available to TBW and CBW could be reduced. 

In response to BPPD’s concerns, Monsanto (April 8, 2010 Submission) provided a rationale for 

low resistance based on geographic limitations for MON 87701. The company intends to plant 

limited acres (<15,000) in areas with high amounts of natural refuge. While Monsanto did not 

provide revised natural refuge calculations or modeling to support MON 87701, BPPD agrees 

that there should be low resistance risk potential for TBW and CBW based on the low acreage 

projections. The areas intended for plantings of MON 87701 have high proportions (>90%) of 

natural refuge including numerous wild hosts and cultivated crops (see reviews in BPPD 2006, 

2007). As described in Monsanto’s submission, 15,000 acres of Bt soybean in these areas would 

represent only a small fraction of total soybean acres -- to illustrate, if all 15,000 acres were 

planted in one state and assuming the lowest USDA acreage figure (430,000), Bt soybean would 

represent 3.5% of total cultivation. Given the small overall amount of MON 87701 projected for 

planting, it is unlikely that the amount of natural refuge available to TBW and CBW will be 

significantly reduced. Further, even if natural refuge was lowered due MON 87701 there would 

still be abundant sources of other plants hosts (e.g., peanuts, tobacco, vegetable crops, and 

weeds) such that it is unlikely the resistance risk of TBW and CBW to Cry1Ac would 

appreciably increase. 
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BPPD’s assessment of the impact of MON 87701 on Bt cotton natural refuge is contingent upon 

the acreage projections provided by Monsanto (as discussed in the previous section). A full 

commercial registration of MON 87701 without acreage limitations would require a more 

detailed assessment that likely would include revised natural refuge calculations and simulation 

modeling. 

d. Stewardship Activities 

BPPD recommends that other aspects of IRM including resistance monitoring, a remedial action 

plan, and annual sales reporting be implemented for MON 87701. A full resistance monitoring 

plan with pest sampling and detection bioassays may not be warranted for a limited registration; 

rather an approach based on investigations and follow-ups on reports of unexpected pest damage 

may be appropriate. A remedial action plan, in the event of document resistance, could mimic 

those in place for Bt corn or cotton or could rely on a “stop sale” approach if resistance develops. 

An annual sales report detailing total acreage by state is also recommended. Provided MON 

87701 is registered without a structured refuge requirement, a compliance assurance plan and 

grower education program should not be necessary. 

References

Barbara, K.A., 2008. University of Florida, Department of Entomology and Nematology 

informational web page for velvetbean caterpillar (Anticarsia gemmatalis): 

http://entnemdept.ufl.edu/creatures/field/velvetbean.htm. Publication Number: EENY151. 

BPPD, 2006. Review of data and support materials submitted by Monsanto to amend the insect 

resistance management requirements for Bollgard II Bt cotton. A. Reynolds and S. 

Matten memorandum to L. Cole, May 16, 2006. 

BPPD, 2007. Scientific review of a proposal by Monsanto to replace structured refuge with 

natural refuge for Bollgard II Bt cotton (resubmitted amendment request to address the 

concerns of the FIFRA SAP). A. Reynolds and S. Matten memorandum to M. 

Mendelsohn, May 3, 2007. 

Capinera, J.L., 2005. University of Florida, Department of Entomology and Nematology 

informational web page for soybean looper (Pseudoplusia includens): 

http://entnemdept.ufl.edu/creatures/veg/leaf/cabbage_looper.htm. Publication Number: 

EENY-116. 

MacRae T.C., M.E. Baur, D.J. Boethel, B.J. Fitzpatrick, A.G. Gao, J.C. Gamundi, L.A. Harrison, 

V.T. Kabuye, R.M. McPherson, J.A. Miklos, M.S. Paradise, A.S. Toedebusch, A. Viegas, 

2005. Laboratory and field evaluations of transgenic soybean exhibiting high-dose 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

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expression of a synthetic Bacillus thuringiensis cry1A gene for control of Lepidoptera. J 

Econ Entomol. 98 (2): 577-87. 

Pedigo, L.P., 1999. Entomology and Pest Management (Third Edition). Prentice Hall: New 

Jersey. 691 pp. 

Scientific Advisory Panel (SAP), 1998. Transmittal of the final report of the FIFRA Scientific 

Advisory Panel Subpanel on Bacillus thuringiensis (Bt) Plant-Pesticides and Resistance 

Management, Meeting held February 9 and 10, 1998. Report dated April 28, 1998. 

Available at http://www.epa.gov/scipoly/sap/meetings/1998/0298_mtg.htm

III. TERMS AND CONDITIONS OF REGISTRATION 

1) The subject registration will automatically expire on midnight September 30, 2012. 

2) The subject registration is limited to seed increase and to a total of 15,000 acres per year in 

the Atlantic Coast States of Georgia, South Carolina, North Carolina, Virginia, and 

Maryland within the Continental United States with no more than 1,000 acres per county 

per year. 

3) Monsanto must submit IRM monitoring and remedial action plans to EPA for approval by 

January 31, 2011 and reports on such annually by August 31st

. 

4) Monsanto must provide EPA annual reports on the acreage and States where MON 88017 

has been grown by January 31st. 

IV. REGULATORY POSITION FOR Cry1Ac 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 time-limited 

registration of Bacillus thuringiensis Cry1Ac Protein and the Genetic Material (Vector PVGMIR9) Necessary for Its Production in MON 87701 (OECD Unique Identifier: MON 877Ø1-2) 

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 period, 

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 Cry1Ac protein, during 

the limited registration period is not expected to be significant. 
Bacillus thuringiensis Cry1Ac in MON 87701 Soybean 

Biopesticide Registration Action Document 

39

The expiration date of this registration has been set to September 30, 2012. The registration is 

limited to seed increase and to a total of 15,000 acres per year in the Atlantic Coast States of 

Georgia, South Carolina, North Carolina, Virginia, and Maryland within the Continental United 

States with no more than 1,000 acres per county per year.