DRAFT BIOPESTICIDES REGISTRATION ACTION DOCUMENT

Bacillus thuringiensis eCry3.1Ab insecticidal protein and the genetic material necessary for its

production (via elements of vector PSYN12274) in 5307 Corn (SYN-537-1)

PC Code: 016483

U.S. Environmental Protection Agency

Office of Pesticide Programs

Biopesticides and Pollution Prevention Division
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) June 2012 DRAFT

2

Table of Contents

I. OVERVIEW

A. Background .................................................................................................3

B. Use Profile ....................................................................................................4

 

II. SCIENCE ASSESSMENT

A. Product Characterization .................................................................................5

 

 B. FQPA Safety Assessment ..............................................................................21

C. Environmental Assessment .............................................................................42

D. Bt11xMIR604xTC1507x5307 Environmental Assesment .........................................60

E. Bt11xMIR162xMIR604xTC1507x5307 Environmental Assessment ...........................67

F. Insect Resistance Management .......................................................................73

G. Public Interest Finding..................................................................................97

III. REGULATORY POSITION FOR 5307 CORN .................................99
Bacillus thuringiensis eCry3.!Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

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Bacillus thuringiensis eCry3.1Ab insecticidal protein and the genetic material necessary for its

production (via elements of vector PSYN12274) in 5307 Corn (SYN-537-1) Corn Regulatory

Action Team

Product Characterization and Human Health

John Kough, Ph.D.

Annabel Waggoner

Environmental Fate and Effects

Gail Tomimatsu, Ph.D.,

Shannon Borges

Insect Resistance Management

Jeannette Martinez

Alan Reynolds

Benefits Assessment

Alan Reynolds

Registration Support

Mike Mendelsohn

I. OVERVIEW

A. Background

Syngenta Seeds, Inc. – Field Crops - NAFTA (Syngenta Seeds, Inc.) is seeking a FIFRA Sec. 3(c)(5)

Registration for Event 5307 maize [OECD Unique ID. SYN-537-1] for use as a plant-incorporated 

protectant (PIP). Syngenta developed PIP Event 5307 maize (Zea mays) through Agrobacteriummediated transformation (via plasmid vector PSYN12274) to express eCry3.1Ab insecticidal crystal 

protein to provide protection against root feeding damage caused by the following coleopteran pest 

species: Diabrotica virgifera (western corn rootworm, WCR), D. longicornis barberi (northern corn 

rootworm, NCR), and D. virgifera zeae (Mexican corn rootworm, MCR). The eCry3.1Ab is an 

engineered chimera protein, composed of portions of modified Cry3A (mCry3A) protein derived from 

the native Cry3A protein from Bacillus thuringiensis (Bt) subsp. tenebrionis and Cry1Ab protein derived 

from Bt subsp. kurstaki HD-1 (Hofte and Whiteley, 1989; Walters et al. 2010). Chimeric cry genes can 

be engineered with novel insecticidal specificities and activities by exchanging various domains that are 

homologous between different cry genes (Hofte and Whiteley, 1989).

Event 5307 corn also contains the pmi gene, which was introduced along with the eCry3.1Ab protein 

with the same PSYN12274 transformation vector. The gene represents the manA gene from Escherichia 

coli and encodes the enzyme phosphomannose isomerase (PMI), which was employed as a selectable 

marker during the process of regenerating plant material following transformation. The PMI protein is a 
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

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common enzyme involved in carbohydrate metabolism to allow for selection of transformants in cell

culture, by only allowing transformed corn cells to utilize mannose as a sole carbon source, while corn 

cells lacking the pmi gene fail to grow (Negrotto, et al. 2000).

In conjunction with the Sec.3 registration of event 5307 maize, Syngenta Seeds, Inc. submitted a petition 

for an exemption from the requirement of a tolerance pursuant to section 408(d)(1) of the Federal Food, 

Drug, and Cosmetic Act with respect to the PIP Bacillus thuringiensis eCry3.1Ab insect control protein 

and the genetic material necessary for its production in food and feed commodities of field corn, sweet 

corn, and popcorn. The genetic material necessary for the production of the PIP active ingredients are 

the nucleic acids (DNA, RNA) which comprise genetic material encoding these proteins and their 

regulatory regions. The genetic material (nucleic acids) that are part of any PIP are exempt from the 

requirement of a tolerance [40 CFR § 174.507]. In addition, an exemption from the requirement of a 

tolerance has been established for PMI enzyme in all food commodities when used as a plantincorporated protectant inert ingredient [40 CFR § 174.527], thereby, the PMI enzyme expressed in 

event 5307 corn is covered in this exemption. 

The Agency previously granted a Sec. 5 Experimental Use Permit (EUP) for event 5307 corn [EPA Reg. 

No. 67979-EUP-8] to conduct experimental field tests, and in conjunction with the EUP, the Agency 

established a temporary exemption from the requirement of a tolerance for Bt eCry3.1Ab protein 

residues on food and feed corn commodities [40 CFR § 174.532], which is set to expire on December 

31, 2013. 

B. Use Profile

Pesticide Name: Bacillus thuringiensis eCry3.1Ab insecticidal protein and the genetic material 

 necessary for its production (via elements of vector PSYN12274) in 5307 Corn 

 (SYN-537-1)

Trade and Other 5307 Corn; AgrisureTM VipteraTM 3222; AgrisureTM NGRW 3122

Names: 

OPP Chemical Code: 016483

Basic Manufacturers: Syngenta Seeds, Inc. – Field Crops – NAFTA

 P.O. Box 12257

 Research Triangle Park, NC 27709-2257

Type of Pesticide: Plant-Incorporated Protectant

Use: Field Corn

Target Pests: western corn rootworm (Diabrotica virgifera virgifera), northern corn 
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

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rootworm (Diabrotica barberi), and Mexican corn rootworm (Diabrotica 

virgifera zeae)

Products Expressing 5307 Corn – EPA File Symbol 67979-EE

This Pesticide: Bt11xMIR162xMIR604XTC1507x5307 Corn – EPA File Symbol 67979-EG

Bt11xMIR604XTC1507x5307 Corn – EPA File Symbol 67979-EU

II. SCIENCE ASSESSMENT

The classifications that are found for each data submission are assigned by Environmental Protection 

Agency (EPA) science reviewers and are an indication of the usefulness of the information contained in 

the documents for risk assessment. A rating of “ACCEPTABLE” indicates the study is scientifically

sound and is useful for risk assessment. A “SUPPLEMENTAL” rating indicates the data provide some 

information that can be useful for risk assessment. The studies may have certain aspects determined not

to be scientifically acceptable (“SUPPLEMENTAL: UPGRADABLE”). If a study is rated as 

“SUPPLEMENTAL: UPGRADABLE,” EPA always provides an indication of what is lacking or what 

can be provided to change the rating to “ACCEPTABLE.” If there is simply a “SUPPLEMENTAL” 

rating, the reviewer will often state that the study is not required by the current 40 Code of Federal

Regulations (CFR) Part 158. Both “ACCEPTABLE” and “SUPPLEMENTAL” studies may be used in 

the risk assessment process as appropriate. An “UNACCEPTABLE” rating indicates that the study is 

not useful for risk assessment and cannot be upgraded.

A. Product Characterization

Syngenta Seeds, Incorporated has developed Event 5307 corn [OECD Unique ID. SYN-537-1] to 

express eCry3.1Ab protein for use as a plant-incorporated protectant (PIP) against root feeding damage 

caused by the following coleopteran pest species: Diabrotica virgifera (western corn rootworm, WCR), 

D. longicornis barberi (northern corn rootworm, NCR), and D. virgifera zeae (Mexican corn rootworm, 

MCR). This proposed PIP is a recombinant fusion protein, composed of portions of modified Cry3A 

(mCry3A) (derived from the native Cry3A protein from Bacillus thuringiensis (Bt) subsp. tenebrionis)

and Cry1Ab protein (derived from Bt subsp. kurstaki HD-1). Event 5307 maize also contains the manA 

gene from Escherichia coli, which encodes phosphomannose isomerase (PMI) as a selectable marker in 

regenerating plant material following transformation (Negrotto et al. 2000). The PMI protein is a 

common enzyme involved in carbohydrate metabolism to allow for selection of transformants in cell 

culture, by only allowing transformed corn cells to utilize mannose as a sole carbon source, while corn 

cells lacking the pmi gene fail to grow (Negrotto, et al. 2000). 
Bacillus thuringiensis eCry3.1Ab Corn

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1. Transformation System

Based on amino acid sequence similarity and crystal structures, known Cry proteins have a similar threedimensional structure comprised of three domains, Domain I, II and III (Nakamura et al., 1990; Li et al., 

1991; Ge et al., 1991; Honee et al., 1991). The toxin portions of Cry proteins are characterized by 

having five conserved blocks (CB) across their amino acid sequence. These are numbered CB1 to CB5 

from the N- terminus (5’ end) to the C-terminus (3’end) (Hofte and Whiteley, 1989). The sequences 

preceding and following these conserved blocks are highly variable and are designated as variable 

regions V1 to V6. Because Cry proteins share structural similarities, chimeric cry genes can be 

engineered via the exchange of domains that are homologous between different cry genes.

eCry3.1Ab was engineered as a chimera protein by exchanging the variable regions (V1 to V6) of 

mCry3A protein between Cry1Ab protein for enhanced toxicity against western corn rootworm 

(Diabrotica virgifera) and other Diabrotica spp. The ecry3.1Ab gene (Entrez Accession Number 

GU327680 [NCBI, 2011]) (Walters et al. 2010) consists of a fusion between the N-terminus (Domain I, 

Domain II, and a portion of Domain III) of the mcry3A geneaand the C-terminus (a portion of Domain

III and Variable Region 6) of the cry1Ab geneb(Hofte and Whiteley, 1989) (see Figure 1). 

Figure 1. Schematic 

Illustrating the Origin of 

the Amino Acid Residues 

Present in eCry3.1Ab

 

Figure 1 was reproduced from Figure 1(A), page 11 in MRID No. 48442501

a

Description of the mcry3A gene: A maize-optimized cry3A gene was synthesized to accommodate the preferred codon 

usage for maize (Murray et al. 1989). The synthetic sequence was based on the native Cry3A protein sequence from 

Bacillus thuringiensis subsp. tenebrionis (Sekar et al. 1987). The maize-optimized gene was then modified to incorporate 

a consensus cathepsin-G protease recognition site within the expressed protein. The mcry3A gene encodes for mCry3A

protein, which is expressed in Event MIR604 maize. Event MIR604 is currently registered for use as a PIP (EPA Reg. 

No.67979-5) and its risk and benefits assessment is found in the Agency’s BRAD for mCry3A corn (US EPA, 2006). An 

exemption from the requirement of a tolerance was established for mCry3A in maize [40 CFR § 174.505].

b

Description of the cry1Ab gene: The cry1Ab gene was originally cloned from Bacillus thuringiensis var. kurstaki strain 

HD-1 (Geiser et al. 1986). Its amino acid sequence has been codon-optimized (Koziel et al. 1997) to accommodate the 

preferred codon-usage for maize (Murray et al. 1989). The cry1Ab gene encodes for Cry1Ab protein, which is expressed in 

Event Bt11 maize. Event Bt11 is currently registered for use as a PIP (EPA Reg. No.67979-1) and its risk and benefits 

assessment is found in the BRAD for the Agency’s Reassessment for Bt crops in 2001 (US EPA, 2001). An exemption 

from the requirement of a tolerance was established for Cry1Ab in all crops [40 CFR § 174.511].
Bacillus thuringiensis eCry3.1Ab Corn

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Transformation of Zea mays to produce event 5307 maize was accomplished using immature embryos of 

a proprietary maize line via Agrobacterium tumefaciens-mediated transformation (Negrotto et al. 2000). 

The region between the left and right borders of the transformation plasmid included ecry3.1Ab and pmi

gene expression cassettes. This T-DNA was transferred into the maize genome during transformation 

with plasmid vector pSYN12274. The ecry3.1Ab expression cassette consisted of the ecry3.1Ab coding 

region regulated by a cestrum yellow leaf curling virus promoter (CMP) and a nopaline synthase (NOS) 

polyadenylation termination sequence. The pmi expression cassette consisted of the pmi coding region 

regulated by a Z. mays polyubiquitin promoter (ZmUbiInt) and the NOS polyadenylation termination 

sequence. The ecry3.1Ab expression cassette consisted of the ecry3.lAb coding region regulated by a 

cestrum yellow leaf curling virus promoter (CMP) and a nopaline synthase (NOS) polyadenylation 

sequence. The pmi expression cassette consisted of the pmi coding region regulated by a Z. mays

polyubiquitin promoter (ZmUbilnt) and the NOS polyadenylation sequence. 

2. Characterization of the DNA Inserted in the Plant and Inheritance and Stability

Characterization of genomic DNA isolated from event 5307 corn leaf tissue using restriction enzyme 

digests and Southern blot analyses indicated that the DNA was inserted in the corn genome at a single 

locus, and the insert contains one copy of the ecry3.1Ab and pmi expression cassettes. There were no 

other detectable genetic elements other than those associated with the respective cassettes. No vector 

backbone sequences from the transformation plasmid pSYN12274 were found in event 5307 maize. 

Segregation patterns over several generations of event 5307 maize confirmed the expected inheritance 

ratio for both the ecry3.1Ab and pmi genes consistent with Mendelian principles. Sequencing data 

demonstrated that the insert is intact with the exception of one single nucleotide change when compared 

to the transformation plasmid pSYN12274. This nucleotide change was located 48 base pairs upstream 

of the CMP promoter, in a non-coding region of the T-DNA, and has no effect on the functionality of 

the T-DNA insert. Therefore, the overall integrity of the insert and the functional elements within the 

insert are contiguous, as present in pSYN12274. 

3. Protein Characterization and Equivalence

Production of microbial-derived eCry3.1Ab protein was chosen in order to obtain sufficient material for 

testing, therefore, its equivalence to the plant-derived eCry3.1Ab protein expressed in event 5307 corn 

was determined. The ECRY3.1AB-0208 test substance, containing purified microbially-produced 

eCry3.1Ab protein, was prepared from an Escherichia coli (E. coli) overexpression system to contain

the same ecry3.1Ab gene that was introduced into Event 5307 corn plants. The eCry3.1Ab protein 

expressed in test substance ECRY3.1AB-0208 is identical to that expressed in transgenic maize Event 

5307 except that it contains one additional methionine and six additional histidine residues at the Nterminus, which were added to aid purification purposes in microbial production. 

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses showed the

eCry3.1Ab proteins from both sources had the same approximate molecular weight (ca. 74.8 kDa) based 

on the predicted molecular weight of ca. 74.8 kDa. Densitometric analyses showed the purity of 

eCry3.1Ab in the test material to be ca. 89.6% by weight. Western blot analysis showed that both plant-
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

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produced and microbially produced eCry3.1Ab cross-reacted with the same antibodies, confirming the 

identity and integrity of the eCry3.1Ab protein. The mass of the eCry3 .1Ab protein contained in 

ECRY3.1AB-0208 measured by mass spectrometry, was 74832.80 Da (74.8 kDa) confirming the 

theoretical mass based on amino acid sequence analysis. In addition, the expected N-terminal peptide 

was confirmed for eCry3.1Ab from both sources. Both proteins produced comparable insecticidal 

activity in insect feeding bioassays with first instar Colorado potato beetle (Leptinotarsa decemlineata) 

larvae, based on overlapping ranges of LC50 values. There was also no evidence of post-translational 

glycosylation of eCry3.1Ab protein from either source. Collectively, the results support the conclusion 

that eCry3.1Ab produced in recombinant E. coli (test substance ECRY3.1AB-0208) is biochemically 

and functionally equivalent to the eCry3.1Ab produced in Event 5307 maize, and is a suitable surrogate 

for eCry3.1Ab produced in Event 5307 maize.

4. Protein Expression

Expression level data were submitted to quantify eCry3.1Ab protein concentrations in different plant 

tissues, sampled at key developmental growth stages in event 5307 corn using ELISA analyses. A

summary of the results are provided in Table 1 below.

Table 1. Protein Expression levels of eCry3.1Ab protein in Event 5307 Corn Tissues

Tissue Type

eCry3.1Ab Protein 

(μg/g dry weight ± SD)a

Leaves <LOQb- 142.96 ± 53.44

Roots 9.13 ±6.38 - 42.72 ±4.67

Whole plants 8.27 ±2.90 - 111.08 ±38.36

Kernels 4.45 ±0.82 - 6.19 ±1.87

Pollenc0.10b

a Range reflects means at different growth stages across field sites

b

LOQ = 0.10 μg/g dry weight

c

Pollen samples were pooled for each location, however, due to insufficient material or samples 

detected at the LOQ, the mean protein concentration for pollen across the field locations was not 

calculated. However, most of the samples were detected at the LOQ (0.10 μg/g dry weight), which is 

sufficient for reporting the eCry3.1Ab protein in pollen.

Table 1 was reproduced from Tables 3-7 in MRID No. 48442509.
Bacillus thuringiensis eCry3.1Ab Corn

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Table 2. Product Characterization Data Submitted for Event 5307 maize

STUDY TITLE STUDY SUMMARY MRID NO. 

Molecular 

characterization 

of Event 5307 

maize a

Transformation of Zea mays (maize) to produce event 5307 was 

described and the presence of the intended transgenic DNA insert, 

containing the ecry3.1Ab and pmi genes into the plant genome, was 

confirmed via PCR analyses. Segregation patterns over several 

generations of 5307 maize confirmed the expected Mendelian 

inheritance ratio for both the ecry3.1Ab and pmi genes were

inherited in the predicted manner consistent with Mendelian 

principles. A single intact insert in 5307 maize and single copies of 

the ecry3.1Ab and pmi genes were identified. Event 5307 maize is 

free of any backbone sequences from the transformation plasmid 

pSYN12274. Sequence analysis of the 5307 maize insert showed 

one single nucleotide change when compared to the transformation 

plasmid pSYN12274. This nucleotide change was located 48 base 

pairs upstream of the CMP promoter, in a noncoding region of the 

T-DNA, and has no effect on the genes encoded by event 5307 

maize.

CLASSIFICATION: ACCEPTABLE; This report is 

superseded by MRID No. 484425-01.

477347-02

Comparison of 

eCry3.1Ab 

protein produced 

in Event 5307-

derived maize 

plants and 

eCry3.1Ab 

protein produced 

in recombinant 

Escherichia coli a

The plant-derived eCry3.1Ab protein (extracted from 5307 maize 

tissue) and the bacterial-derived eCry3.1Ab protein (produced in a 

recombinant E. coli over-expression system) revealed similar 

molecular weights (73.7 and 74.8 kDa, respectively). Both 

eCry3.1Ab plant- and bacterial- derived proteins were 

immunologically cross-reactive with antibodies capable of 

detecting the eCry3.lAb protein. Both eCry3.1Ab plant- and 

bacterial- derived proteins showed comparable insecticidal activity 

against Colorado potato beetle larvae and no evidence of posttranslational glycosylation. Peptide mass mapping analysis showed 

that the identified peptides corresponded to regions throughout the 

sequence of eCry3.1Ab from both eCry3.1Ab plant- and bacterialderived proteins, except that the microbial-derived version contains 

one additional methionine and six histidine residues at the Nterminus.

CLASSIFICATION: ACCEPTABLE

477347-03

Characterization 

of Test Substance 

ECRY3.1ABECRY3.1AB-0208 test substance (containing bacterial-derived 

eCry3.1Ab protein) was tested at various buffers and was readily

soluble at 10 mg/mL; ECRY3.1AB-0208 was determined to 

477347-04
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STUDY TITLE STUDY SUMMARY MRID NO. 

0208 and 

Certificate of 

Analysis a

contain 92.4% protein (estimated by absorbance at 280 nm via 

spectrophotometry) and densitometry analysis after SDS-PAGE 

determined the test substance to contain 97.0% eCry3.1Ab protein. 

Thereby, the overall purity of test substance ECRY3.1AB-0208 

was calculated to be 89.6% eCry3.1Ab (w/w). Western blot 

analysis revealed an immunoreactive band representing a molecular 

weight of ~74.8 kDa; The total mass of the protein was measured 

by a quadrupole time-of-flight mass spectrometer and confirmed 

the molecular weight of ECRY3A.1AB-028 as 74832.80 Da (74.8 

kDa). 

CLASSIFICATION: ACCEPTABLE

Bioactivity of 

Test Substance 

ECRY3.1AB0208 a

The bioactivity of the bacterial-produced eCry3.1Ab protein in test 

material ECRY3.1AB-0208 was assessed in three independent 

insect feeding bioassays with first instar Colorado potato beetle 

(Leptinotarsa decemlineata) larvae. The larvae were provided diets 

containing various dose concentrations of eCry3.1Ab μg/mL of diet 

for six days. The larvae demonstrated a dose response to the test 

material, with 100% mortality at the highest dose. Mortality in the 

buffer control and the assay control group was 14% and 17%, 

respectively. The LC50 for the test was 2.3 μg/mL (95% c.i., 1.4 -

3.3 μg/mL).

CLASSIFICATION: ACCEPTABLE

477347-05

Evaluation of 

Transgenic 

Protein Levels in 

Multiple 

Generations of 

Plants Derived 

from 

Transformation 

Event 5307 Maize

a

The concentration of eCry3.1Ab and PMI protein expression were 

measured via ELISA in plant tissues collected from five Event 

5307 maize plants, representing 4 generations (at plant growth 

stages VT-R1) in a greenhouse study. The mean eCry3.1Ab 

protein concentration in leaves, roots, and pollen of the four 

generations ranged from 83.40 to 93.67 μg/g dry weight, 23.88 to 

35.39 μg/g dry weight, and <LOD (0.08 μg) to 0.15 μg/g dry

weight, respectively. The mean PMI protein concentration in 

leaves, roots, and pollen of the four generations ranged from 1.77 to 

1.95 μg/g dry weight, 1.05 to 1.19 μg/g dry weight, and 18.96 to 

25.58 μg/g dry weigh, respectively. Therefore, the concentrations 

of eCry3.1Ab and PMI protein were comparable across four 

generations of event 5307 maize at VT-R1 growth stages were 

comparable, indicating consistency of protein expression across 

multiple generations.

CLASSIFICATION: SUPPLEMENTAL- Data deficiencies 

477347-07
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STUDY TITLE STUDY SUMMARY MRID NO. 

were addressed in MRID No. 484425-09

Molecular 

characterization 

of Event 5307 

maize in support 

of the EUP 

application; 

Amended Report 

No. 1: Replaces 

Original Report 

Dated February

17, 2009; MRID 

No. 47734702

Transformation of Zea mays (maize) with plasmid pSYN12274 to 

produce event 5307 was described. The presence of the intended 

transgenic DNA insert, (containing the ecry3.1Ab and pmi genes) 

in the plant genome was confirmed via PCR analyses. Segregation 

patterns over several generations of 5307 maize confirmed the 

expected inheritance ratio for both the ecry3.1Ab and pmi genes 

consistent with Mendelian principles. Southern blot analyses 

demonstrated that the T-DNA insert in event 5307 maize carries a 

single copy of the ecry3.1Ab and pmi genes. In addition, event 5307 

maize is free of any backbone sequences from the transformation 

plasmid pSYN12274. Sequence analysis of the 5307 maize insert 

showed one single nucleotide change when compared to the 

transformation plasmid pSYN12274. This nucleotide change was 

located 48 base pairs upstream of the CMP promoter, in a noncoding region of the T-DNA, and has no effect on the genes 

encoded by event 5307 maize.

CLASSIFICATION: ACCEPTABLE

484425-01

[This 

report 

supersedes 

MRID No. 

47734702]

Characterization 

of 

Phosphomannose 

Isomerase Test 

Substance PMI0105 and 

Certificate of 

Analysis

The purpose of this study was to characterize microbially produced 

test substance PMI-0105 for its solubility, purity, protein identity, 

integrity and enzymatic activity. The PMI-0105 test substance was 

produced by the manA gene in the recombinant Escherichia coli

over-expression system and encodes the same amino acid sequence 

as the plant-expressed PMI protein present in maize event 5307 as a 

PIP inert ingredient. The results of the solubility of PMI-0105 in 

various buffers were appropriate. Spectrophotometric and 

densitometric analysis showed the overall purity/concentration of 

the PMI protein in PMI-0105 test substance as 89.5% PMI by

484425-02
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STUDY TITLE STUDY SUMMARY MRID NO. 

weight. SDS-PAGE analyses showed a molecular weight of 42.8 

kDa. Western blot analysis revealed a dominant immunoreactive 

band corresponding to the expected PMI molecular weight of 42.8 

kDa. The predicted PMI molecular weight was also confirmed as 

42,850 Da by quadrupole time-of-flight mass spectrometry. Nterminal sequencing analyses confirmed that the first 15 amino 

acids of PMI in test substance PMI-0105 corresponded to the 

predicted N-terminal sequence of PMI protein encoded by the 

manA gene. Results of the enzymatic activity assay showed the 

PMI protein in test substance PMI-0105 was active, with an 

average specific activity of 503.77 U/mg PMI. In conclusion, the 

results confirmed the solubility, purity, protein identity, integrity 

and enzymatic activity of PMI protein in the microbial-expressed 

PMI-0105 test substance, and its suitability as biochemically and 

functionally equivalent to the plant-expressed PMI protein in event 

5307.

CLASSIFICATION: ACCEPTABLE

Comparison of 

phosphomannose 

isomerase 

produced in event 

5307-derived 

maize plants and 

phosphomannose 

isomerase 

produced in 

recombinant 

Escherichia coli.

The purpose of this study was to demonstrate the biochemical and 

functional equivalence between the plant-expressed 

phosphomannose isomerase (PMI) protein in event 5307 maize and 

PMI-0105 test substance (the microbially-produced PMI protein 

prepared from a recombinant E. coli over-expression system).

PMI from event 5307 plant leaf material (pedigree NP2171 x 

BC5F3) and from PMI-0105 test substance showed the same 

mobility in the Western blot analysis and confirmed an apparent 

molecular weight consistent with the predicted molecular weight of 

approximately 42.8 kDa. Western blot analyses showed the PMI

protein from both sources were immunologically cross-reactive

with anti-PMI antibodies. In an enzymatic activity assay, both the 

plant-produced PMI and the microbially-produced test substance 

were active. The specific activity for plant produced PMI was 

455.67 Units/mg PMI and the specific activity for the microbiallyproduced PMI was 526.26 Units /mg PMI. These results support 

the conclusion that PMI produced PMI-0105 test substance is 

biochemically and functionally equivalent to the PMI produced in 

5307 maize. Therefore, the PMI produced in recombinant E. coli 

(test substance PMI-0105) is suitable surrogate for PMI produced 

in event 5307 maize.

CLASSIFICATION: ACCEPTABLE

484425-03

Event 5307 

maize: Copy

Southern blot analyses of 5307 maize demonstrated that (1) 5307 

maize contains a single copy of ecry3.1Ab, pmi, the CMP promoter 

484425-04
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STUDY TITLE STUDY SUMMARY MRID NO. 

number 

functional 

element southern 

blot analysis

sequence, the ZmUbiInt promoter sequence and two copies of the 

NOS terminator sequence, as expected for a single insertion site; 

(2) there are no extraneous DNA fragments of the functional 

elements inserted elsewhere in the maize genome; and (3) 5307 

maize is free of backbone sequence from the transformation 

plasmid pSYN12274.

CLASSIFICATION: ACCEPTABLE

Event 5307 

maize: Genetic 

stability analysis

Southern blot analyses of 5307 maize demonstrated that (1) the 

5307 maize carries a single, complete copy of the T-DNA insert 

with no extraneous DNA fragments of plasmid pSYN12274 TDNA inserted elsewhere in the maize genome, (2) the transgenic 

locus is stable across all the 5307 maize generations analyzed, and 

(3) all generational samples of 5307 maize examined were negative 

for the vector backbone sequence from the transformation plasmid 

pSYN12274.

CLASSIFICATION: ACCEPTABLE

484425-05

Event 5307 

maize: Genome to

insert junction 

analysis for 

translated open 

reading frames 

with a minimum 

size of 30 amino 

acids: Assessment 

of amino acid 

sequence 

similarity to 

known or putative 

toxins.

Bioinformatic analysis of the DNA sequences spanning the 

junctions between the maize genomic sequence and the 5307 maize 

insert were used to identify any putative open reading frames 

(ORFs) with a minimum putative translation size of 30 amino 

acids. One putative ORF spanning the junction between the maize 

genomic sequence and the 3' region of the 5307 maize insert was 

determined. The Basic Local Alignment Search Tool for Proteins 

(BLASTP) program was used to search the National Center for 

Biotechnology Information (NCBI) Entrez® Protein Database 

(2010) to determine whether the translations of this putative ORF 

showed significant similarity to known and putative toxins. Results 

showed the amino acid translation of the putative ORF shares no 

biologically relevant amino acid sequence similarity to any known 

or putative protein toxins.

CLASSIFICATION: ACCEPTABLE

484425-06

Event 5307 

maize: Genome to 

insert junction 

analysis for 

translated open 

reading frames 

with a minimum 

Bioinformatic analysis of the DNA sequences spanning the 

junctions between the maize genomic sequence and the 5307 maize 

insert were used to identify any putative open reading frames 

(ORFs) with a minimum putative translation size of 30 amino 

acids. One putative ORF spanning the junction between the maize 

genomic sequence and the 3' region of the 5307 maize insert was 

identified. The translation of the putative ORF was systematically 

484425-07
Bacillus thuringiensis eCry3.1Ab Corn

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14

STUDY TITLE STUDY SUMMARY MRID NO. 

size of 30 amino 

acids: Assessment 

of amino acid 

sequence to 

known or putative 

allergens. 

compared against the protein sequences in the Food Allergy

Research and Resource Program AllergenOnline database (2010; 

version 10.0) using a FASTA search algorithm (2010, version 

3.45). No amino acid sequence similarities were observed after 

comparing 80 sequential amino acid peptides of this translated 

putative ORF with any known allergen in the AllergenOnline 

database spanning either junction of the maize genomic DNA and 

event 5307 maize insert. Also, no matches of eight or more 

contiguous amino acids were observed between the translated 

putative ORF and any entry in the AllergenOnline database. 

Therefore, the amino acid translations of the putative ORF 

spanning the junction between the maize genomic sequence and the 

3' region of the 5307 maize insert have no biologically relevant 

amino acid sequence similarity to known or putative protein 

allergens.

CLASSIFICATION: ACCEPTABLE

Validation of the 

ELISA method 

for quantitative 

detection of 

eCry3.1Ab 

protein in maize 

grain, broiler 

diets, and rat 

diets. 

A previously validated enzyme-linked immunosorbent assay

(ELISA) method (Beacon Analytical Systems, Portland, ME) was 

evaluated for its performance as a suitable analytical method for the 

quantification of eCry3.1Ab protein in maize grain, broiler chicken 

diets, and rat diets. The parameters for validation included 

accuracy, quantitative range, intermediate and repeatability

precision, sensitivity (minimum dilution factor, limit of detection 

(LOD), limit of quantitation (LOQ)), short-term and freeze/thaw 

stability, specificity of the assay based on cross-reactivity with nontarget transgenic proteins. In addition, the ECRY3.1AB-020 test 

substance (a microbially-derived eCry3.1Ab recombinant protein 

prepared from an over-expression E. coli system) used in the 

ELISA method as a positive assay control sample (PACS) for 

eCry3.1Ab was analyzed to establish a new specification range for 

the eCry3.1Ab. 

Results showed the performance parameters evaluated in this study

for the ELISA method were within the acceptance criteria defined 

for each parameter; therefore, the validity of the ELISA method 

used for determining eCry3.1Ab protein concentrations in event 

5307 maize grain, broiler diets, and rat diets is confirmed. In 

addition, the dilution factors, LODs, LOQ, and extraction 

efficiencies can be used for subsequent studies using ELISA 

analyses for quantifying eCry3.1Ab in maize grain, broiler chicken 

484425-08
Bacillus thuringiensis eCry3.1Ab Corn

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15

STUDY TITLE STUDY SUMMARY MRID NO. 

and rat diets.

In a separate report, the accuracy of the values reported in this 

study were verified based on the review of individual data used in 

calculating the accuracy, precision, percent recoveries, coefficient 

of variations, dilution factors, extraction efficiencies, and assay

specificity (MRID No. 48802604).

CLASSIFICATION: ACCEPTABLE

Quantification of 

eCry3.1Ab and 

phosphomannose 

isomerase in 

maize tissues 

derived from 

transformation 

event 5307. 

Field studies were conducted to quantify eCry3.1Ab and PMI

protein concentrations in event 5307 maize plant tissue matrices 

(leaves, roots, whole plants, pollen, and kernels) at key

developmental growth stages (whorl, anthesis, maturity, and 

senescence) via enzyme-linked immunosorbent assay (ELISA)

analyses across four different locations in the 2008 growing season. 

The mean concentrations of eCry3.1Ab across all locations and 

plant stages on a dry-weight basis ranged from <LOQ to 142.96 ± 

53.44 g/g in leaves, from 9.13 ±6.38 to 42.72 ±4.67 g/g in roots, 

and from 8.27 ±2.90 to 111.08 ±38.36 g/g in whole plants. The 

mean concentrations of eCry3.1Ab across all locations on a dryweight basis ranged from 4.45 ±0.82 to 6.19 ±1.87 g/g in kernels 

at maturity and senescence stages. The mean concentration of 

eCry3.1Ab in pooled pollen samples (collected only at the anthesis 

stage) was not calculated across locations, because some of the 

eCry3.1Ab samples were not quantifiable or unavailable. However, 

most of the samples were detectable at the LOQ (0.10 g/g); 

therefore, the 0.10 g/g can be designated as the eCry3.1Ab protein 

concentration in pollen for the purposes of this study.

The mean concentrations of PMI across all locations and plant 

developmental stages on a dry-weight basis ranged from <LOQ to 

4.83 ±1.47g/g in leaves, <LOQ to 2.11 ±0.60 g/g in roots and 

0.97 ±0.37 g/g to 4.38 ±2.43 g/g in whole plants. The mean 

concentrations of PMI across all locations on a dry-weight basis 

ranged from 1.11 ±0.05 g/g to 2.08 ±0.49 g/g in kernels at 

maturity and senescence stages, while the mean concentration of 

PMI across locations in pooled pollen samples was 6.51 ±0.72 

g/g.

484425-09

[This 

report 

supersedes 

MRID No. 

47734707]
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

16

STUDY TITLE STUDY SUMMARY MRID NO. 

The results show the protein concentrations of eCry3.1Ab and PMI

protein extracted from various plant tissue matrices collected from 

four field sites of event 5307 maize were either detectable or 

quantifiable in all tissues analyzed with the exception of some leaf 

tissue samples for PMI protein at the senescence stage. Moreover, 

the ranges of concentrations for eCry3.1Ab and PMI proteins 

expressed in various plant tissue matrices of event 5307 maize were 

similar for each developmental stage across multiple field sites. 

These results indicate the stability of the transgenic insertion of 

ecry3.1Ab and pmi gene cassette by encoding consistent levels of 

eCry3.1Ab and PMI protein expression in event 5307 maize.

CLASSIFICATION: ACCEPTABLE

Validation of the 

ELISA method 

for quantitative 

detection of 

eCry3.1Ab. 

This study was conducted to validate the performance of an 

enzyme-linked immunosorbent assay (ELISA) method as a suitable 

analytical method for the quantitative analysis of eCry3.1Ab 

protein. The ELISA is a double-antibody sandwich assay specific to 

eCry3.1Ab developed by Beacon Analytical Systems, Inc. (BAS). 

Analysis of eCry3.1 Ab reference protein standard curves was 

performed to evaluate the precision of the assay, evaluate the 

goodness of fit of a four-parameter logistic model, determine the 

quantitative range of the assay, evaluate lot-to-lot variability of the 

ELISA test kits, and cross-reactivity with non-target transgenic 

proteins (AMY797E, Cry1Ab, PAT, mCry3A, PMI as expressed in 

event MIR604 corn, Vip3Aa20, and mEPSPS). 

Results show the performance parameters evaluated in the BAS 

ELISA method were within the acceptance criteria defined for each 

performance parameter. Therefore, the validity of the ELISA 

method used for determining eCry3.1Ab protein concentrations in 

event 5307 maize is confirmed and the BAS eCry3.1Ab ELISA kit 

is suitable as a quantitative analytical detection method for 

eCry3.1Ab. In addition, the dilution factors, LODs, LOQ, and 

extraction efficiencies can be used for subsequent studies using 

ELISA analyses for quantifying eCry3.1Ab in maize.

In a separate report, the accuracy of the values reported in this study

were verified based on the review of individual data used in 

calculating the intra- and inter-assay precision, goodness-of-fit, 

assay background, quantitative range, lot-to-lot variability, and 

cross reactivity with non-target proteins (MRID No. 48802604).

CLASSIFICATION: ACCEPTABLE

484425-14
Bacillus thuringiensis eCry3.1Ab Corn

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17

STUDY TITLE STUDY SUMMARY MRID NO. 

Validation of a 

quantitative 

ELISA method 

for indication of 

eCry3.1Ab 

protein stability

and qualification 

of the eCry3.1Ab 

positive control 

for use in the 

eCry3.1 Ab 

quantitative 

ELISA method. 

This study was conducted to test the suitability of a previously 

validated enzyme-linked immunosorbent assay (ELISA) kit 

(Beacon Analytical Systems, Inc. (BAS)) for use as a protein 

stability indicating method. The BAS ELISA is a double-antibody

sandwich assay specific for the quantitative analysis of eCry3.lAb 

protein. The suitability was evaluated by the ability of the ELISA 

method to detect a decrease in the amount of eCry3.lAb protein due 

to destabilization, which may occur during long term storage. 

Using accelerated protein destabilization through forced 

denaturation, ELISA analyses of the eCry3.lAb protein treated with 

sodium dodecyl sulfate (SDS) demonstrated decreasing eCry3.lAb 

recovery with increasing SDS concentration. Thus, the BAS 

eCry3.1Ab ELISA method is suitable for indicating the protein 

stability of eCry3.1Ab protein. 

Additionally, the qualification of ECRY3.1AB-0208 test substance 

(a microbially-derived eCry3.1Ab protein prepared from an 

Escherichia coli overexpression system) for use as the eCry3.1Ab 

positive control sample material was conducted. Results showed 

the concentration of the eCry3.lAb positive control sample material 

is 116.73 ug/ml, within an acceptable range of 72.82 ng/ml to 

160.64 g/ml. Therefore, the eCry3.1Ab positive control sample 

material is qualified for use in the BAS eCry3.1Ab ELISA method.

CLASSIFICATION: ACCEPTABLE

484425-15

Effect of 

temperature on 

the bioactivity of 

eCry3.1Ab 

protein as 

contained in test 

substance. 

The study showed that eCry3.1Ab (ECRY3.1AB-0208) prepared 

from an E. coli over-expression system pre-incubated at 25oC, 

37oC, and 65oC for 30 minutes and tested for activity in a bioassay

with L. decemlineata (Colorado potato beetle) larvae retained 

activity relative to the protein pre-incubated at 4oC. No activity was 

observed when eCry3.1Ab was pre-incubated at 95oC for 30 

minutes. Thus, the bacterial-expressed eCry3.1 Ab protein becomes 

denatured and loses its biological activity after heat treatment for 

30 minutes at 95°C.

CLASSIFICATION: ACCEPTABLE

484425-16

Validation of an 

ELISA method 

for quantification 

of eCry3.1Ab 

protein in maize 

An enzyme-linked immunoabsorbent assay (ELISA) method was 

re-validated for its use as a suitable analytical method for the 

quantitation of eCry3.1Ab protein in maize kernels because minor 

modifications to the previously validated ELISA method have 

resulted in increased sensitivity of eCry3.1Ab protein quantitation 

484425-17
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

18

STUDY TITLE STUDY SUMMARY MRID NO. 

kernel. in kernel matrix. The method performance characteristics evaluated 

included sensitivity (minimum dilution factor, limit of detection), 

accuracy, quantitative range (upper and lower limits), linearity, 

method specificity (cross-reactivity), and extraction efficiency. 

Results show the performance parameters evaluated for the BAS 

ELISA method were within the acceptance criteria defined for each 

performance parameter. Therefore, the validity of the ELISA 

method used for determining eCry3.1Ab protein concentrations in 

event 5307 maize kernel is confirmed and the BAS eCry3.1Ab 

ELISA kit is suitable as a quantitative analytical detection method 

for eCry3.1Ab in maize kernels. In addition, the dilution factors, 

LODs, LOQ, and extraction efficiencies can be used for subsequent 

studies using ELISA analyses for quantifying eCry3.1Ab in maize

kernels.

In a separate report, the accuracy of the values reported in this 

study were verified based on the review of individual data used in 

calculating the accuracy, precision, percent recoveries, coefficient 

of variations, dilution factors, extraction efficiencies, and assay

specificity (MRID No. 48802604).

CLASSIFICATION: ACCEPTABLE

Independent 

Laboratory

Validation/ Kit 

for eCry3.1Ab 

Testing in Leaf, 

Seed, and Bulk 

Grain Corn

Two commercial qualitative lateral flow strip test kits were 

developed by EnviroLogixTM to determine the presence or absence 

of eCry3.1Ab protein in corn grain, leaf or seed samples. The 

QuickStixTM Kit for eCry3.1Ab Corn Bulk Grain (Cat. No. AS 

TBD BG) detects eCry3.1Ab protein at a level of 1% or greater 

(one kernel in 100). In grains samples prepared from two separate 

seed lots of event 5307 corn, eCry3.1Ab protein tested showed 

100% accuracy in its detection and demonstrated its precision 

based on using test kits taken from three different manufactured 

lots. The QuickStixTM Kit for eCry3.1Ab Leaf and Seed (Cat. No. 

AS TBD LS) also detects eCry3.1Ab protein at a level of 1%. 

Results from the cross-reactivity test using the bulk grain test kit 

using samples from event MIR604 transgenic corn (known to 

contain a protein with similar homology to eCry3.1Ab protein) 

were negative. Additional cross reactivity analyses were negative 

for leaf tissue and seed samples from transgenic corn containing 

other unrelated Cry proteins. Collectively, these results demonstrate

the test kits are able to detect eCry3.1Ab protein residues in corn 

grain, leaf and seed with sufficient accuracy, precision, and 

477347-17
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

19

STUDY TITLE STUDY SUMMARY MRID NO. 

sensitivity. Thus, the EnviroLogixTM QuickStixTM Kit for 

eCry3.1Ab Corn Bulk Grain and the QuickStixTM Kit for 

eCry3.1Ab for leaf and seed are suitable analytical methods for the 

qualitative detection of eCry3.1Ab protein residues. 

EnviroLogixTM also obtained USDA GIPSA performance 

certification of the QuickStixTM Kit for eCry3.1Ab Corn Bulk 

Grain (Certificate of Performance No. GIPSA 2009-011), which is 

also acceptable as confirmation of the test kit’s performance in lieu 

of an ILV study. 

CLASSIFICATION: ACCEPTABLE

Validation of a 

quantitative 

eCry3.1Ab 

ELISA for 

analysis of maize 

leaf, root, kernel, 

and pollen 

extracts

The study was conducted to validate the use of an enzyme-linked 

immunosorbent assay (ELISA) method for the quantitative analysis 

of eCry3.1Ab protein expression levels in event 5307 maize leaf, 

root, kernel, and pollen tissue matrices. Recoveries of eCry3.1Ab 

from fortified extracts of leaf, root, kernel, and pollen matrices 

ranged from 85.42% to 113.88%, indicating that the assay is 

accurate across the range of the eCry3.1Ab reference protein 

standard curve. Extraction efficiencies of 77.09% and 74.66% were 

achieved for leaf and root matrices, respectively. Recoveries of 

eCry3.1Ab from fortified extracts spiked with mCry3A and 

Cry1Ab proteins ranged from 75.81% to 112.87% for the matrices 

tested, indicating that the assay is specific for the quantitative 

analysis of eCry3.1Ab protein. These results show the ELISA 

method is sufficient for accurately quantifying eCry3.1Ab protein 

concentrations in various corn tissue matrices. In addition, the 

LODs, LOQ, and extraction efficiencies values can be used for 

subsequent studies using ELISA analyses.

CLASSIFICATION: ACCEPTABLE

477539-02

Event 5307 

Maize: Insert 

Sequence 

Analysis; 

Amended Report 

No. 2

This study was conducted to determine the deoxyribonucleic acid 

(DNA) sequence of the event 5307 maize insert and to assess the 

intactness of the insert, the organization of the functional elements, 

and the presence of any rearrangements, deletions, and/or base pair 

changes within the event 5307 maize insert.

Two overlapping fragments that span the 5307 maize insert 

were amplified from genomic DNA extracted from 5307 maize 

using polymerase chain reaction. These fragments were cloned, 

and sequences of the clones were aligned to create a consensus 

of the transferred- DNA (T-DNA) sequence. This sequence was 

488026-01
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

20

STUDY TITLE STUDY SUMMARY MRID NO. 

compared to plasmid pSYN12274, the transformation plasmid 

used to create 5307 maize. Therefore, DNA sequence analysis 

demonstrated that the 5307 maize insert was intact and the 

organization of the functional elements within the insert is 

contiguous, as present in pSYN12274. 

CLASSIFICATION: SUPPLEMENTAL; No DER preparedThe data in this report were used to support the results reported in 

MRID No: 484425-01.

Event 5307 

Maize: Flanking 

Sequence 

Determination: 

Amended Report 

No. 1

This study was conducted to determine the maize genomic 

sequence flanking both sides of the 5307 maize insert. The 

sequence of each flanking region was amplified using polymerase 

chain reaction analysis. These amplification products were cloned, 

and multiple clones were sequenced and compared to generate a 

consensus sequence for each flanking region. Results showed for 

each flanking region, 1,000 base pairs were reported.

CLASSIFICATION: SUPPLEMENTAL; No DER preparedThe data in this report were used to support the results reported in 

MRID No: 484425-01.

488026-02

5307 Corn: 

Supplemental 

ELISA Validation 

Data for MRIDs 

484425-08, 

484425-10, 

484425-11, 

484425-14, and 

484425-17

During the Agency’s review of Syngenta Seeds, Inc. event 5307 

data submission, the Agency identified data deficiencies in the 

ELISA method validation studies for eCry3.1Ab (specifically in 

MRID No. 484425-08, 484425-14, and 484425-17). The Agency

requested the raw data used to calculate ELISA method sensitivity, 

accuracy, precision, quantitative range, linearity, specificity, and 

extraction. In response, Syngenta submitted a compilation of raw 

data for each data deficiency identified from each study report in a 

single volume. Review of the individual data for each study verified 

the accuracy of the values reported for the ELISA validation 

studies.

CLASSIFICATION: SUPPLEMENTAL; No DER preparedThe raw data compiled for each study were used to support the 

results reported in MRID No. 484425-08, 484425-14 and 484425-

17.

488026-04

a

Previously Reviewed in US EPA 2010b
Bacillus thuringiensis eCry3.1Ab Corn

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21

B. FQPA FOOD SAFETY ASSESSMENT 

1. Background and Statutory Findings

Section 408(c)(2)(A)(i) of the FFDCA allows EPA to establish an exemption from the requirement for a 

tolerance (the legal limit for a pesticide chemical residue in or on a food) only if EPA determines that 

the exemption is “safe.” Section 408(c)(2)(A)(ii) of the FFDCA defines “safe” to mean that “there is a 

reasonable certainty that no harm will result from aggregate exposure to the pesticide chemical residue, 

including all anticipated dietary exposures and all other exposures for which there is reliable 

information.” This includes exposure through drinking water and in residential settings, but does not 

include occupational exposure. Section 408(b)(2)(C) of the FFDCA requires EPA to give special 

consideration to exposure of infants and children to the pesticide chemical residue in establishing a 

tolerance and to “ensure that there is a reasonable certainty that no harm will result to infants and 

children from aggregate exposure to the pesticide chemical residue”... Additionally, section 

408(b)(2)(D) of the FFDCA requires that the Agency consider “available information concerning the 

cumulative effects of a particular pesticide's residues and other substances that have a common 

mechanism of toxicity.” 

EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide 

residues. First, EPA determines the toxicity of pesticides. Second, EPA examines exposure to the 

pesticide through food, drinking water, and through other exposures that occur as a result of pesticide 

use in residential settings. 

2. Toxicological Profile 

Consistent with section 408(b) (2) (D) of the FFDCA, EPA has reviewed the available scientific data 

and other relevant information in support of this action and considered its validity, completeness and 

reliability and the relationship of this information to human risk. EPA has also considered available 

information concerning the variability of the sensitivities of major identifiable subgroups of consumers, 

including infants and children.

a) Product Characterization Overview

Based on amino acid sequence homology and crystal structures, known Cry proteins have a similar 

three-dimensional structure comprised of three domains, Domain I, II, and III (Nakamura et al. 1990; Li 

et al. 1991; Ge et al. 1991; Honee et al. 1991). The toxin portions of Cry proteins are characterized by

having five conserved blocks (CB) across their amino acid sequence. These are numbered CB1 to CB5 

from the N-terminus to the C-terminus (Hofte and Whiteley 1989). The sequences preceding and 

following these conserved blocks are highly variable and are designated as variable regions V1 to V6. 

Because Cry proteins share structural similarities, chimeric cry genes can be engineered via the 

exchange of domains that are homologous between different cry genes.

Syngenta Seeds, Inc. – Field Crops - NAFTA (Syngenta) has developed Event 5307 maize (Zea mays)

[OECD Unique ID. SYN-537-1] to express Bacillus thuringiensis (Bt) eCry3.1Ab insecticidal 
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

22

crystal protein for use as a plant-incorporated protectant against root feeding damage from Western corn 

rootworm (WCR, Diabrotica virgifera) and related Diabrotica species. This proposed PIP is an 

engineered chimera protein, composed of portions of modified Cry3A (mCry3A) protein, a protein 

derived from the native Cry3A protein from Bt subsp. tenebrionis, and on the Cry1Ab protein from Bt 

thuringiensis subsp. kurstaki HD-1.The ecry3.1Ab gene (Entrez Accession Number GU327680 [NCBI, 

2011]) (Walters et al. 2010) consists of a fusion between the N-terminus (Domain I, Domain II, and a 

portion of Domain III) of a mcry3A gene and the C-terminus (a portion of Domain III and Variable 

Region 6) of a cry1Ab gene (Hofte and Whiteley, 1989). The eCry3.1Ab protein is 654 amino acid 

residues in size and is approximately 74.8 kilodaltons.

Event 5307 maize also contains the manA (also known as pmi) gene from Escherichia coli, which 

encodes phosphomannose isomerase (PMI) as a selectable marker in regenerating plant material 

following transformation (Negrotto et al. 2000). An exemption from the requirement of a tolerance was 

established for PMI in all crops when used as a PIP inert ingredient [40 CFR § 174.527, effective April 

25, 2007].

b) Mammalian Toxicity Assessment

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

levels of exposure to the pure eCry3.1Ab protein. These data demonstrate the safety of the product at a 

level well above maximum possible exposure levels that are reasonably anticipated in the crop. 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 158.2130(d)(1)(i) and 158.2140(d)(7)). For microbial products, further toxicity testing and 

residue data are triggered by significant adverse acute effects in studies such as the mouse oral toxicity

study, to verify and quantify the observed adverse effects and clarify the source of these effects (Tiers II 

& III). 

An acute oral toxicity study in mice (MRID No. 477539-01) indicated that eCry3.1Ab is non-toxic. Two 

groups of ten male and ten female mice were orally dosed (via gavage) with 2,000 milligrams/kilograms 

bodyweight (eCry3.1Ab protein mg/kg bwt) of the ECRY3.1AB-0208 test substance, a biochemically

and functionally equivalent microbially-produced eCry3.1Ab protein. All treated animals gained weight 

and had no test material-related clinical signs and no test material-related findings at necropsy. Since 

there were no significant differences between the test and control groups related to the oral 

administration of ECRY3.1AB-0208 test material; the eCry3.1Ab protein does not appear to cause any

significant adverse effects at an exposure level of up to 2000 mg/kg bwt and supports the finding that the 

eCry3.1Ab protein would be non-toxic to mammals.

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 eCry3.1Ab, even at 

relatively high dose levels, the eCry3.1Ab protein is not considered toxic. Further, amino acid sequence 

comparisons showed no similarities between the eCry3.1Ab protein and known toxic proteins in protein 

databases that would raise a safety concern.
Bacillus thuringiensis eCry3.1Ab Corn

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c) Allergenicity Assessment

Since eCry3.1Ab is a protein, allergenic sensitivities were considered. Currently, no definitive tests exist 

for determining the allergenic potential of novel proteins. Therefore, EPA uses a “weight-of-the 

evidence” approach where the following factors are considered: source of the trait; amino acid sequence 

similarity with known allergens; prevalence in food; and biochemical properties of the protein, including 

in vitro digestibility in simulated gastric fluid (SGF), and glycosylation (as recommended by CAC,

2003). Current scientific knowledge suggests that common food allergens tend to be resistant to 

degradation by acid and proteases; may be glycosylated; and present at high concentrations in the food. 

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 eCry3.1Ab with known 

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

contiguous amino acid residues. This is the appropriate level of sensitivity to detect possible IgE 

epitopes without high false positive rates.

3. Prevalence in food. Expression level analysis of eCry.1Ab protein is present at relatively low 

levels. The expression has been shown to be in the parts per million range. Thus, dietary exposure 

is expected to be correspondingly low. 

4. Digestibility. The eCry3.1Ab protein was rapidly digested in less than 30 seconds in simulated 

mammalian gastric fluid containing pepsin (pH 1.2) after incubation at 37°C. 

5. Glycosylation. The eCry3.1Ab protein expressed in corn was shown not to be glycosylated.

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

for eCry3.1Ab to be a food allergen is minimal.

3. Aggregate Exposures

In examining aggregate exposure, EPA considers available information concerning exposures from the 

pesticide residue in food and all other non- occupational exposures, including drinking water from 

ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings 

(residential and other indoor uses). 

The Agency has considered available information on the aggregate exposure levels of consumers (and 

major identifiable subgroups of consumers) to the pesticide chemical residue and to other related 

substances. First, with respect to other related substances, the eCry3.1Ab protein is a chimeric Bacillus 

thuringiensis protein, composed of portions of Cry1Ab and mCry3A proteins, both of which are 

registered PIPs that were previously assessed as having a lack of mammalian toxicity at high levels of 

exposure. Exemptions from the requirement of a tolerance already have been established for Cry1Ab in 

food and mCry3A in maize [see 40 CFR § 174.511, effective Apr. 25, 2007 and 40 CFR § 174.505, 

effective Apr. 25, 2007, respectively]. Second, and specific to the eCry3.1Ab protein, these 

considerations include dietary exposure under the tolerance exemption and all other tolerances or 

exemptions in effect for the plant-incorporated protectant chemical residue and exposure from non-
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

24

occupational sources. Exposure via the skin or inhalation is not likely since the plant-incorporated 

protectant is contained within plant cells, which essentially eliminates these exposure routes or reduces 

these exposure routes to negligible. The amino acid similarity assessment included similarity to known 

aeroallergens. It has been demonstrated that there is no evidence of occupationally related respiratory

symptoms, based on a health survey on migrant workers after exposure to Bt pesticides (Berstein et al. 

1999). Exposure via residential or lawn use to infants and children is also not expected because the use 

sites for the eCry3.1Ab protein are all agricultural for control of insects. Oral exposure, at very low 

levels may occur from ingestion of processed corn products and, potentially, drinking water. 

However, oral toxicity testing done at a dose of 2 gm/kg showed no adverse effects. Furthermore, the 

expected dietary exposure from corn is several orders of magnitude lower than the amounts of 

eCry3.1Ab protein shown to have no toxicity. Therefore, even if negligible aggregate exposure should 

occur, the Agency concludes that such exposure would present no harm due to the lack of mammalian 

toxicity and the rapid digestibility demonstrated for the eCry3.1Ab protein. 

4. Cumulative Effects from Substances with a Common Mechanism of Toxicity

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, or 

revoke a tolerance, the Agency consider “available information” concerning the cumulative effects of a 

particular pesticide’s residues and “other substances that have a common mechanism of toxicity.”

Since eCry3.1Ab is not considered toxic, EPA has not found eCry3.1Ab to share a common mechanism 

of toxicity with any other substances, and eCry3.1Ab does not appear to produce a toxic metabolite 

produced by other substances. For the purposes of this tolerance action, therefore, EPA has assumed that 

eCry3.1Ab does not have a common mechanism of toxicity with other substances. Following from this, 

therefore, EPA concludes that there are no cumulative effects associated with eCry3.1Ab that need be 

considered. For information regarding EPA’s efforts to determine which chemicals have a common 

mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see EPA’s website at 

http://www.epa.gov/pesticides/cumulative.

5. Determination of Safety for U.S. Population, Infants and Children 

The data submitted and cited regarding potential health effects for the eCry3.1Ab protein include the 

characterization of the expressed eCry3.1Ab protein in corn, as well as the acute oral toxicity, heat 

stability, and in vitro digestibility of the proteins. The results of these studies were used to evaluate 

human risk, and the validity, completeness, and reliability of the available data from the studies were

also considered. 

As discussed more fully in section B above, the acute oral toxicity data submitted supports the 

prediction that the eCry3.1Ab protein would be nontoxic to humans. Moreover, eCry3.1Ab showed no 

sequence similarity to any known toxin. Because of this lack of demonstrated mammalian toxicity, no 

protein residue chemistry data for eCry3.1Ab were required for a human health effects assessment. 

Even so, preliminary expression level analysis showed eCry3.1Ab protein is present at relatively low 

levels. Dietary exposure is expected to be correspondingly low. 
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

25

Since eCry3.1Ab is a protein, its potential allergenicity is also considered as part of the toxicity

assessment. Data considered as part of the allergenicity assessment include that the eCry3.1Ab protein 

came from Bacillus thuringiensis which is not a known allergenic source, showed no sequence similarity 

to known allergens, was readily degraded by pepsin, and was not glycosylated when expressed in the 

plant. Therefore, there is a reasonable certainty that eCry3.1Ab protein will not be an allergen. 

Considered together, the lack of mammalian toxicity at high levels of exposure to the eCry3.1Ab protein 

and the minimal potential for that protein to be a food allergen demonstrate the safety of the product at 

levels well above possible maximum exposure levels anticipated in the crop.

Finally, and specifically in regards to infants and children, FFDCA section 408(b)(2)(C) provides that 

EPA shall assess the available information about consumption patterns among infants and children, 

special susceptibility of infants and children to pesticide chemical residues, and the cumulative effects 

on infants and children of the residues and other substances with a common mechanism of toxicity. In 

addition, FFDCA section 408(b)(2)(C) provides that EPA shall apply an additional tenfold margin of 

safety for infants and children in the case of threshold effects to account for prenatal and postnatal 

toxicity and the completeness of the data base unless EPA determines that a different margin of safety

will be safe for infants and children. 

Based on its review and consideration of all the available information, as discussed in more detail above, 

the Agency concludes that there is a reasonable certainty that no harm will result to the U.S. population, 

including infants and children, from aggregate exposure to residues of the eCry3.1Ab protein and the 

genetic material necessary for its production in corn. This includes all anticipated dietary exposures and 

all other exposures for which there is reliable information. The Agency has also concluded, again for 

the reasons discussed in more detail above, that there are no threshold effects of concern and, as a result, 

that an additional margin of safety for infants and children is unnecessary in this instance.

6. Other Considerations 

a) Analytical Method(s)

EPA has determined that an analytical method is not required for enforcement purposes when

establishing an exemption from the requirement of a tolerance without any numerical limitation. 

Nonetheless, Syngenta has submitted validation method studies on two qualitative lateral flow strip kits 

for the analytical detection of eCry3.1Ab protein in corn grain, leaf and seed corn matrices. Results 

showed the test kits are able to detect eCry3.1Ab protein residues in corn with sufficient accuracy, 

precision, and sensitivity. A validation study conducted by an independent 3rd party laboratory

confirmed the performance of EnviroLogixTM QuickStixTM Kit for eCry3.1Ab Corn Bulk Grain (Cat. 

No. AS TBD BG) and EnviroLogixTM QuickStixTM Kit for eCry3.1Ab Corn Leaf and Seed (Cat. No. AS 

TBD LS). Lastly, Syngenta has submitted documentation that the EnviroLogixTM QuickStixTM Kit for 

eCry3.1Ab Corn Bulk Grain (Cat. No. AS TBD BG) received USDA GIPSA certification of the test 
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

26

kit’s performance and manufacturer claims (Certificate of Performance No. GIPSA 2009-011). In lieu of 

an independent laboratory validation study, GIPSA certification is acceptable to the Agency as 

confirmation of the test kit’s use as a suitable analytical residue method for the qualitative detection of 

eCry3.1Ab protein in bulk grain. 

b) Codex Maximum Residue Level

In making its tolerance decisions, EPA seeks to harmonize U.S. tolerances with international standards 

whenever possible, consistent with U.S. food safety standards and agricultural practices. In this context, 

EPA considers the international maximum residue limits (MRLs) established by the Codex Alimentarius

Commission (Codex), as required by FFDCA section 408(b)(4). The Codex Alimentarius is a joint U.N. 

Food and Agriculture Organization/World Health Organization food standards program, and it is 

recognized as an international food safety standards-setting organization in trade agreements to which 

the United States is a party. EPA may establish a tolerance that is different from a Codex MRL; 

however, FFDCA section 408(b)(4) requires that EPA explain the reasons for departing from the Codex 

level.

The Codex has not established a MRL for Bacillus thuringiensis eCry3.1Ab protein in corn. 

Table 3. Toxicological and Allergenicity Data for Event 5307 Maize

STUDY TITLE STUDY TITLE MRID 

NO.

ECRY3.1AB-0208: 

Single-dose Oral 

(Gavage) Toxicity

Study in Mice with a 

14-Day Observation 

Period a

There was no evidence of toxicity resulting from a single oral 

dose of ECRY3.1AB-0208 test substance to mice via oral 

gavage after 14 days. No adverse effects were seen and the 

LD50 for males, females, and combined sexes in mice was

greater than 2000 mg/kg (TOXICITY CATEGORY III, 

classified by dose amount only).

CLASSIFICATION: ACCEPTABLE

477539-01

eCry3.1Ab: 

Assessment of amino 

acid sequence 

homology with known 

toxins a

No significant sequence similarities with eCry3.1Ab protein

were identified with any proteins known to be toxins.

CLASSIFICATION: ACCEPTABLE; Updated analysis 

presented in MRID No. 48442519.

477347-09

eCry3.1Ab: 

Assessment of amino 

acid sequence 

homology with known 

or putative allergens a

No significant sequence similarities of eCry3.1Ab protein were 

identified with any known proteins or putative allergens.

CLASSIFICATION: ACCEPTABLE; Updated analysis 

presented in MRID No. 48442518.

477347-10

In vitro digestibility of The eCry3.1Ab protein from either microbial or plant sources 477347-11
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

27

STUDY TITLE STUDY TITLE MRID 

NO.

eCry3.1Ab protein as 

contained in test 

substance 

ECRY3.1AB-0208 and 

event 5307 maize under 

simulated mammalian 

gastric conditions b

was completely digested within ten minutes in simulated 

human gastric fluid containing pepsin. No digestion occurred 

in simulated gastric fluid that did not contain pepsin, showing 

it was responsible for the digestion. However, several data 

deficiencies and anomalous results were identified in this study

and must be addressed by the registrant.

CLASSIFICATION: SUPPLEMENTAL; Deficiencies 

addressed in MRID No. 47734713.

Phosphomannose 

isomerase (Entrez 

Database Accession No 

AAA24109): 

Assessment of amino 

acid sequence 

homology with known 

toxins a

No significant sequence similarities of PMI were identified 

with any proteins known to be toxins.

CLASSIFICATION: ACCEPTABLE; Updated analysis 

presented in MRID No. 48442521

477347-12

Phosphomannose 

isomerase (Entrez 

Database Accession No 

AAA24109): 

Assessment of amino 

acid sequence 

homology with known 

or putative allergens a

No significant sequence similarities of PMI protein were 

identified with any known or putative proteins that are 

allergens.

CLASSIFICATION: ACCEPTABLE; Updated analysis 

presented in MRID No. 48442521

477347-13

Supplemental 

Information for Study 

SSB-024-08: In Vitro

Digestibility of 

eCry3.1Ab Protein as 

Contained in Test 

Substance 

ECRY3.1AB-0208 and 

in 5307 Maize Under 

Simulated Mammalian 

Gastric Conditions 

[EPA MRID 

47734711] b

Using an enzyme kinetic approach, densitometric analysis of 

the SDS-PAGE gels from the previously conducted in vitro 

digestibility study in SGF for the microbial-derived and the 

plant-derived eCry3.1Ab test substances showed rapid pepsin 

degradation rates (DT50= <1 minute and DT50= <2 minutes, 

respectively). In addition, the data deficiencies that were 

identified in MRID No. 477347-11 were adequately addressed.

CLASSIFICATION: ACCEPTABLE- for the purposes of 

the EUP, but not Sec. 3 Registration; Deficiencies 

addressed in MRID No. 48442523, 48442524 and 48442525.

480384-01

eCry3.1Ab (Entrez No significant sequence similarities in the overall amino acid 484425-18
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

28

STUDY TITLE STUDY TITLE MRID 

NO.

database accession 

number ADC30135): 

Assessment of amino 

acid sequence 

similarity to known or 

putative allergens

sequence of eCry3.1Ab were identified when compared with 

sequences from known or putative protein allergens in the 

2011 Food Allergy Research and Resource Program (FARRP) 

AllergenOnline database using alignments of 80 or more 

amino acids. There were also no matches of eight of more 

contiguous amino acids in eCry3.1Ab with allergen entries in 

the FARRP AllergenOnline database.

CLASSIFICATION: ACCEPTABLE

eCry3.1Ab (Entrez 

database accession 

number ADC30135): 

Assessment of amino 

acid sequence 

similarity to known or 

putative toxins

Bioinformatic searches were conducted to compare the amino 

acid sequence of eCry3.1Ab with that of known or putative 

protein toxins. The analysis was conducted using the BLASTP 

search tool to compare the amino acid sequence of eCry3.1Ab 

with 13,043,846 protein sequences in the NCBI Entrez®

Database . The threshold value (E-value>0.079) for 

determining significance of matches was based on searches 

conducted with randomly shuffled sequences of the amino 

acids comprising eCry3.1Ab. Although 505 protein sequences 

with significant similarity to that of eCry3.1Ab were found, 

further evaluation from the records in the NCBI Entrez® 

Protein Database for identity and biological function, showed 

none were associated with known or putative human or 

mammalian toxins. 

CLASSIFICATION: ACCEPTABLE

484425-19

Phosphomannose 

isomerase (Entrez 

database accession 

number AAA24109): 

Assessment of amino 

acid sequence 

similarity to known or 

putative allergens

Bioinformatic searches were conducted to determine if the 

amino acid sequence of PMI shared any significant sequence 

similarities with known or putative allergen entries in the 

FARRP AllergenOnline database (2011). Overlapping 

stretches of 80 amino acids were used as query sequences, as 

were stretches of the PMI sequence containing 8 or more 

contiguous amino acids. The current search showed no 

additional amino acid similarities other than the previously

reported 2004 serum screening analysis study showing a lack 

of immune recognition to PMI protein after one 8 amino acid 

alignment that was common to PMI and a food allergen αparvalbumin from Rana species CH2001(reported in Rabe 

2004; MRID No. 464252-01) was identified. This study

reconfirms the conclusion that PMI shows no biologically

relevant amino acid sequence similarity to any known or 

484425-20
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

29

STUDY TITLE STUDY TITLE MRID 

NO.

putative protein allergens.

CLASSIFICATION: ACCEPTABLE

Phosphomannose 

isomerase (Entrez 

database accession 

number AAA24109): 

Assessment of amino 

acid sequence 

similarity to known or 

putative toxins 

No significant sequence similarities of PMI were identified 

with any proteins known to be toxins.

CLASSIFICATION: ACCEPTABLE

484425-21

In vitro digestibility of 

eCry3.1Ab protein as 

contained in test 

substance 

ECRY3.1AB-0208 

under simulated 

mammalian intestinal 

conditions

Sodium dodecyl sulfate polyacrylamide gel electrophoresis 

(SDS-PAGE) and Western blot analyses were used to evaluate 

the in vitro digestibility of eCry3.1Ab protein (produced from 

recombinant Escherichia coli) in simulated mammalian 

intestinal fluid (SIF) containing pancreatin. SDS-PAGE 

results showed no bands representative of intact eCry3.1Ab 

protein (Molecular weight: 74.8 kDa) following incubation for 

1 minute at 35°C with SIF containing pancreatin (pH 7.5). 

The limit of detection for the SDS-PAGE method was 

determined to be 8 ng. Western blot results also showed no 

bands representative of intact eCry3.1Ab protein in SIF (pH 

7.5) after 1 minute incubation at 35°C. Immunoreactive 

fragments of eCry3.lAb with molecular weights of 

approximately 56, 40, and 5 kDa were present at the 

conclusion of the 48 hour time course of the study. The 

protein bands were identified as degradation products of 

eCry3.1Ab. The limit of detection for the method was 

determined to be 0.25 ng. The pancreatin stability and test 

material stability controls gave appropriate responses in both 

assays. In conclusion, both assays demonstrated the eCry3.1Ab 

protein is susceptible to the proteolytic activity of pancreatin 

and rapidly degrades after one minute of incubation in 

simulated mammalian intestinal fluid.

CLASSIFICATION: ACCEPTABLE

484425-22

In vitro digestibility of 

eCry3.1Ab protein 

under simulated 

mammalian gastric 

Sodium dodecyl sulfate polyacrylamide gel electrophoresis 

(SDS-PAGE), Western blot analyses and densitometric 

analyses were used to evaluate the in vitro digestibility of 

eCry3.1Ab protein (produced from recombinant Escherichia 

484425-23

[This 

report 
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

30

STUDY TITLE STUDY TITLE MRID 

NO.

conditions coli) in simulated mammalian gastric fluid (SGF), containing 

pepsin. SDS-PAGE results showed no bands representative of 

intact eCry3.1Ab protein (Molecular weight: 74.8 kDa) 

following incubation for 30 seconds at 37°C with SGF 

containing pepsin (pH 1.2). Two faint bands with molecular 

weights of approximately 4 kDa and 5 kDa, respectively, were 

visible after incubation in SGF for 15 seconds; however these 

two bands diminished in intensity over time and were no 

longer detectable after incubation in SGF for 10 minutes. The 

limit of detection for the SDS-PAGE method was determined 

to be 0.025 g. Densitometry analysis of the stained gels from 

SDS-PAGE showed eCry3.l Ab is readily digested in SGF in 

less than 30 seconds. After incubation of eCry3.1Ab in SGF 

for 15 and 30 seconds, 3% and 0% of the eCry3.1Ab remained, 

respectively. Western blot analysis showed no immunoreactive 

intact or fragmented eCry3.1Ab protein degradates after 30 

seconds of incubation at 37°C with SGF containing pepsin (pH 

1.2). The limit of detection for the SDS-PAGE method was 

determined to be 0.4 ng. The pepsin stability and test material 

stability controls gave appropriate responses in both assays. 

Collectively, the results demonstrated the eCry3.1Ab protein is 

susceptible to the proteolytic activity of pepsin and rapidly

degrades in less than 30 seconds after in vitro digestion in 

simulated mammalian gastric fluid.

CLASSIFICATION: ACCEPTABLE; The data in this 

report were used to address the data deficiencies reported in 

MRID No: 477347-11 and 480384-01.

supersede

s MRID 

No. 

477347-

11]

Effect of buffer ionic 

strength on the in vitro

digestibility of

eCry3.1Ab protein 

under simulated 

mammalian gastric 

conditions

Sodium dodecyl sulfate polyacrylamide gel electrophoresis 

(SDS-PAGE), Western blot analyses and densitometric 

analyses were used to evaluate the effect of buffer ionic 

strength on the in vitro digestibility of eCry3.1Ab protein 

(produced from recombinant Escherichia coli) in simulated 

mammalian gastric fluid (SGF) containing pepsin. The 

eCry3.1Ab protein was prepared in 10 mM ammonium 

bicarbonate buffer (0.01 M, low ionic strength) or 95 mM 

glycine, 50mM sodium phosphate buffer (0.15 M, high ionic 

strength). 

SDS-PAGE results showed no bands representative of intact 

484425-24
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

31

STUDY TITLE STUDY TITLE MRID 

NO.

eCry3.1Ab protein (MW: 74.8 kDa) following incubation for 

30 seconds at 37°C with SGF containing pepsin (pH 1.2) for 

the low ionic strength buffer, whereas the high ionic strength 

buffer showed slower degradation. Densitometry analysis of 

the stained gels from SDS-PAGE showed the eCry3.1Ab 

protein prepared in low ionic strength buffer was readily 

digested by pepsin in SGF with only 6% of the intact protein 

remaining after 15 seconds and none remaining after 30 

seconds. The eCry3.1Ab prepared in high ionic strength buffer 

was digested at a much slower rate with 15% intact protein 

remaining after incubation with pepsin for 1 minute. Western 

blot results showed a similar finding of no bands 

representative of intact eCry3.1Ab protein (MW: 74.8 kDa) 

following incubation for 30 seconds at 37°C with SGF 

containing pepsin (pH 1.2) for the low ionic strength buffer, 

while the high ionic strength buffer showed slower 

degradation. After incubation in the high ionic strength buffer 

for 1 minute, 15% of the eCry3.1Ab remained undigested. The 

pepsin stability and test material stability controls gave 

appropriate responses in both assays. Collectively, these 

results demonstrated the buffer strength affects the rate at 

which the eCry3.1Ab protein is digested by pepsin.

CLASSIFICATION: ACCEPTABLE; The data in this 

report were used to address the data deficiencies reported in 

MRID No: 477347-11 and 480384-01.

A comprehensive 

overview of the 

assessment of the in 

vitro digestibility of 

eCry3.1Ab protein 

under simulated 

mammalian gastric 

conditions.

This report summarizes the studies conducted to assess the 

digestibility of eCry3.1Ab in simulated gastric fluid (SGF) 

containing pepsin. The proteolytic degradation of eCry3.1Ab 

was assessed by sodium dodecyl sulfate polyacrylamide gel 

electrophoresis (SDS-PAGE), Western blot, and densitometric 

analysis. The initial studies showed that the microbially- and 

plant-expressed purified eCry3.1Ab protein samples were 

moderately digested in SGF containing pepsin. These protein 

samples were diluted with a high ionic strength buffer, 95 mM 

glycine, 50 mM sodium phosphate, since it was not feasible to 

dilute the plant-derived eCry3.1Ab in 10 mM ammonium 

phosphate buffer. Each assay showed the eCry3.1Ab protein 

band was present at the 5 min. time point, but no longer visible 

at the 10 min. time point. 

484425-25
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

32

STUDY TITLE STUDY TITLE MRID 

NO.

Additional studies were conducted to compare the digestibility

of the microbially-produced eCry3.1Ab in low and high ionic 

strength buffer. Although, eCry3.1Ab is rapidly digested in 

both buffers, digestion occurs more rapidly in the low ionic 

strength buffer. Densitometric analyses showed only 6% 

remained after 15 seconds and 0% intact eCry3.1Ab remained 

after incubation in SGF for 30 seconds. After incubation in the 

high ionic strength buffer for 1 minute, 15% of the eCry3.1Ab 

remained undigested. These studies support the conclusion the 

buffer strength affects the rate at which the eCry3.1Ab protein 

is digested by pepsin. Moreover, the additional studies using 

the lower strength ionic buffer demonstrate the eCry3.1Ab 

derived from transformation Event 5307 maize is expected to 

be rapidly digested under typical mammalian gastric 

conditions.

CLASSIFICATION: ACCEPTABLE; The previously

conducted studies compiled in this report were used to address 

the data deficiencies identified in MRID No. 477347-11 and 

480384-01 reviewed in this memorandum.

a

Previously Reviewed in US EPA 2010a

b

Previously Reviewed in US EPA 2010b

7) Bt11 x MIR162 x MIR604 x TC1507 x 5307 Corn

The molecular characterization of combination PIP Bt11 x MIR162 x MIR604 x TC1507 x 5307 

corn product and the genetic material necessary for their production in the plant demonstrated the 

stability and integrity of the intended transgenic DNA insertions of cry1Ab, vip3Aa20, mCry3A, cry1F

and eCry3.1Ab from Bt11, MIR162, MIR604, TC1507 and 5307 corn events, respectively, when 

combined through traditional plant breeding. In addition, the combination PIP product protein 

confirmed comparable protein expression levels in various plant tissues matrices at select 

developmental growth stages for the combination PIP plant tissues compared to its parental events. 

These results confirm the equivalence of the biochemical characteristics, thus demonstrating no 

increase in exposure to the presence and amounts of Cry1Ab, Vip3Aa20, mCry3A, Cry1F and 

eCry3.1Ab proteins. 

Laboratory assays using sensitive target pest species showed no enhanced toxicity to the target pest, 

demonstrating the lack of synergism among the Cry proteins expressed in the combination PIP product 

(US EPA 2012a). In addition, previously investigated protein interaction studies using various subsets 

of protein combinations showed a lack of synergism, which includes: Cry1Ab + mCry3A for Bt11 x 
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

33

MIR604 (MRID No. 467956-04; reviewed in US EPA 2007); Cry1Ab + Vip3Aa20 + Cry1F for Bt11 x 

MIR162 x TC1507 (MRID No. 482352-04, reviewed in US EPA 2011a); and a protein mixture of 

Cry1Ab + Vip3Aa20 + Cry1F against a protein mixture of mCry3A + Cry34/35Ab1 for Bt11 × DAS59122-7 × MIR604 × TC1507 corn (MRID No. 482023-17; reviewed in US EPA 2011b). In addition, 

Vip proteins are known to act individually to affect a typical midgut pathology in susceptible insects 

like previously studied Bt delta-endotoxins (Lee, et al. 2003). Thus, no synergistic action or interaction 

of these proteins is known or expected to occur from exposure to the combination PIP product. These 

results, when combined with the results of previous studies that show no synergism among the 

components in each mixture, and the molecular characterization, and protein expression analyses 

confirm the biochemical functional equivalence of the Cry proteins expressed in the combination PIP 

and its constituent parental maize events. 

In conclusion, the submitted data provide justification for bridging the existing results and conclusions 

from the individual PIP events that are already contained in the Agency’s database (US EPA 2001a, 

2001b, 2006, 2007, 2011a, 2011b and 2012b)

Since the mode of action for these proteins does not suggest a synergistic activity in combination for 

mammalian species, the individual parental event data for Cry1Ab, Vip3Aa, Cry1F, eCry3.1Ab and 

mCry3A proteins support the inclusion of the Cry1Ab [40 CFR § 174.511], Vip3Aa [40 CFR § 

174.501], Cry1F [40 CFR § 174.504], eCry3.1Ab [40 CFR § 174.532] and mCry3A [40 CFR 

§174.505] PIP proteins expressed in the combination PIP product under the existing exemptions from 

the requirement of a tolerance for residues on food. Therefore, the toxicological and allergenicity data 

can be bridged to support the finding of reasonable certainty of no harm to exposure of Cry1Ab, 

Vip3Aa20, Cry1F, eCry3.1Ab and mCry3A proteins and the genetic material necessary for their 

production in Bt11 x MIR162 x MIR604 x TC1507 x 5307 to the U.S. population, including infants 

and children. This includes all anticipated dietary exposures and all other exposures for which there is 

reliable information. 

TABLE 4. Summary of Submitted Data in support for Sec. 3 Registration of Combination 

PIP 

Bt11 x MIR162 x TC1507 Corn Product

Study Type/Title Summary MRID #

Comparison of 

Transgenic 

Protein 

Concentrations in 

Event Bt11, Event 

MIR162, Event 

MIR604, Event 

TC1507, Event 

The purpose of this study was to compare protein expression 

by enzyme-linked immunosorbent assays (ELISA) in a 

combination PIP product (Bt11 x MIR162 x MIR604 x 

TC1507 x 5307 x GA21) with expression in corresponding, 

near-isogenic hybrids derived from the individual 

transformation events: Bt11, MIR162, MIR604, TC1507, 

5307, and GA21. 

484429-01
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

34

TABLE 4. Summary of Submitted Data in support for Sec. 3 Registration of Combination 

PIP 

Bt11 x MIR162 x TC1507 Corn Product

Study Type/Title Summary MRID #

5307, Event 

GA21, and Bt11 × 

MIR162 × 

MIR604 x 

TC1507 × 5307 x 

GA21 Maize 

Hybrids

Tissue samples were collected from each of five replicate 

planted blocks of maize of each individual hybrid line and 

the combination PIP product, at different developmental 

stages. Tissues analyzed included leaves, roots, kernels, 

whole plants, and pollen. Results showed eleven statistically

significant differences (out of 33 statistical analyses) were 

seen between the concentrations of the transgenic proteins 

expressed in the maize plant tissues of the individual-event 

hybrids and the combination PIP product, however these 

differences were generally small and were not consistent 

across tissue types or developmental stages. Overall, the 

majority of the transgenic protein concentrations were 

similar between the combination PIP product and the 

corresponding individual event hybrid with the exception for 

the statistically significant difference (~ 60-80% greater) in 

the concentrations of Cry1Ab protein in the combination 

stack corn leaf tissue compared to the corresponding leaf 

tissue of Event Bt11. The data provided by the registrant on 

the statistically significant difference (~50-70% greater) 

indicate no biological significance.

The concentration of PMI and PAT were significantly

higher in the combination PIP product. However these 

results were expected due to the insertions of multiple copies 

of the pmi and pat gene in the combination PIP due to the 

conventional crossing of the single parental events 

containing these genes.

Classification: ACCEPTABLE
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

35

TABLE 4. Summary of Submitted Data in support for Sec. 3 Registration of Combination 

PIP 

Bt11 x MIR162 x TC1507 Corn Product

Study Type/Title Summary MRID #

Bt11 × MIR162 × 

MIR604 x 

TC1507 × 5307 x 

GA21 Maize: 

Comparitive 

Southern Blot 

Analysis

Molecular analyses (restriction enzyme digests and Southern 

blots) were performed to compare the integrity of the 

transgenic inserts in the individual maize lines (Event Bt11, 

Event MIR162, Event MIR604, Event TC1507, and Event 

5307) with the transgenic inserts in the Combination PIP 

Product, Bt11 x MIR162 x MIR604 x TC1507 x 5307 x 

GA21, which was created through conventional plant 

breeding techniques.

Plants from Bt11, MIR162, MIR604, TC1507, 5307, the 

combination PIP product, and a non-transgenic near-isogenic 

control maize were grown under standard greenhouse 

conditions. All plants were individually analyzed by realtime Polymerase Chain Reaction (PCR) to confirm the 

presence of the appropriate genes for the transgenic lines and 

the absence of these genes from the non-transgenic, nearisogenic, negative control plants. Leaves from each 

confirmed maize line were then pooled and genomic DNA 

was extracted for further analyses. 

Data from the Southern blot analyses of the individual 

events Bt11, MIR162, MIR604, TC1507, 5307, and the 

combination PIP product confirmed the integrity of the 

inserts from the individual events as they were incorporated 

into the combination PIP product during conventional 

breeding.

Classification: ACCEPTABLE

484429-02
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

36

TABLE 4. Summary of Submitted Data in support for Sec. 3 Registration of Combination 

PIP 

Bt11 x MIR162 x TC1507 Corn Product

Study Type/Title Summary MRID #

Bt11 × MIR162 × 

MIR604 x 

TC1507 × 5307 x 

GA21 Maize: Test 

and Control 

Substance 

Characterization 

of Event Bt11 

Maize, Event 

MIR162 Maize, 

Event 

MIR604 Maize, 

Event TC1507 

Maize, Event 

5307 Maize, 

Event GA21 

Maize, Bt11 × 

MIR162 × 

MIR604 x 

TC1507 × 5307 x 

GA21 Maize, and 

Nontransgenic 

Maize

The purpose of this study was to determine the identity, 

purity, and stability of the test materials and the identity and 

purity of the control material, where the test materials are 

seeds from Bt11 maize, MIR162 maize, MIR604 maize, 

TC1507 maize, 5307 maize, GA21 maize, and the 

recombinant PIP product (Bt11 × MIR162 × MIR604 × 

TC1507 × 5307 × GA21 maize).The control material is seed 

from a non-transgenic maize hybrid (5XH751/NP2222 

maize), nearly isogenic to the combination PIP product. 

Individual plants grown from the test and control substances 

were analyzed for the presence or absence of transgenic 

inserts by real-time Polymerase Chain Reaction (PCR) 

analysis. Results obtained from real-time PCR analysis were 

confirmed by gel-based, event-specific, PCR analysis. The 

resulting PCR products were analyzed by agarose gel 

electrophoresis to confirm that amplicons of the expected 

band size were produced, thereby confirming the identity, 

purity, and stability of each batch of recombinant seeds.

Classification: ACCEPTABLE

484429-03

Bt11 x MIR162 x 

MIR604 x 

TC1507 x 5307 

Corn: 

Supplemental 

Information for 

MRID 48442901

The purpose of the submitted data was to provide 

supplemental information to address identified data 

deficiencies noted in MRID 48442901. The increase in 

Cry1AB protein expressed in Bt11 x MIR162 x MIR604 x 

TC1507 x 5307 x GA21 leaf tissues was noted in Raybould 

(2011). The maximum increase in exposure to Cry1AB 

from cultivation of Bt11 x MIR162 x MIR604 x TC1507 x 

5307 x GA21 maize is predicted to be about 1.8X the EECs 

from Event Bt11 maize, which is well within the margins of 

exposure for foliar and soil organisms. 

Classification: Supplemental

48802801
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

37

8) Bt11 x MIR604 x TC1507 x 5307

The molecular characterization data of combination PIP Bt11 x MIR604 x TC1507 x 5307 corn product 

demonstrated the stability and integrity of the intended transgenic DNA insertions of cry1Ab, mcry3A,

cry1F and ecry3.1Ab from the constituent parental events Bt11, MIR604, TC1507 and 5307 corn events, 

respectively, when combined through traditional plant breeding. 

In addition, ELISA analyses of the combination PIP plant and its parental events tissues showed 

comparable levels of CryAb, mCry3A, Cry1F and eCry3.1Ab protein expression in various plant tissue 

matrices tested at selected developmental growth stages. These results confirm the genetic stability and 

similar expression levels of the Cry proteins in the combination PIP and its constituent parental maize 

events.

Diet incorporation feeding assays were conducted to determine whether various doses of lepidopteranactive protein mixtures (Cry1AB, Vip3a20, and Cry1F) and coleopteran-active protein mixture (mCry3a 

and eCry3.1Ab) interact when used in combination against European corn borer (ECB) and Colorado 

potato beetle (CPB) (MRID NO. 48442908; reviewed in US EPA 2012a). Results showed the 

coleopteran-active protein mixture does not affect the potency of the lepidopteran-active mixture against 

ECB, and the lepidopteran-active mixture does not affect the potency of the coleopteran-active against 

CPB. 

In addition, previously investigated protein interaction studies using various subsets of protein 

combinations showed a lack of synergism, which includes: Cry1AB + mCry3A for Bt11 x MIR604 

(MRID No. 467956-04; reviewed in US EPA 2007); and a protein mixture of Cry1Ab + Vip3Aa20 + 

Cry1F against protein mixture of mCry3A + Cry34/35Ab1 for Bt11 x DAS-59122-7 x MIR604 x TC1507 

corn (MRID No. 482023-17; reviewed in US EPA 2011). In addition, Vip proteins are known to act 

individually to affect a typical midgut pathology in susceptible insects like previously studied Bt deltaendotoxins (Lee et al. 2003 and Mostafa et al. 2003). Therefore, no synergistic activity of these proteins 

is known or expected from exposure to the combination PIP product on susceptible pest species. These 

results, when combined with the results of previous studies that show no synergism among the 

components in each mixture, and the molecular characterization, and protein expression analyses confirm 

the biochemical functional equivalence of the Cry proteins expressed in the combination PIP and its 

constituent parental maize events.

In conclusion, the submitted data provide justification for bridging the existing results and conclusions

from the individual PIP events that are already contained in the Agency’s database (US EPA 2001a, 

2001b, 2006, 2007, 2011, and 2012b). The data also support the inclusion of the PIP Cry proteins 

expressed in the combination PIP product under the existing exemptions from the requirement of a 

tolerance for Cry1Ab, mCry3A, Cry1F and eCry3.1Ab protein residues in corn [40 CFR § 174.511, 

174.505, 174.504, 174.532, respectively]. Thus, the toxicological and allergenicity data from the 

individual food safety assessments of events Bt11, MIR604, TC1507 and 5307 corn can be bridged to 
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

38

support the finding of reasonable certainty of no harm to the combined exposure of these Cry proteins and 

the genetic material necessary for their production in Bt11× MIR604 × TC1507 and 5307 maize to the 

U.S. population, including infants and children. This includes all anticipated dietary exposures and all 

other exposures for which there is reliable information. 

TABLE 5. Summary of Submitted Data in support for Sec.3 Registration of Combination PIP Bt11 x MIR604 

x TC1507 x 5307

Study Type/ Title Summary MRID#

Comparison of 

Transgenic Protein 

Concentrations in 

Event Bt11, Event 

MIR162, Event 

MIR604, Event 

TC1507, Event 5307, 

Event GA21, and 

Bt11 × MIR162 × 

MIR604 x TC1507 × 

5307 x GA21 Maize 

Hybrids

The purpose of this study was to compare protein expression by enzymelinked immunosorbent assays (ELISA) in a combination PIP product

(Bt11 x MIR162 x MIR604 x TC1507 x 5307 x GA21) with expression in 

corresponding, near-isogenic hybrids derived from the individual 

transformation events: Bt11, MIR162, MIR604, TC1507, 5307, and 

GA21. 

Tissue samples were collected from each of five replicate planted blocks 

of maize of each individual hybrid line and the combination PIP product, 

at different developmental stages. Tissues analyzed included leaves, roots, 

kernels, whole plants, and pollen. Results showed 11 statistically 

significant differences (out of 33 statistical analyses) were seen between 

the concentrations of the transgenic proteins expressed in the maize plant 

tissues of the individual-event hybrids and the combination PIP product, 

however these differences were generally small and were not consistent 

across tissue types or developmental stages. Overall, the majority each 

transgenic protein concentration was similar between the combination PIP 

product and the corresponding individual event hybrid.

The exception was the statistically significant difference (~ 50-70% 

greater) in the concentrations of eCry3.1Ab protein expressed in Bt11 x 

MIR604 x TC1507 x 5307 root issues compared to the corresponding root 

tissue results of Event 5307. The eCry3.1Ab protein is reported in root 

samples (V5 stage) at a mean of 73.48 μg/g DW (52.03 – 111.06 μg/g 

DW) for the combination PIP Bt11 x MIR604 x TC1507 x 5307 product 

and at a mean of 40.29 μg/g DW (25.78-77.85 μg/g DW) for single 

parental event 5307. The data provided by the registrant on the 

statistically significant difference (~50-70% greater) indicate no biological 

significance .

While no statistical comparison of PAT protein concentrations between 

the combination PIP product and the corresponding individuals- event 

hybrids (Bt11 and TC1507) was conducted, the concentrations of PAT in 

the tissues of the combination PIP product were generally similar to the 

TC1507 or Bt11 hybrids. Similarly, no statistical comparison of PMI 

protein concentrations between the combination PIP Product and the 

corresponding individual-event hybrids (MIR604 and 5307) was 

conducted, but the concentrations of PMI in the combination PIP product 

were always higher than either the individual MIR604 or 5307hybrids, as 

expected. 

Classification: ACCEPTABLE

48443001
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

39

TABLE 5. Summary of Submitted Data in support for Sec.3 Registration of Combination PIP Bt11 x MIR604 

x TC1507 x 5307

Comparative 

Southern Blot 

Analysis of Bt11 x 

MIR604 x TC1507 x 

5307 with the 

individual Events Bt 

11 Maize, MIR604 

Maize, TC1507 

Maize, 5307 Maize 

and GA21 Maize

Molecular analyses (restriction enzyme digest and Southern blots) were 

performed to compare the integrity of the transgenic inserts in the 

individual maize lines (Event Bt11, Event MIR604, Event TC1507, and 

Event 5307) with the transgenic inserts in the combination PIP product, 

which was produced through conventional plant breeding techniques.

Plants from Bt11, MIR604, TC1507, 5307, the combination PIP product, 

and non-transgenic near-isogenic control maize were grown under 

standard greenhouse conditions. All plants were individually analyzed by 

real-time Polymerase Chain Reaction (PCR) to confirm the presence of 

the appropriate genes for the transgenic lines and the absence of these 

genes from the non-trangenic, near-isogenic, negative control plants. 

Leaves from each confirmed maize line were then pooled and genomic 

DNA was extracted for further analysis.

Data from the Southern analyses of the individual events Bt11, MIR604, 

TC1507, 537, and the combination Pip product confirmed the intergrity of 

the inserts from the individual events as they were incorporated into the 

combination Pip product during conventional breeding. 

Classification: ACCEPTABLE

48443002

Bt11 x MIR604 x 

Tc1507 x 5307 x 

GA21 Maize: Test 

and Control 

Substance 

Characterization of 

Event Bt11 Maize, 

Event MIR604 

Maize, Event TC1507 

Maize, Event 5307 

Maize, Event GA21 

Maize, Bt11 x 

MIR604 x TC1507 x 

5307 x GA21 Maize, 

and Nontransgenic 

Maize

The purpose of this study was to determine the identity, purity, and 

stability of the test materials and the identity and purity of the control 

material, where the test materials are seeds from Bt11 maize, MIR604 

maize, TC 1570 maize, 5307 maize, GA21 maize, and the recombinant 

Pip product (Bt11 x MIR604 x TC1507 x 5307 x GA21 maize). The 

control material is seed from a nontransgenic maize hybrid 

(5XH751/NP2222 maize), nearly isogenic to the recombinant PIP Product. 

Individual plants grown from the test and control substances were 

analyzed for the presence or absence of transgenic inserts by real-time 

Polymerase Chain Reaction (PCR) analysis. Results obtained from realtime PCR analysis were confirmed by gel-based, event-specific, PCR 

analysis. The resulting PCR products were analyzed by agarose gel 

electrophoresis to confirm that amplicons of the expected band size were 

produced, thereby confirming the identity, purity and stability of each 

batch of recombinant seeds. 

Classification: ACCEPTABLE

48443003

Bt11 x MIR604 x 

TC1507 x 5307 Corn: 

Supplemental 

Information for 

MRID 48443001

The purpose of the submitted data was to provide supplemental 

information to address identified data deficiencies noted in MRID 

48443001. Review of the data indicate that significant increased exposure 

of nontarget organisms to the Cry1Ab, mCry3A, Cry1F, and 3Cry3.1Ab 

proteins is not expected as a result of cultivation of Bt11 x MIR604 x 

TC1507 x 5307 corn plants.

Classification: Supplemental

48802701
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

40

REFERENCES

Bernstein, I.L., Bernstein, J.A., Miller, M., Tierzieva, S., Bernstein, D.I., Lummus, Z., Selgrade, M.K., 

Doerfler DL, Seligy VL. (1999) Immune responses in farm workers after exposure to Bacillus 

thuringiensis pesticides. Environ Health Perspect. 107(7):575-82.

CAC (2003) Alinorm 03/34: Joint FAO/WHO Food Standard Programme. Codex Alimentarius 

Commission, Twenty-Fifth Session, 30 July 2003. Rome, Italy. Appendix III: Guideline for 

Conduct of Food Safety Assessments of Foods Derived from Recombinant-DNA Plants; Appendix 

IV: Annex on Assessment of Possible Allergenicity. CAC, 47–60.

Ge, A., D. Rivers, R. Milne, and D.H. Dean. (1991) Functional Domains of Bacillus thuringiensis 

Insecticidal Crystal Proteins. Refinement of Heliothis virescens and Trichoplusia ni Specificity

Domains on Cry1A(c). J. Biol. Chem. 266: 17954-17958.

Geiser M., Schweizer S, Grimm C. (1986) The hypervariable region in the genes coding for 

entomopathogenic crystal proteins of Bacillus thuringiensis: nucleotide sequence of the kurhd1

gene of subsp. kurstaki HD-1 Gene 48:109-118.

Herman R.A., Storer N.A., Gao Y. (2006) Digestion assays in allergenicity assessment of transgenic 

proteins. Environmental Health Perspectives. 114:1154–1157.

Hofte, H., and Whitley H.R. (1989) Insecticidal Crystal Proteins of Bacillus thuringiensis. Microbiol. Rev.

53: 242-255.

Honee, G., D. Convents, J. Van Rie, S. Jansens, M. Peferoen, and B. Visser. (1991) The C-Terminal Domain 

of the Toxic Fragment of a Bacillus thuringiensis Crystal Protein Determines Receptor Binding. Mol. 

Microbiol. 5: 2799-2806.

Li, J., J. Carroll, and D.J. Ellar. (1991) Crystal Structure of Insecticidal δ-Endotoxin from Bacillus 

thuringiensis at 2.5 A resolution. Nature 353: 815-821.

Murray EE, Lotzer J, Eberle M. (1989) Codon usage in plant genes. Nucleic Acids Res 17:477–498.

Nakamura, K., K. Oshie, M. Shimizu, Y. Takada, K. Oeda, and H. Ohkawa. (1990) Construction of 

Chimeric Insecticidal Proteins Between the 130-kDa and 135-kDa Proteins of Bacillus thuringiensis 

subsp. aizawai for Analysis of Structure-Function Relationship. Agric. Biol. Chem. 54: 715-724.

NCBI (2011) Entrez Nucleotide database. Bethesda, MD: National Center for Biotechnology

Information, National Library of Medicine, National Institutes of Health, 

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide.
Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

41

Negrotto, et al. (2000) The use of phosphomannose-isomerase as a selectable marker to recover 

transgenic maize plants (Zea mays L. via Agrobacterium transformation). Plant Cell Reports, 19: 

798-803.

Sekar V, Thompson DV, Maroney MJ, Bookland RG, Adang MJ. (1987) Molecular cloning and 

characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var. tenebrionis. 

Proc Natl Acad Sci USA 84:7036–7040.

Sjoblad, R. D., McClintock, J.T., and Engler, R. (1992) Toxicological Considerations for Protein 

Components of Biological Pesticide Products, Reg. Toxicol. Pharmacol. 15(1): 3-9.

US EPA (2001) Biopesticides Registration Action Document for Bacillus thuringiensis (Bt) PlantIncorporated Protectants, dated October 21, 2001. US Environmental Protection Agency, 

Washington, D.C.

US EPA (2006) Biopesticides Registration Action Document for Bacillus thuringiensis (Bt) Modified 

Cry3A Protein and the Genetic Material Necessary for its Production (Via Elements of pZM26) in 

Event MIR604 Corn (OECD Unique Identifier: SYN-IR6Ø4-8), updated September 2010. US 

Environmental Protection Agency, Washington, D.C.

US EPA (2010a) Review of Product Characterization and Human Health Data for Plant- Incorporated 

Protectant Bacillus thuringiensis (Bt) eCry3.1Ab insect control protein and the genetic material 

necessary for its production in Event 5307 maize (Zea mays) [EPA Reg. No. 67979-EUP-I] in 

support for an Experimental Use Permit and Temporary Exemption from the Requirement of a 

Tolerance; submitted by Syngenta Seeds, Inc. Memorandum from A. Waggoner through J. Kough 

to M. Mendelsohn, dated May 25, 2010. US Environmental Protection Agency, Washington, D.C.

US EPA (2010b) Review of Human Health Data in support for an Experimental Use Permit for Field 

Testing of Event 5307 corn [EPA Reg. No. 67979-EUP-I] expressing Bacillus thuringiensis (Bt)

eCry3.1Ab protein and a Petition for Temporary Tolerance Exemption from the Requirement of a 

Tolerance for eCry3.1Ab protein; submitted by Syngenta Seeds, Inc. Memorandum from A. 

Waggoner through J. Kough to M. Mendelsohn, dated March 12, 2010. US Environmental 

Protection Agency, Washington, D.C.

Walters FS, deFontes CM, Hart H, Warren GW, Chen JS. (2010) Lepidopteran-active variable-region 

sequence imparts coleopteran activity in eCry3.1Ab, an engineered Bacillus thuringiensis hybrid 

insecticidal protein. Appl Environ Microb 76:3082-3088.
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C. Environmental Risk Assessment for eCry3.1Ab as Expressed in Event 5307 Corn 

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 costc. 

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 nontarget 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.

 

c 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. 
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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)d. 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 strain-improved, 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)e. Tier I tests 

serve to identify potential hazards and are conducted in the laboratory at high dose levels which increase 

the statistical power to test the hypotheses. Elevated test doses, therefore, add certainty to the 

assessment, and such tests can be well standardized. The Guidelines call for initial screening testing of a 

single group or several groups of test animals at the maximum hazard dose level. The Guidelines call for 

testing of one treatment group of at least 30 animals or three groups of 10 test animals at the screening 

test concentration. The Guidelines further state that the duration of all Tier I tests should be 

approximately 30 days. 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.f A less than 50% mortality

effect at the MHD is taken to indicate minimal risk. However, greater than 50% mortality does not 

necessarily indicate the existence of unacceptable risk in the field, but it does trigger the need to collect 

additional dose-response information and a refinement of the exposure estimation before deciding if the 

risk is acceptable or unacceptable. Where potential hazards are detected in Tier I testing (i.e. mortality is 

greater than 50%), additional information at lower test doses is required which can serve to confirm 

whether any effect might still be detected at more realistic field [1X EEC] concentrations and routes of 

exposureg. 

 

d OPPTS Testing Guidelines, Series 850 and 885 website: 

http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/885Microbial_Pesticide_Test_Guidelines/Series

e 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.

f

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. 

g 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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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 shows less than 50 % mortality at the maximum hazard dosage level – (i.e. LC50, ED50, or 

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

testing unnecessary. It is also notable that the recommended >10X EEC maximum hazard dose level is 

a highly conservative factor. The published EPA Level of Concern [LOC] is 50% mortality at 5X EEC h

(USEPA 1998). 

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 (USEPA 

2000, 2001, 2002, and 2004). 

Chronic studies: Delayed adverse effects and/or accumulation of toxins through the food chain are not 

expected to result from exposure to Bt PIP proteins, and these protein toxins are not routinely tested for 

chronic effects on non-target organisms. However, the 30 day test duration requirement does amount to 

subchronic testing when performed at field exposure test doses. Proteins also do not bioaccumulate. The 

biological nature of proteins makes them readily susceptible to metabolic, microbial, and abiotic 

degradation once they are ingested or excreted into the environment. Although there are reports that 

 

h 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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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. 

The 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. However, if long range adverse 

effects must be ascertained, then higher-tier longer-term field testing will be required. As noted above, 

the October 2000 SAP and the National Academy of Sciences (NAS 2000) recommended testing nontarget organisms directly in the field. This approach, with an emphasis on testing invertebrates found in 

corn fields, was also recommended by the August 2002 SAP and was supported by several public 

comments. Based on these recommendations, the Agency has required field studies on long term 

invertebrate population/community and 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-analysisiof the data collected from 42 field studies indicated that nontarget 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 

large-scale 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. 

 

i

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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1. Environmental 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 in which the protein(s) will be 

expressed. Following SAP recommendations, the EPA determined that non-target organisms with the 

greatest exposure potential to Cry protein in transgenic cotton fields are beneficial insects that feed on 

cotton pollen and nectar, particularly lepidopteran insects, and soil invertebrates. While EPA’s risk 

assessments of Bt cotton 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 on adverse effects at the field exposure rates, which are typically

based on protein expression levels within the plant for PIPs. Although it is recommended that nontarget 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 

eCry3.1Ab expressed in Event 5307 corn, the test material dose levels were based on an estimated 

concentration of eCry3.1Ab protein expressed in the tissue(s) with the highest concentration based on 

protein expression studies (MRID 44842509). 

2. Ecological Effects Data for eCry3.1Ab Expressed in Event 5307 Corn

In the absence of PIP-specific risk assessment guidance, 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 on representative organisms from several taxa 

was performed in support of the eCry3.1Ab Section 3 FIFRA registrations. As stated above, BPPD’s risk 

assessments focus more greatly on beneficial nontarget invertebrates, since they are most closely related to 

organisms susceptible to the insecticidal action of Bt toxins. The eCry3.1Ab protein is meant to target 

species within the order Coleoptera (e.g., beetles). 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. Earthworm studies are also recommended. The toxicity of the eCry3.1Ab protein has been evaluated 

on several species of invertebrates including the lady beetle (Coleomegilla maculata), carabid beetle 

(Poecilus cupreus), rove beetle (Aleochara bilineata), predatory bug (Orius laevigatus), an aquatic 

invertebrate shredder species (Gammarus fasicatus), honey bee (Apis mellifera), and earthworm (Eisenia 

fetida). Reproductive and/or developmental observations were also examined in the lady beetle, rove 

beetle, carabid beetle, predatory bug, and honey bee studies. 
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Since exposure may also occur to other nontarget organisms, EPA has received data to comply with the 

Agency’s published data requirements on other nontarget organisms. A study to determine soil 

degradation of eCry3.1Ab has also been submitted. The individual results for nontarget organism and 

soil degradation testing for eCry3.1Ab are summarized in Table 1. The studies are described in more 

detail below, and full reviews of each study 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 cotton plant tissue and where large amounts of Cry protein are needed for maximum 

hazard dose testing. In support of the Event 5307 registrations, test substances used in the submitted 

studies included bacteria-produced purified eCry3.1Ab protein as well as plant tissue (grain) from Event 

5307 corn. Comparative analyses that were previously submitted for the EUP showed the equivalence of 

the bacterially-produced and purified eCry3.1Ab and the eCry3.1Ab proteins produced in Event 5307 

corn. 

Table 6. Summary of environmental effects studies and scientific rationales for eCry3.1Ab as expressed in

Event 5307 corn submitted to comply with data requirements published in 40 CFR § 158.2150.

Data 

Requirement 

OPPTS

Guideline

Test Substance Results Summary and Classification MRID 

No. 

Avian dietary 

testing, 

broiler chicken, 

Gallus 

domesticus 

885.4050 Event 5307 corn

grain 

A 49-day dietary study showed no adverse

effects to broiler chickens when fed starter, 

grower, and finisher diets composed of 54%, 

58%, and 63% Event 5307 corn containing 

eCry3.1Ab, respectively. The amount of protein 

contained in the diets was lower than expected.

Classification: Supplemental

48442527

Avian 

inhalation 

testing

885.4100 N/A Not required. Avian oral testing indicates that 

eCry3.1Ab is not toxic to birds.

N/A

Avian oral 

testing

850.2100 Microbiallyproduced 

eCry3.1Ab

No adverse effects were observed in Northern 

bobwhite (Colinus virginianus) exposed to an 

acute oral dose of 900 mg eCry3.1Ab/kg-bw

after 14 days of observation. The oral LD50 is 

>900 mg eCry3.1Ab/kg-bw.

Classification: Acceptable

48442529

Wild mammal 

testing

885.4150 N/A Not required. Adverse effects were not observed 

in an acute oral toxicity study in which mice 

were dosed with eCry3.1Ab at 2000 mg/kg body 

weight. This laboratory study is sufficient for 

use in determining risk to wild mammals.

47753901

Freshwater fish 

testing, Channel 

catfish

885.4200 Event 5307 corn

grain

Consumption of Event 5307 grain at the 

exposure level tested (41% of the diet) had no 

adverse effects on survival, body weight, and 

feed:weight gain ratio of channel catfish 

(Ictalurus punctatus) after 28 days of exposure.

Classification: Acceptable

48442531

Freshwater 885.4240 Lyophilized No adverse effects on survival or reproduction 48442533
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Data 

Requirement 

OPPTS

Guideline

Test Substance Results Summary and Classification MRID 

No. 

invertebrate 

testing, 

Gammarus 

fasicatus

Event 5307 corn 

leaves, V6-V8 

stage

of Gammarus fasicatus exposed to Event 5307 

corn leaf material for 5 days. 

Classification: Acceptable

Estuarine and 

marine animal 

testing 

885.4280 N/A Not required. Exposure in marine and/or 

estuarine environments is not expected to be 

significant.

N/A

Non-target plant 

testing

885.4300 N/A Not required. Bt and its proteins are not known 

plant pathogens or toxins, and adverse effects in 

plants are not anticipated. Exposure to nontarget 

plants is expected to be minimal.

N/A

Non-target 

insect testing, 

carabid beetle, 

Poecilus 

cupreus

885.4340 Microbiallyproduced

eCry3.1Ab

No adverse effects on survival were observed in 

carabid beetle larvae fed 400 μg eCry3.1Ab/g 

diet. Emerged adults fed eCry3.1Ab had 

significantly lower body weight than controls. 

The LC50 was >400 μg eCry3.1Ab/g diet. 

Classification: Acceptable

48442539

Non-target 

insect testing, 

lady beetle, 

Coleomegilla 

maculata

885.4340 Microbiallyproduced 

eCry3.1Ab or 

Event 5307 

maize pollen

No adverse effects on survival, development 

time, or adult weight in C. maculata fed 500 μg 

eCry3.1Ab/g diet (nominal) for 21 days. Results 

of groups fed pollen are inconclusive. The LC50

is >500 μg eCry3.1Ab/g diet.

Classification: Acceptable

48442532

Non-target 

insect testing, 

rove beetle, 

Aleochara 

bilineata

885.4340 Microbiallyproduced 

eCry3.1Ab

No adverse effects on adult survival or number 

of F1 progeny were observed in adult A. 

bilineata fed 400 μg eCry3.1Ab/g diet (nominal) 

for 35 days. The LC50 is >400 μg eCry3.1Ab/g 

diet

Classification: Acceptable

48442536

Non-target 

insect testing, 

predatory bug, 

Orius

laevigatus

885.4340 Microbiallyproduced 

eCry3.1Ab

No adverse effects on survival of nymphs fed 

400 μg eCry3.1Ab/g diet (nominal) until 

adulthood in a 12-day laboratory study. The 

LC50 is >400 μg eCry3.1Ab/g diet

Classification: Acceptable

48442537

Honeybee 

testing, adults

Apis mellifera

885.4380 Microbiallyproduced 

eCry3.1Ab

No adverse effects on survival or brood 

development were observed when honey bee 

adults fed on a sucrose solution of 50 μg 

eCry3.1Ab/g for 5 days in a 21-day semi-field 

bioassay.

Classification: Acceptable

48442534

Honeybee 

testing, adults

Apis mellifera

885.4380 Microbiallyproduced 

eCry3.1Ab

No adverse effects on brood development were 

observed when honey bee adults fed on a 

sucrose solution of 50 μg eCry3.1Ab/g for 5 

days in a 24-day semi-field bioassay. Results for 

cumulative mortality in adults are uncertain due 

to an uncontrolled factor in the study.

Classification: Supplemental

48442538
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Data 

Requirement 

OPPTS

Guideline

Test Substance Results Summary and Classification MRID 

No. 

Earthworm 

toxicity, 

Eisenia fetida

850.6200 Microbiallyproduced 

eCry3.1Ab

No adverse effects on survival or body weight 

were observed in earthworms fed 50 μg 

eCry3.1Ab/g of soil dry weight after 14 days. 

The LC50 was determined to be > 50 μg 

eCry3.1Ab/g of soil and the NOAEC was 50 μg 

eCry3.1Ab/g of soil.

Classification: Acceptable

48442530

Soil fate and 

degradation

885.5200 Microbiallyproduced 

eCry3.1Ab

Based on the measured bioactivity in Colorado 

potato beetle (Leptinotarsa decemlineata), 

eCry3.1Ab was shown to lose approximately 

half of its insecticidal activity between 7 and 14 

days. Little activity was apparent after 14 days; 

eCry3.1Ab is not likely to persist in soil. 

Classification: Supplemental

48442535

a. Non-target Organism Study Summaries for eCry3.1Ab

 1. Avian Wildlife

Avian Dietary Toxicity

Heritage broiler chicks (Gallus domesticus) were fed diets prepared from either Event 5307 corn grain 

(expressing eCry3.1Ab protein), a near-isoline nontransgenic corn grain, or a commercially-available 

North Carolina corn grain (NCSU 2007). Feeding continued for 49 days, through provision of starter, 

grower, and finisher diets, to evaluate if diet prepared with Event 5307 corn grain caused adverse effects 

on survival, body weight, feed consumption, feed conversion, or carcass yield. The diets were 

standardized to a similar nutrient content, with the corn content of the diets ranging from 52% to 64% by 

weight. Based on a separate diet analysis (MRID 48442528, which was determined to be acceptable), 

the starter, grower, and finisher 5307 diet contained a mean of 2.13, 0.34, and 0.38 μg eCry3.1Ab/g diet, 

respectively, which is less than the amount expected. At test end, there were no treatment-related 

statistically significant differences in mortality, body weight, feed consumption, feed conversion, or 

carcass yield among birds fed the 5307, nontransgenic, or NCSU 2007 corn grain diets. This study

indicates that relatively long-term dietary exposure to eCry3.1Ab at these levels is not expected to result 

in adverse effects in birds; however, the exposure levels are very low. MRID 48442527.

Avian Oral Toxicity 

A laboratory study was conducted to determine the acute toxicity of a single oral dose of eCry3.1Ab 

protein to Northern bobwhite (Colinus virginianus). Birds received either a nominal dose of 900 mg 

eCry3.1Ab/kg body weight via a gelatin capsule or an empty gelatin capsule only (control group). The 

birds were observed for 14 days, and body weight and feed consumption were determined. There were 

no mortalities, and all birds appeared normal in appearance and behavior during the test. There were no 
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apparent treatment-related effects on body weight or feed consumption. The acute oral LD50 was >900 

mg a.i./kg. MRID 48442529.

Avian Inhalation Toxicity

Avian inhalation toxicity testing is typically not required for Bt corn event registrations, since exposure 

to Cry proteins via inhalation is not expected. Avian inhalation testing is not required for eCry3.1Ab.

 2. Wild mammals

An acute oral toxicity study with mice (MRID 47753901) has been submitted to EPA in support of the 

Event 5307 experimental use permit. In this study, mice were dosed with microbially produced 

eCry3.1Ab at 2000 mg/kg body weight. Treatment related mortalities and clinical signs of treatmentrelated effects were not observed during the study or at necropsy. This study has been determined to be 

acceptable. There is no indication that laboratory species would not be representative of wild mammals 

in this case; therefore, this information is suitable for use in assessment of risk to wild mammals, and no

additional testing with wild species is necessary. 

 3. Aquatic species

Freshwater animals

A laboratory study was conducted to determine the effects of consumption of grain from Event 5307 

maize on survival, body weight and feed:weight gain ratio of channel catfish (Ictalurus punctatus). Grain 

was incorporated into diet at 41% and fed to 20 juvenile channel catfish for 28 days; control fish were fed 

a diet containing near-isogenic corn grain incorporated at the same percentage. Fish were fed an amount 

of diet daily that was approximately equal to 8% of their total initial biomass for 14 days. The fish were 

then weighed and the amount fed was adjusted to 8% of the day 14 total biomass for the remainder of the 

study. No mortalities or physical or behavioral abnormalities were observed among the treatment or 

control fish during the 28-day exposure. At test termination, no significant differences were observed in 

total biomass, weight gain, or feed:weight gain ratio. Based on the results, effects to freshwater fish are 

not expected at the level of exposure tested. MRID 44842531.

Because of concerns raised in a study with the leaf shredding (caddis fly) trichopteran, Lepidostoma liba

(Rosi-Marshall, et al. 2007), EPA is requiring registrants of Bt corn and cotton to submit freshwater 

invertebrate toxicity studies to reduce uncertainty regarding this issue. A study with a leaf shredding 

freshwater isopod, Gammarus fasciatus, was submitted to determine the effects of ingestion of Event 

5307 leaf material collected from corn plants at the V6-V8 growth stage (based on information in MRID 

48802603) on the survival of adults. Percent survival of gammarids at test termination after five days 

was 88% for insects receiving Event 5307 leaf material and 80% for controls receiving near isoline leaf 

material. Consumption of the leaf material also did not differ between these two groups. Based on the 

results, consumption of Event 5307 leaf material is not expected to have adverse effects on freshwater 

invertebrates. MRID 48442533.
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Estuarine and marine animals

No significant direct exposures of estuarine and marine animals to eCry3.1Ab are anticipated as a result 

of agricultural production of corn containing eCry3.1Ab. Studies with estuarine and/or marine fish and 

invertebrates are not required for eCry3.1Ab. 

 4. Terrestrial and aquatic plant species

The active ingredient, eCry3.1Ab is part of a larger group of insect toxins produced a naturally-occuring 

Bacillus thuringiensis (Bt -endotoxins) that have not shown toxicity to plants. Effects to nontarget 

plants in the terrestrial and aquatic environments are not expected and testing is not required. 

 5. Invertebrate species 

A laboratory toxicity study was performed with larvae of a carabid ground beetle, Poecilus cupreus. 

Beetle larvae (n=40 per treatment) were fed an artificial meat-based diet containing either 400 μg 

eCry3.1Ab protein/g diet, water (negative control), or teflubenzuron (a chitin synthesis inhibitor, 

positive control) for 59 days. The exposure level tested is approximately 10 times the average expression 

rate of eCry3.1Ab in leaves of Event 5307 corn plants. Overall mortality was 10% in the control, 8% in 

the eCry3.1Ab treatment, and 100% in the positive control. Corrected mortality did not differ 

significantly between the groups receiving eCry3.1Ab and the water control; however, the mean weights 

of emerged adults in the control and eCry3.1Ab treatments were 65.7 mg and 52.7 mg, respectively, and 

these differed significantly. Development time was slightly longer for beetles receiving eCry3.1Ab, 

though this was not statistically significant. Because of the significant difference in adult body weight, 

this study indicates adverse sublethal effects at ~8X EEC. MRID 48442539.

Another laboratory study was conducted to determine the dietary toxicity of microbially produced 

eCry3.1Ab and Event 5307 corn pollen (which expresses eCry3.1Ab) to the predatory ladybird beetle 

(Coleomegilla maculata). Groups of C. maculata larvae were provided diet containing 500 μg 

eCry3.1Ab/g diet for 21 days. The test also included a group fed Event 5307 corn pollen, an assay

control group (diet only), a positive control group (potassium arsenate), and a non-transgenic corn pollen 

control group. Compared to the assay control group, the weight of adults in the eCry3.1Ab group was 

significantly higher (~20%), and development time (days to pupation, days to adulthood) was 

significantly shorter, but these differences were not considered to be adverse effects. Results with the 

test groups fed pollen indicate a possible problem with this portion of the study; however, the results of 

the experiment with the microbially-produced eCry3.1Ab are sufficient to show that eCry3.1Ab does not 

have adverse effects on this insect species at the exposure level tested. The LC50 for this species is >500 

μg eCry3.1Ab/g diet. MRID 48442532.

A laboratory study was conducted with a third Coleopteran species, the rove beetle (Aleochara 

bilineata). The purpose of the study was to assess the effects of eCry3.1Ab protein on the survival and 

reproduction of adult beetles. Adults of this beetle are predominantly entomophagous and their larvae 
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are endoparasites of fly pupae. In this study, eCry3.1Ab was incorporated into an artificial meat-based 

diet at 400 μg eCry3.1Ab/g diet. A negative control group received diet treated with water, and a 

positive control group received diet treated with teflubenzuron (a chitin synthesis inhibitor). Adults were 

fed their respective diet for five weeks, during which survival was observed. Afterward, observations 

were made on the number of F1 progeny that emerged from fly pupae placed in the test arenas. Mortality

of adults after 35 days was 17.5% in the controls and 20% in the eCry3.1Ab groups, and did not differ 

significantly. No difference in the number of F1 progeny produced was observed between these groups. 

Mortality was only 18.8% in the positive control group; however, no offspring were produced in this 

group, which was expected. The study indicates that at the level tested, eCry3.1Ab does not produce 

adverse effects. The LC50 for this species is >400 μg eCry3.1Ab/g diet. MRID 48442536. 

An additional study was conducted with the predatory bug, Orius laevigatus. Nymphs were provided an 

artificial meat-based diet containing a nominal concentration of 400 μg eCry3.1Ab/g diet. An untreated 

control diet and a toxic reference diet containing teflubenzuron were also included in the test. All 

nymphs developed to adults or died by test day 12. Pre-imaginal mortality was 0% and 5% in the control 

and test groups, respectively, which was not significantly different. Mortality was 100% in the toxic 

reference group. Based on the results, eCry3.1Ab had no adverse effects on O. laevigatus when provided 

at a rate of 400 μg/g diet. The LC50 for this species is >400 μg eCry3.1Ab/g diet. MRID 48442537.

A semi-field study with honey bee (Apis mellifera) was also conducted to determine the effects of 

eCry3.1Ab on adults and on brood development. Adults were fed a sucrose solution containing 50 μg 

eCry3.1Ab/g for 5 days. A sucrose solution-only assay control and a sucrose solution containing

fenoxycarb as a positive control were also included. Mortality was observed for eight days prior to 

feeding. The test diets were provided to the bees daily within the hive for five days and then 

observations of mortality and brood development were made up to 22 days after initial feeding. 

Mortality was monitored with a dead bee trap placed on the hive, and flight intensity (the number of 

worker bees entering and leaving the hive) was also observed. Brood development was monitored after 

the initial feeding day and assessed using a brood development index based on expected and observed 

development stage at the time of observation (brood index). An additional development index 

(compensation index) was calculated to account for eggs or larvae that were placed in cells as a result of 

a previous mortality. Mortality and development indices did not differ significantly between the negative 

control group and the group exposed to eCry3.1Ab. Significant mortality and brood losses were 

observed in the positive control compared to the negative control. Based on the results adverse effects 

on honey bee adults and brood development are not expected when hives are exposed to eCry3.1Ab at 

50 μg eCry3.1Ab under these conditions. MRID 44842534.

The effects of eCry3.1Ab on honey bees was also observed in another semi-field study in which bees 

were similarly exposed to 50 μg eCry3.1Ab/g in a sucrose solution for five days. A negative control and 

a positive control containing diflubenzuron were also included. The endpoints evaluated were: a) the 

pre-imaginal mortality of pre-marked broods; b) the number of dead adult bees, larvae and pupae 

expelled from the hive; and c) an assessment of whether the treatments affected the relative proportions 

of brood and food cells within the hives over the duration of the test (24 days). For cells that contained 

eggs at the time of initial feeding, mortality was 25.5% in the (negative) control, compared with 38.2% 
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(17.0% corrected mortality) in the eCry3.1Ab treatment group and 99.8% in the toxic reference 

treatment group. For cells that contained larvae, mortality was mortality was 16.8% in the control group, 

compared with 2.3% (0.0% corrected mortality) in the eCry3.1Ab treatment group and 100% in the toxic 

reference treatment group. Mortality between the negative control and eCry3.1Ab treated groups was 

not significant. The numbers of dead bees and brood deposited in the dead bee traps during the treatment 

and post-treatment periods were 22.4, 12.0 and 25 .3 per hive for the control, eCry3.1Ab and toxic 

reference treatments, respectively. The study authors noted that wasps were taking cadavers out of the 

bee traps, which may have affected the results. However, the study reliably indicates that overall hive 

condition and brood development are not affected by eCry3.1Ab at the level tested under the exposure 

conditions. MRID 48442538.

Toxicity to a non-arthropod invertebrate was also examined in a study to determine the toxicity of 

eCry3.1Ab to the earthworm (Eisenia fetida). Artificial soil was treated at a rate of 50 μg eCry3.1Ab/g 

of soil dry weight, which was intended to represent approximately 10X the average expression rate of 

eCry3.1Ab in roots of Event 5307 plants. Control artificial soil was treated with purified water only. Ten 

adult earthworms were placed on the soil surface in each jar, and the jars were maintained under 

controlled conditions for 14 days. At test end, none of the earthworms in either group had died, and all 

showed normal condition and behavior. The change in mean earthworm weight was -9.1% in the control 

group, and -7.2% in the test material group, and the difference between these was not statistically

significant. Worms likely lost weight because no food was included in the treatment. The no-observedeffect concentration in this study was 50 μg eCry3.1Ab/g soil dry weight. The presence and activity of 

eCry3.1Ab in the artificial soil used in the test were confirmed in a separate study. MRID 48442530.

b. Soil Fate Studies for eCry3.1Ab 

Soil organisms may be exposed to eCry3.1Ab through contact with corn roots (by direct feeding), plant 

root exudates, incorporation of above-ground plant tissues into soil following harvest, or by soildeposited pollen. Some evidence suggests that soils that are high in clays and humic acids are more 

likely to bind Cry protein. However, neutral pH soils tend to have high microbial activity and microbes 

contribute to Cry protein degradation. In addition, a study on the release of Cry proteins in the root 

exudates of Bt cotton has shown that no Cry proteins were detected immunologically or by larvicidal 

assay in any soil or hydroponic solution in which Bt cotton had been grown (Saxena and Stotzky, 2001). 

The weight of evidence indicates that Cry proteins do not accumulate in soil to arthropod-toxic levels. 

Because Cry proteins are toxins derived from soil-inhabiting bacteria, Bacillus thuringiensis, the 

degradation of eCry3.1Ab from Event 5307 corn is expected to be similar to that of these proteins that 

are produced naturally from soil bacteria. Nonetheless, BPPD required the following soil fate 

evaluations to support this registration. 

A bioassay system (first instar larvae of the Colorado potato beetle, CPB) was used to investigate the 

loss of insecticidal activity of eCry3.1Ab protein in soil over 120 days under laboratory conditions. The 

test soil, characterized as a loam, was collected from the top six inches of soil over an area of 

approximately four acres in Newtown, IN. The site had been used for growing corn over the three 

previous years, and was currently being used for soybeans. Soil samples were dosed with a 
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concentration of approximately 3.125 mg eCry3.1Ab/mL. Controls were dosed with 1 mL of the buffer. 

Additional incubation bottles, containing dry weight equivalents of soil, were designated for biomass 

measurements. All samples were mixed well, and maintained in an incubation chamber under aerobic 

conditions at a temperature of 15-30°C. Each incubation bottle was connected to hydration bottle and 

airflow system. The bottles were checked every one to two weeks, and moisture levels were adjusted 

with purified water to maintain proper hydration levels. Treated soils were incubated for 0, 1, 3, 7, 14, 

29, 60, 90, and 120 days (NOTE: the 90-day treated soils were dosed incorrectly, and results from the 

sample were not used). Untreated controls were incubated for 0, 60 and 120 days. Results from the 60 

and 120 day control incubations were also not available. Following incubation, the soil samples were 

incorporated into a commercially available CPB diet at a concentration of 10% (w/v). A total of 150 μL

of soil/diet mixture was fed to each of 24 larvae. The containers with larvae were sealed and in the dark 

at ambient temperature. Mortality readings were taken daily starting at 72 hours and continuing through 

120 hours. Corrected mortality was 72%, 63%, 48%, and 40% on Days 0, 1, 3, and 7, and then were -

4%, 3%, 1% and 0% for Days 14, 29, 60, and 120. The data are not sufficient to calculate a half-life. 

However, based on the data provided, the half-life of the eCry3.1Ab in the soil tested appears to be 

between 7 and 14 days. This is comparable to half lives observed with other Cry proteins (e.g., see 

MRID 48442526). The study design could be improved to provide a more robust estimate of soil half 

life, and a range of soil types should have been tested, including those with high clay and humic acid 

content, due to their known binding affinity for proteins. The data do show that eCry3.1Ab is not likely

to persist in loam soils. However, additional studies would be useful in evaluating insecticidal protein 

degradation, accumulation, and persistence in a variety of soil types and also for refining the estimate of 

the DT50 for eCry3.1Ab in soil.

Based on FIFRA Scientific Advisory Panel recommendations and public comments, the Agency has 

required three year soil fate studies for the currently registered Cry protein producing crops grown in a 

variety of soils and environmental conditions, as a condition of registration. , A comprehensive review 

of all available scientific data on ecological effects of commercially grown GM crops over ten years was 

published in 2007 (Sanvido, et al. 2007). The review concluded “none of the laboratory or field studies 

suggest accumulation of Bt-toxins in soil over several years of cultivation” and “experience from 

commercial cultivation indicates that Bt-toxin will not persist for long periods under natural conditions.” 

The Agency agrees with these conclusions. Collectively, the long-term field studies for Bt crops also 

confirm the previous SAP conclusion that “bioaccumulation is not expected to occur with transgenic 

proteins because biodegredation mechanisms for proteins are ubiquitous” (US EPA, 2000). More 

importantly, the numerous laboratory studies that demonstrated rapid protein degradation in soil of Bt 

proteins produced in Bt crops (when performed under realistic environmental conditions) can be 

considered predictive that Bt protein in soil is not likely to persist or accumulate in soil after continuous 

cultivation. 

In light of these published findings and the relatively rapid degradation of eCry3.1Ab protein in soil as 

demonstrated in the insect bioassay described above, there is no indication that the eCry3.1Ab protein 

expressed in Event 5307 corn is likely to persist in soil after continuous cultivation. Therefore, no 

additional long-term field studies are required for these PIP products.
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c. Supplemental Information

Syngenta submitted a summary of nontarget organism testing with eCry3.1Ab (MRID 44842526), which 

summarized the information above, and included an assessment of ecological risk and some discussion of 

environmental fate. These summaries were determined to be supplemental, and relevant information was 

utilized in this risk assessment. 

3. Environmental Risk Assessment for eCry3.1Ab 

a. Effects to Nontarget Wildlife, Invertebrates, and Plants

 1. Avian Wildlife

Birds are known to forage in corn fields, and may be exposed to eCry3.1Ab. An estimate of dietary

exposure for birds is provided in MRID 44842526, which is based on estimates of daily dietary dose 

based on consumption of Event 5307 grain and mean expression levels (fresh weight) in this plant tissue 

using a method developed by Crocker et al. (2002). The worst case exposure, based on 100% 

consumption of grain was estimated to be 1.94 mg eCry3.1Ab/kg bodyweight for birds with body

weights ranging from 22g (e.g., a sparrow) to 953g (e.g., a pheasant). Exposure through other foods is 

not well characterized, however, and to account for uncertainties related to their possible consumption, a

more conservative estimate of exposure may be determined using estimates of protein expression in 

tissues that express the highest amounts (e.g., leaves). Based on the method and mean fresh weight 

expression levels used in MRID 44842526, assuming 100% consumption of a diet containing material 

with an eCry3.1Ab concentration equivalent to the mean fresh weight expression level in leaves (51.74 

μg/g), a worst-case exposure would range from 5.69 to 18.11 mg eCry3.1Ab/kg bodyweight for birds 

with the same body weights. 

An avian oral toxicity study was submitted, wherein the LD50 for eCry3.1Ab was determined to be >900 

mg eCry3.1Ab/kg bodyweight. This exposure level is much higher than the maximum exposure 

estimates discussed above, and minimizes any uncertainties related to exposure in the field. Additional 

data provided in the study with broiler chickens shows that longer-term dietary exposure at low levels is 

unlikely to affect the parameters examined in that study. Based on these data, cultivation of Event 5307 

corn expressing eCry3.1Ab is not anticipated to cause adverse effects in birds.

 2. Wild mammals

Wild mammals also forage in corn fields and may be exposed to eCry3.1Ab. Syngenta provided an 

estimated maximum dose of 1.82 mg eCry3.1Ab/kg bodyweight for mammals, based on the same 

methods as described above for birds. Using these methods and the mean fresh weight expression in 

leaves as given above, a maximum exposure for mammals would be 17.07 mg eCry3.1Ab/kg 

bodyweight. 
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Toxicity testing with laboratory mice showed no adverse effects with exposure to eCry3.1Ab at a dose

of 2000 mg/kg bodyweight. This amount is much higher than concentrations that wild mammals are 

expected to encounter in the field; therefore, any uncertainties related to exposure are minimized by

testing at this level. Based on this information, adverse effects to wild mammals are not expected as a 

result of the registration and use of eCry3.1Ab as expressed in Event 5307 corn. 

3. Freshwater animals

BPPD previously determined that exposure to Cry proteins in the freshwater environment were expected 

to be low, based on the assumption that exposure in aquatic environments primarily resulted from pollen 

deposition. In light of published studies showing reduced growth in caddis flies exposed to antilepidopteran Cry1A protein (Rosi-Marshall et al. 2007), concerns were raised regarding the potential for 

exposure to shredder invertebrate species via consumption of corn plant litter in aquatic environments. 

EPA determined (see USEPA 2010a) that post-harvest crop residue is the most likely route of exposure 

to aquatic organisms (Carstens et al. 2011); however, their exposure to biotech crops is likely to be low, 

since the concentrations of Cry proteins in post-harvest crop tissues are limited temporally and spatially

(Swan et al. 2009, Jensen et al. 2010, Wolt and Peterson 2010, Carstens et al. 2011). Nonetheless, 

because of concerns that have been raised over this issue, EPA has required aquatic invertebrate testing.

Studies lasting 7-10 days with Daphnia spp. have typically been required. Studies with shredder species 

would test directly the invertebrate species expected to be affected by Bt corn plant litter that may enter 

streams. A study with a shredder species (Gammarus fasicatus) was submitted that showed that this 

species of isopod is not affected when exposed to Event 5307 corn leaves. The leaves were collected 

during a growth stage in which leaves express higher amounts of the eCry3.1Ab protein compared to 

other growth stages. Event 5307 corn leaves also express greater amounts of eCry3.1Ab compared to 

other tissues (based on MRID 48442509). Therefore, the isopods in this study were exposed to 

eCry3.1Ab at or near a maximum EEC. Additionally the study with channel catfish described above 

found no effects of eCry3.1Ab in this species when fed Event 5307 corn grain at 41% in the diet. Based 

on the results of this study, adverse effects to freshwater fish and invertebrates are not anticipated as a 

result of the registration and of eCry3.1Ab as expressed in Event 5307 corn.

4. Estuarine and marine animals

As discussed above, significant amounts of eCry3.1Ab use of Event 5307 corn are not expected to reach 

marine/estuarine areas. Therefore, BPPD concludes that adverse effects to fish and invertebrates in these 

environments as a result of the registration and cultivation of corn containing eCry3.1Ab are not expected

because exposure is expected to be minimal. 

 5. Terrestrial and aquatic plant species

The active ingredient, eCry3.1Ab is part of a larger group of insect toxins produced a naturally-occuring 

Bacillus thuringiensis (Bt -endotoxins) that have not shown toxicity to plants. Effects to nontarget 
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plants in the terrestrial and aquatic environments are not expected, and BPPD concludes that adverse 

effects to terrestrial and aquatic plants as a result of the registration and use of eCry3.1Ab in Event 5307 

corn are not anticipated.

 6. Invertebrate species 

Nontarget insects are expected to receive exposure primarily through consumption of Bt cotton pollen 

and/or nectar, though they may also consume pest insects that feed on cotton plant tissue or rarely other 

corn plant tissues themselves. The principal exposure route of soil-dwelling invertebrates, such as 

collembola, earthworms, and rove beetles, to eCry3.1Ab is assumed to be from decomposing plant tissue 

and plant exudates in soil. Studies submitted to EPA that examined the effects of eCry3.1Ab in lady 

beetle, rove beetle, predatory bug, and earthworm showed no adverse effects at concentrations well 

above those that would be encountered by nontarget insects in the environment, even if directly

consuming tissues with the highest levels of eCry3.1Ab protein expression. No mortality was observed 

in carabid beetles fed eCry3.1Ab at 400 μg eCry3.1Ab/g diet (~8X EEC); however, adult body weight 

was significantly reduced at this exposure level. Effects in this study are not necessarily unexpected, 

since eCry3.1Ab is a coleopteran-active protein. This sublethal effect could affect individual 

reproductive success and possibly population size for sensitive species, and additional testing to 

establish a sublethal endpoint (e.g., EC50 or NOAEC) would reduce any uncertainty related to its 

observation in this study. Since other studies with nontarget coleopteran species showed no adverse 

effects with exposure to eCry3.1Ab, and because exposure is expected to be much lower in the field, 

BPPD does not anticipate commercial cultivation of Event 5307 corn plants to cause significant effects 

to populations of nontarget insects and other invertebrates in the field, including coleopteran species. 

Two studies examining the effects of eCry3.1Ab on adult honey bees, honey bee brood development, 

and overall hive health showed no adverse effects to honey bees at exposure levels to eCry3.1Ab above 

those expected to be encountered in the environment. Therefore risk to honey bees as a result of the 

proposed registrations is also expected to be minimal. 

It should be noted that effects to invertebrates that are recognized as corn pests (i.e., insects that feed 

detrimentally on corn plants) that are not targeted by the eCry3.1Ab protein (but are controlled by other 

means) are not included in BPPD’s assessment of risk to nontarget insects. 

b. Effects on Soil Microorganisms

Numerous published studies indicate that exposure to Cry proteins 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 proteins. In addition, the soil degradation study

discussed above shows that the eCry3.1Ab degrades quickly in soil. 
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With regard to the impact of genetically engineered crops on soil, EPA has previously noted (USEPA 

2010a) 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. 

c. 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, or have their origin, in soil-inhabiting bacteria. Soil is also 

the habitat of other toxin-producing bacteria, and 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 toxin engineered into Event 5307 is derived from 

soil-inhabiting bacteria, the EPA has concluded that the risk of HGT of transgenes found in Event 5307 

corn expressing eCry3.1Ab is low.

d. Gene Flow and Weediness Potential 

The movement of transgenes from the host plant into weeds has been a significant concern for EPA due

to the possibility of novel exposures to the pesticidal substance. This concern has been considered for 

each of the Bt plant-incorporated protectants currently registered, and was extensively reexamined in 

2010 (see USEPA 2010a), and EPA believes that these concerns have been satisfactorily addressed. The 

Agency has determined that there is no significant risk of gene capture and expression of any Bt 

endotoxin by wild or weedy relatives of corn in the U.S., its possessions or territories. In addition, the 

USDA/APHIS has made this same determination under its statutory authority under the Plant Pest Act. 

The FIFRA EPA Scientific Advisory Panel meeting held on October 18-20, 2000, further discussed the 

matter of gene flow and offered some issues for consideration in this matter. The panel agreed that the 

potential for gene transfer between corn (maize) and any receptive plants within the U.S., its possessions 

and territories was of limited probability and nearly risk free. 

Based on these extensive analyses, the potential for gene flow and development of weediness is 

expected to be low for Event 5307 corn expressing eCry3.1Ab protein.

e. Conclusions

BPPD concludes that significant adverse effects on birds, mammals, nontarget insects, honey bees, 

freshwater and marine/estuarine fish and invertebrates, and terrestrial and aquatic plants are not expected 
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as a result of the use of Event 5307 corn expressing eCry3.1Ab. EPA has also determined that there is 

no significant risk of gene capture and expression of eCry3.1Ab protein by wild or weedy relatives of 

corn within the U.S. or its territories. Available data do not indicate that Cry proteins have any 

measurable adverse effect on microbial populations in the soil, nor has horizontal transfer of genes from 

transgenic plants to soil bacteria been demonstrated. 

f. Impacts on Endangered Species 

The primary route of exposure to eCry3.1Ab protein in corn is through ingestion of corn tissue. Since 

eCry3.1Ab protein is not expected to have adverse effects on mammals, birds, plants, freshwater and 

estuarine/marine fish and invertebrates, nontarget insects (see discussion below) and other invertebrate 

species at the Estimated Environmental Concentration (EEC), a “No Effect” determination is made for 

direct and indirect effects to federally listed threatened and endangered (“listed”) species. In addition, 

EPA does not expect that any threatened or endangered plant species will be affected by outcrossing to 

wild relatives or by competition with such entities. Hybrid corn does not exist in the wild, nor are there 

wild plants that can interbreed with corn in the United States.

Because of the selectivity of eCry3.1Ab protein for some coleopteran species, there exists some concern 

for listed species in the order Coleoptera. The potential risk to listed threatened or endangered 

coleopteran species has been previously addressed for Bt Cry proteins in corn (USEPA 2009, 2010b, 

2010c). At present, 18 coleopteran species are listed as threatened or endangered, and 16 of these occur 

in counties in which corn is grown. Eight of these species are subterranean, and exposure to Cry proteins 

expressed in plant tissues is not expected in these environments. The remaining species existing above 

ground, in terrestrial or aquatic habitats, are habitat specialists that are largely excluded from agricultural 

areas due to their specific habitat requirements. While habitats of some of these species may occur 

nearby to agricultural areas where Event 5307 corn may be cultivated, exposure via consumption of 

Event 5307 corn tissue is not anticipated due to their specific food requirements that would exclude 

consumption of corn plant material (i.e., they are predators/scavengers or consume specific food plants). 

BPPD also has previously determined that exposure to Cry proteins in pollen outside of areas where Bt 

corn is cultivated is limited (USEPA 2010a). Furthermore, the protein expression study for Event 5307 

corn (MRID 48442509), which is pending review, shows that very little eCry3.1Ab is expressed in 

Event 5307 pollen, so exposure to eCry3.1Ab resulting from pollen that lands on food items within or 

outside of areas where Event 5307 corn is grown is expected to be very low. 

As discussed in previous assessments of coleopteran-active Cry proteins, the American burying beetle 

(Nicrophorus americanus) is the only species that may potentially utilize areas where corn is grown. 

Exposure to this species has been discussed previously, and BPPD determined that this species is not 

expected to be exposed because it largely feeds on carrion (USEPA 2010c). 

Based on the available information, exposure of listed coleopteran species to eCry3.1Ab protein 

produced by Event 5307 corn is not expected. Therefore, a “No Effect” determination is made for direct 

effects to these species. Since effects on other insects or other animal or plant taxa are not anticipated, as 
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discussed above, a “No Effect” determination is also made for indirect effects to listed coleopterans and 

for effects to their critical habitat.

D. Environmental Risk Assessment for eCry3.1Ab, Cry1Ab, mCry3A, and Cry1F Expressed 

 Bt11 x MIR604 x TC1507 x 5307 Corn Hybrid 

1. Background 

Syngenta has applied for a FIFRA Section 3 Registration for a combined trait corn product Bt11 x

MIR604 x TC1507 x 5307 (EPA File Symbol 67979-EU) that confers lepidopteran and coleopteran 

insect resistance and herbicide tolerance (with the Event GA21 trait). The combined trait corn Bt11 x

MIR604 x TC1507 x 5307 product was developed by crosses (via traditional breeding methods) with 

each of the following individual PIP events: Bt11 corn (expressing Cry1Ab), MIR604 (expressing 

mCry3A), TC1507 (expressing Cry1F) and 5307 (expressing eCry3.1Ab). This registration is supported 

by acceptable data approved previously for the individual parental lines, and data for the eCry3.1Ab 

protein expressed in Event 5307.,. A synergism study involving the Cry1Ab, Vip3Aa20, and Cry1F 

proteins was previously submitted and reviewed by EPA (MRID No. 48235204; reviewed in USEPA

2011) . To support the current applications for these stacks, a synergism study was submitted to study

potential interactions in between mCry3A and eCry3.1Ab proteins. Another study was also submitted 

that tested the interaction of the combination of Cry1Ab, mCry3A, Vip3Aa20, Cry1F and eCry3.1Ab

proteins in susceptible lepidopteran and coleopteran species. These studies provide support for bridging 

to data for the individual PIP events that are already contained in the Agency’s database.

2. Environmental Assessment

The hazard of each of the individual events in Bt11 x MIR604 x TC1507 x 5307 has been addressed for 

previous registrations and for Event 5307 as discussed above. EPA has previously determined that the 

database for ecological risk assessment is complete for the Cry1Ab, Cry1F, and mCry3A proteins 

expressed in this hybrid, and also for eCry3.1Ab based on the assessment above. Synergism is a unique 

concern with the environmental risk assessment of stacked PIPs is the potential for synergistic effects 

that may result from interaction between the Cry proteins contained in the hybrid plants. Additionally, 

stacked hybrids are assessed for protein expression comparability with their individual parent lines to 

ensure that exposure is not significantly higher in the hybrid. Therefore, this assessment contains an 

evaluation of the potential for synergism in the Bt11 x MIR604 x TC1507 x 5307 corn hybrid and a 

discussion of protein expression comparability. 

a. Event Bt11 (lepidopteran active) Environmental Risk Assessment Summary

Potential adverse effects to non-target organisms by the Cry1Ab protein expressed by event Bt11 have 

been previously reviewed and was recently updated (USEPA 2010a). The following is a summary of 

the Bt11 environmental risk assessment. 
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The Cry1Ab protein expressed in Bt11 corn is intended to control lepidopteran pests. The hazard of the 

Cry1Ab protein was evaluated for the ladybird beetle, green lacewing, parasitic hymenoptera, 

collembolan, Daphnia spp., honey bee, earthworm, Monarch butterfly, and birds, mammals, fish, and 

non-target plants. Soil degradation/persistence studies were also evaluated. Additionally, gene flow 

and weediness assessments via pollen and Cry protein DNA uptake by plants and soil microorganisms 

previously have been performed and are discussed extensively in the update to the Cry1Ab BRAD 

(USEPA 2010a). This document also discusses field surveys of effects on nontarget insects and effects 

on nontarget lepidopterans that were conducted for several Bt Cry protein registrations in corn. Based 

on this information, EPA concluded that risk to non-target wildlife, aquatic, and soil organisms is not 

expected from Cry1Ab expressed in Event Bt11 corn. 

At present, the Agency is aware of no identified significant adverse effects of Cry1Ab on the abundance 

of non-target organisms in any population in the aquatic or terrestrial field environment. In addition, no 

direct or indirect effects on Federally listed endangered and threatened species or effects on their critical 

habitat are expected. Further, the EPA has determined that there is no significant risk of gene capture 

and expression of Cry1Ab protein by wild or weedy relatives of corn in the U.S., its possessions, or 

territories, available data do not indicate that Cry proteins have any measurable adverse effect on 

microbial populations in the soil, nor has horizontal transfer of genes from transgenic plants to soil 

bacteria been demonstrated. 

In conclusion, the risk assessment found no risk to the environment from cultivation of Event Bt11 corn 

expressing Cry1Ab. 

b. Event TC1507 (lepidopteran active) Environmental Risk Assessment Summary

Potential adverse effects to non-target organisms by Cry1F protein were reviewed and updated in the 

Cry1F BRAD, in 2010 (USEPA 2010a). The following is a summary of the Cry1F environmental risk 

assessment. EPA performed risk assessments on plants, wild mammals, birds, fish, aquatic 

invertebrates, earthworms, terrestrial non-target insects (including honey bee, parasitic wasps, green 

lacewings, ladybird beetle, springtails [Collembola toxicity/reproduction], and monarch butterflies), as 

well as field evaluations of the effects of Cry1F exposure on non-target insects in corn fields, soil 

degradation/persistence studies, and an endangered species impact assessment, particularly for 

Lepidoptera. In addition, gene flow and weediness assessments via pollen and Cry protein DNA 

uptake by plants were also performed. Cry1F protein in soil has been shown to degrade rapidly to very 

low levels. EPA concluded that there is sufficient information to believe that there is no risk from the 

uses of Cry1F corn to non-target wildlife, aquatic, and soil organisms. 

At present, the Agency is aware of no identified significant adverse effects of Cry1F protein on the 

abundance of non-target organisms in any population in the aquatic or terrestrial field environment. 

Field testing and field census data submitted to the Agency show minimal to undetectable changes in 

the beneficial insect abundance or diversity. To date the available field test data show that compared to 

crops treated with conventional chemical pesticides, the transgenic crops have no detrimental effect on 
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the abundance of non-target invertebrate populations. In addition, no direct or indirect effects on 

Federally listed endangered and threatened species or effects on their critical habitat are expected. The 

EPA has reviewed the potential for gene capture and expression of Cry1F protein by wild or weedy 

relatives of corn in the United States, its possessions or territories and has found that there is no 

significant risk in the United States, its possessions or territories (USEPA 2010a).

In conclusion, the risk assessment found no risk to the environment from cultivation of Event TC1507 

corn expressing Cry1F protein.

c. Event MIR604 (coleopteran active) Environmental Risk Assessment Summary

A risk assessment was performed for modified Cry3A (mCry3A) protein expressed in Event MIR604 

corn when it was originally registered, and the BRAD for this PIP was also updated in 2010 (USEPA 

2010b). EPA examined data on birds (Northern bobwhite, broiler chicken), mammals (from laboratory

studies submitted for toxicology review), freshwater fish (rainbow trout), lady beetle, carabid beetle, 

rove beetle, insidious flower bug, honey bee, and earthworm. Data were not required for 

estuarine/marine fish and invertebrates due to expected lack of exposure. Similarly, data were not 

required for freshwater invertebrates because mCry3A was not detectable in MIR604 corn pollen, and 

exposure was expected to be minimal in these environments. A soil degradation study was also 

submitted. Based on these data, EPA performed an assessment of the risk of mCry3A to nontarget 

organisms. In addition, gene flow and weediness assessments were performed when MIR604 was 

initially registered, which was extensively updated in 2010. Based on the data submitted, EPA 

concluded that adverse effects to nontarget organisms, including listed species, are not expected as a 

result of the cultivation of Event MIR604 corn plants expressing mCry3A protein. Based on 

recommendations from a Science Advisory Panel meeting that was convened to comment on EPA’s 

risk assessment and proposed findings for the mCry3A registration, EPA required a full-scale field 

study to examine long-range effects of cultivation of corn expressing mCry3A on invertebrate 

communities as a condition of registration (USEPA 2010b). This study has been submitted and a 

review is pending. 

At present, the Agency is aware of no identified significant adverse effects of mCry3A protein on the 

abundance of non-target organisms in any population in the aquatic or terrestrial field environment. In 

addition, no direct or indirect effects on Federally listed endangered and threatened species or effects 

on their critical habitat are expected. The EPA has reviewed the potential for gene capture and 

expression of mCry3A protein by wild or weedy relatives of corn in the United States, its possessions 

or territories and has found that there is no significant risk in the United States, its possessions or 

territories (USEPA 2010b).

In conclusion, the risk assessment found no risk to the environment from cultivation of Event MIR604 

corn expressing mCry3A protein.

d. Event 5307 (coleopteran active) Environmental Risk Assessment Summary
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The full risk assessment for eCry3.1Ab is provided above; a summary of this assessment is presented 

here. The eCry3.1Ab protein expressed in Event 5307 corn is a new active ingredient that has not been 

registered. Application for its registration has been made concurrently with that for this Bt11 x MIR604 

x TC1507 x 5307 corn hybrid. Syngenta has submitted studies with Northern bobwhite, broiler chicken, 

mammals (from laboratory studies), channel catfish, a Gammarus species (an aquatic shredder 

invertebrate), ladybird beetle, rove beetle, carabid beetle, predatory bug, honey bee, and earthworm to 

assess the effects to nontarget organisms. A soil persistence study was also submitted. Based on the data 

provided, BPPD found that no adverse effects to nontarget birds, mammals, fish, aquatic invertebrates, 

nontarget insects and honey bees, and nontarget plants were assessed are expected as a result of the 

cultivation of Event 5307 corn plants expressing eCry3.1Ab. Additionally, no direct or indirect effects 

on Federally listed endangered and threatened species or effects on their critical habitat are expected. 

The potential for gene capture and expression of Cry proteins has been extensively examined by EPA 

(USEPA 2010a). This analysis is relevant to the eCry3.1Ab protein as well, and it was determined that 

the risk of gene flow and development of weediness is low. 

In conclusion, the risk assessment found no risk to the environment from cultivation of Event 5307 corn 

expressing eCry3.1Ab protein.

e. Synergism Studies

The purpose of synergism studies is to characterize the potential for interaction between the Bt Cry 

proteins contained in combined trait corn event Bt11 x MIR604 x TC1507 x 5307. To bridge the 

ecological effects and environmental fate data of the individual parental events to the combined PIP 

products, the combined trait PIP product must be demonstrated as biochemically and functionally 

equivalent to their respective parental PIP events. The functional equivalence is established by 

demonstrating that the effects of the pesticidal mixture of the combined PIP product on a susceptible 

pest species are comparable to the effects of each protein tested individually. Interactions between the 

test materials can be assessed by comparing the larval mortality observed for the mixed proteins with the 

predicted responses based on the bioassay of each protein individually, via diet-incorporation sensitive 

insect bioassays. If there is no greater mortality than expected over the range of concentrations in a 

sensitive pest species, it is likely that there will be no synergism of the mixture against non-target 

organisms and the effect of a mixture of proteins on non-target organisms can be predicted from the 

effects of the individual proteins alone.

The hypothesis of no synergism of the insecticidal proteins when combined in Bt11 x MIR604 x 

TC1507 x 5307 was tested in three stages: 1) a test for synergism among the lepidopteran-active proteins 

(Cry1Ab and Cry1F), 2) a test for synergism among the coleopteran-active proteins (mCry3A and 

eCry3.1Ab), and 3) a test for synergism between the mixture Cry1Ab + Cry1F and the mixture mCry3A 

+ eCry3.1Ab. 

1. Bioassays for Interaction Between Cry1Ab and Cry1F
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Tests for synergism among Cry1Ab and Cry1F were previously conducted in two bioassays (MRID 

48235204): one using European corn borer (Ostrinia nubilalis, ECB), which is sensitive to Cry1Ab and 

Cry1F, and one using fall armyworm (Spodoptera frugiperda, FAW), which is sensitive to Cry1F only. 

Both bioassays also included an additional component, Vip3Aa20, which is present in Event MIR162 

corn, and is active against FAW but not ECB. Vip3Aa20 was included because interactions among 

Cry1Ab, Vip3Aa20, and Cry1F were also being investigated as part of the development of a different 

stacked hybrid corn. This study was previously reviewed and found to be acceptable by EPA (USEPA 

2011).

In each bioassay, freshly-hatched larvae were exposed to the proteins incorporated into commercial insect 

diet. The treatments consisted of various individual concentrations of microbially-produced Cry1Ab, 

Vip3Aa20, and Cry1F, and of mixtures of microbially-produced Cry1Ab + Vip3Aa20 + Cry1F. The 

protein concentrations used were chosen to give a range of predicted responses to the mixtures, based on 

preliminary tests and historical data. The presence of synergism or antagonism among the proteins was 

assessed by comparing the observed mortality of larvae on diets containing the protein mixtures with the 

predicted mortality based on bioassays using each protein individually. The predicted responses were 

calculated based on an assumption of independent action, using an extension of the Colby method 

(Colby, 1967) as described in the study report. 

Based on the results for ECB and FAW, there was no tendency for an excess or deficit in observed 

mortality in any test at any time, indicating that there is no synergism or antagonism among Cry1Ab, 

Vip3Aa20, and Cry1F.

2. Bioassays for Interaction Between mCry3A and eCry3.1Ab

Tests for interactions between coleopteran-active mCry3A and eCry3.1Ab insecticidal proteins were 

conducted with similar bioassays using the Colorado potato beetle (Leptinosara decemlineata, CPB) 

(MRID 48442909). Microbially-produced mCry3A and eCry3.1Ab protein test substances were used in a 

series of concentrations chosen to give a range of predicted responses to the combined proteins based on 

preliminary tests and historical data. The presence of synergism or antagonism among the proteins was 

assessed by comparing the observed mortality of larvae on diets containing the protein mixtures with the 

predicted mortality based on bioassays using each protein individually. As in the bioassays previously 

performed to assess the interaction of Cry1Ab, Vip3Aa20, and Cry1F (MRID 48235204, discussed 

above), the predicted responses were calculated based on an assumption of independent action using an 

extension of the Colby method (Colby 1967). This study was classified as acceptable.

The observed mortality results were lower than expected in the combined protein mixture bioassays in 

comparison to the predicted mortality calculations, which presumed an additive effect due to the 

independent action of mCry3A and eCry3.1Ab proteins. However, the results of the combined protein

mixtures were comparable to the observed mortality for the proteins when tested individually against 

CPB, which corroborates the hypothesis synergism is not expected between mCry3A and eCry3.1Ab 

insecticidal proteins when expressed in combination. The results also support the functional equivalence 

of the mCry3A and eCry3.1Ab insecticidal proteins expressed in combined PIP Bt11 x MIR604 x 
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MIR162 x TC1507 x 5307 corn product with its respective parental corn events MIR604 and 5307 corn.

Therefore, this combination of proteins is not likely to cause unexpected increased effects in nontarget 

organisms.

3. Bioassays for Interaction of Cry1Ab, Cry1F, mCry3A, and eCry3.1Ab

Diet incorporation feeding assays were conducted to determine whether a mixture of lepidopteran-active 

proteins (Cry1Ab and Cry1F, which also included Vip3Aa20) and a mixture of coleopteran-active 

proteins (mCry3A and eCry3.1Ab) interact when used in combination (MRID 48442908). A study had 

been previously conducted to determine the dose-response relationships of the lepidopteran-active and 

coleopteran-active proteins using ECB and CPB, and establish appropriate dosage levels for the 

interaction study (MRID 48202317). Using these dosage levels, an interaction study was performed in 

which ECB and CPB were exposed to a mixture containing all of the lepidopteran- and coleopteranactive proteins. The effect of the coleopteran-active mixture on the insecticidal activity of the 

lepidopteran-active mixture was determined using ECB assays. Two doses of the lepidopteran-active 

mixture (ECB 1 [lower dose] and ECB 2 [higher dose]) were used alone and in combination with the 

higher dose of the coleopteran-active mixture (CPB 2). Mortality results for the lepidopteran-active 

mixture alone were compared to those for the combined lepidopteran-active and coleopteran-active 

mixtures. The effect of the lepidopteran-active mixture on the insecticidal activity of the coleopteranactive mixture was determined using CPB assays. Two doses of the coleopteran-active mixture (CPB 1 

[lower dose] and CPB 2 [higher dose]) were used alone and in combination with the higher dose of the 

lepidopteran-active mixture (ECB 2). Mortality results for the coleopteran-active mixture alone were 

compared to those for the combined coleopteran-active and lepidopteran-active mixtures. For each 

interaction bioassay, F-tests were used to assess the statistical significance of the effects of dose, inactive 

ingredient, and dose x inactive ingredient interaction at the 5% probability level.

In the interaction studies with ECB, the ECB 2 dose produced higher mortality than the ECB 1 dose, as 

expected. There were no statistically significant differences in ECB mortality from addition of the 

coleopteran-active (ECB-inactive) ingredient (CPB 2) and no statistically significant dose x ECB-inactive 

ingredient interactions. Similarly, the CPB 2 dose produced higher mortality than the CPB 1 dose in 

CPB, as expected. There were no statistically significant differences in CPB mortality from addition of 

the lepidopteran-active (CPB-inactive) ingredient (ECB 2) and no statistically significant dose x CPBinactive ingredient interactions. 

The study reviewer noted that there was some increase in the activity in the CPB assays that was related 

to the high ECB dose, but it is not statistically significant. Based on the results of the study, the 

combination of these insecticidal proteins does not indicate potential for synergistic activity in the two 

sensitive insect species tested. The results also support the functional equivalence of the combined PIP 

Bt11 x MIR604 x TC1507 x 5307 corn product with its respective parental corn events Bt11, MIR604, 

TC1507 and 5307 corn, respectively. Therefore, the combination of these proteins is unlikely to produce 

unexpected effects in nontarget organisms as a result of the cultivation of Bt11 x MIR604 x TC1507 x

5307 corn plants.
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f. Protein Expression in Bt11 x MIR604 x TC1507 x 5307 Corn Plants

The expression of Cry1Ab, mCry3A, Cry1F, and eCry3.1Ab individually in single-trait Bt11, MIR604, 

TC1507, and 5307 plants, respectively, was compared with the expression of each of these proteins in 

the combined trait Bt11 x MIR604 x TC1507 x 5307 corn plants (MRID 48443001). This study is also 

discussed in the environmental risk assessment submitted by Syngenta for Bt11 x MIR604 x TC1507 x

5307 corn (MRID 48443004). The data show that several proteins are expressed at significantly higher 

amounts in some tissues of the stacked hybrid corn plants compared to the individual trait plants. In the 

hybrid, Cry1Ab is expressed at levels that are significantly higher in R1 leaves and in R1 whole plants, 

mCry3A expression is significantly higher in pollen (also see discussion below), Cry1F expression is 

significantly higher in V5 leaves, and eCry3.1Ab is significantly higher in V5 roots. EPA 

communicated concerns for these increased levels of expression in Bt11 x MIR604 x TC1507 x 5307

corn plants in a letter to Syngenta dated March 15, 2012, and asked Syngenta to provide further 

information to justify equivalence of protein expression. Syngenta provided a response (MRID 

48802701), which discussed the relevance of these increased levels of expression to margins of exposure 

for nontarget organisms and overall exposure during the life of the plant. 

In the letter, EPA also raised concern over the presence of mCry3A protein in pollen of the Bt11 x

MIR604 x TC1507 x 5307 stack. In the original risk assessment for this protein, mCry3A was not 

detectable in MIR604 corn pollen, and tests for effects of mCry3A in aquatic invertebrates were not 

required due to the expected lack of exposure. MRID 48802701 also contains Syngenta’s response for 

these concerns, which is based on a discussion of new detection methods that enhanced the sensitivity of 

detection assays below the previous level of quantitation. Syngenta additionally discussed the exposure 

of aquatic invertebrates, and concluded that the previous findings are still relevant to the risk assessment 

for Bt11 x MIR604 x TC1507 x 5307 corn.

The comparative protein expression study for Bt11 x MIR604 x TC1507 x 5307 corn and Syngenta’s 

response to the Agency’s concerns is currently under review, and conclusions of its biochemical 

equivalence to its respective parental events are to be provided in the product characterization memo for 

Bt11 x MIR604 x TC1507 x 5307 corn (USEPA 2012a). Provided that the expression study and 

supplemental information are found to be acceptable, a conclusion can be made that significant 

increased exposure of nontarget organisms to the Cry1Ab, mCry3A, Cry1F, and eCry3.1Ab proteins is 

not expected as a result of cultivation of Bt11 x MIR604 x TC1507 x 5307 corn plants. 

g. Risk Assessment Conclusions for Cry1Ab, mCry3A, Cry1F, and eCry3.1Ab as Expressed 

in Bt11 x MIR604 x TC1507 x 5307 Corn

EPA has concluded that the individual proteins expressed in Bt11 x MIR604 x TC1507 x 5307 corn will 

have no adverse effect on nontarget organisms, do not persist in the soil, and pose no risk of gene flow 

and development of weediness in wild relatives. Synergism studies submitted to EPA also show that 

increased toxicity is not expected from the combination of these proteins. While protein expression data 

are currently under review, a significant increase in nontarget exposure is not anticipated. Therefore, a 

conclusion is made that cultivation of Bt11 x MIR604 x TC1507 x 5307 corn will not result in adverse 
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effects to nontarget organisms or the environment, and additionally will have no effect, direct or 

indirect, on Federally listed threatened and endangered species or their designated Critical Habitats. This 

conclusion is dependent on a finding that the expression study for Bt11 x MIR604 x TC1507 x 5307 

corn and its associated supplemental information are found to be acceptable and to support the 

conclusion of no significant increase in exposure. 

E. Environmental Risk Assessment for Cry1Ab, Vip3Aa20, mCry3A, Cry1F, and eCry3.1Ab

 Expressed Bt11 x MIR162 x MIR604 x TC1507 x 5307 Corn Hybrid 

1. Background 

Syngenta has applied for a FIFRA Section 3 Registration for a combined trait corn product Bt11 x

MIR162 x MIR604 x TC1507 x 5307 (EPA File Symbol 67979-EG) that confers lepidopteran and 

coleopteran insect resistance and herbicide tolerance (with the Event GA21 trait). The combined trait 

corn Bt11 x MIR162 x MIR604 x TC1507 x 5307 product was developed by crosses (via traditional 

breeding methods) with each of the individual events. The data to support this registration are based on 

data submitted and approved previously for the individual parental lines, as well as the data for the 

eCry3.1Ab protein expressed in Event 5307, as discussed above. Synergism studies involving these 

proteins were also submitted. These studies provide support for bridging to data for the individual PIP 

events that are already contained in the Agency’s database.

2. Environmental Assessment

The hazard of each of the individual events in Bt11 x MIR162 x MIR604 x TC1507 x 5307 has been 

addressed for previous registrations. Event 5307 is a new active ingredient, and the risk assessment 

performed for this active ingredient also applies to this stack. EPA has previously determined that the 

database for ecological risk assessment is complete for the Cry1Ab, Vip3Aa20, Cry1F, and mCry3A 

proteins expressed in this hybrid, and also for eCry3.1Ab based on the assessment above. Summaries of 

the risk assessments for the individual Cry1Ab, mCry3A, Cry1F, and eCry3.1Ab are provided above in 

the risk assessment for the Bt11 x MIR604 x TC1507 x 5307 combined trait corn product. A summary

of the environmental assessment of Vip3Aa20 is provided below. This assessment also addresses the 

potential for synergism in the Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn hybrid, as well as 

protein expression comparability between the combined trait hybrid and corn plants expressing the 

individual traits only. 

a. Event MIR162 (lepidopteran active) Environmental Risk Assessment Summary

Potential adverse effects to non-target organisms by the Vip3Aa20 protein expressed by event MIR162 

have been previously reviewed and was recently updated (USEPA 2009). The following is a summary

of the MIR162 environmental risk assessment. 

The Vip3Aa20 protein expressed in MIR162 corn is a Bt-derived vegetative insecticidal protein 

intended to control lepidopteran pests; however, it is not active against all primary lepidopteran corn 
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pests. This protein is known to act individually to affect a typical midgut pathology in susceptible

insects like previously studied Bt delta-endotoxins (Lee, et al. 2003). Although the general symptoms

displayed by sensitive lepidopteran larvae following ingestion of Vip proteins resembles that caused by

Cry1Ab proteins (i.e., cessation of feeding, loss of gut peristalsis, overall paralysis of the insect, and 

death) (Yu, et al. 1997; and Lee et al. 2008), the Vip3Aa protein variants share no homology with

Cry1Ab and other known Cry proteins and the modes of action differ between the Vip and Cry

proteins. Therefore, while event MIR162 is active against a wide spectrum of lepidopteran pests, it is 

intended for use in creating combinations with other PIPs instead of commercial distribution in singletrait seed. The potential hazard of Vip3Aa20 protein was evaluated for Northern bobwhite, broiler 

chicken, mammals (based on acute oral toxicological laboratory studies on mice), channel catfish, 

Daphnia magna, insidious flower bug, lady beetle, green lacewing, rove beetle, collembolan, honey 

bee, and earthworm. In support of the Event MIR162 registration, test substances used in the submitted 

studies included bacterial-produced purified Vip3Aa19 and Vip3Aa1 protein, plant-expressed 

Vip3Aa20 protein as expressed in Event Pacha maize leaves, in addition to plant-expressed Vip3Aa20 

protein as expressed in Event MIR162 maize grain, pollen, and leaves. Since biochemical and 

functional equivalence was shown among Vip3Aa1, Vip3Aa19 and Vip3Aa20 proteins, some of the 

studies conducted with Vip3Aa1 and Vip3Aa19 were bridged to the assessment for Vip3Aa20. A soil 

degradation study was also submitted that allowed EPA to conclude that Vip3Aa20 is not likely to 

persist in soil. Additionally, gene flow and weediness assessments were performed, and the potential 

for these events has also been discussed extensively for Cry proteins in corn in updated Bt corn 

BRADs (e.g., see USEPA 2010). Based on this information, EPA concluded that risk to non-target 

wildlife, aquatic, and soil organisms is not expected from Vip3Aa20 expressed in Event MIR162 corn. 

At present, the Agency is aware of no identified significant adverse effects of Vip3Aa20 on the 

abundance of non-target organisms in any population in the aquatic or terrestrial field environment. In 

addition, no direct or indirect effects on Federally listed endangered and threatened species or effects on 

their critical habitat are expected. Further, the EPA has determined that there is no significant risk of 

gene capture and expression of Vip3Aa20 protein by wild or weedy relatives of corn in the U.S., its 

possessions, or territories, available data do not indicate that Cry proteins have any measurable adverse 

effect on microbial populations in the soil, nor has horizontal transfer of genes from transgenic plants to 

soil bacteria been demonstrated. 

In conclusion, the risk assessment found no risk to the environment from cultivation of Event MIR162 

corn expressing Vip3Aa20.

b. Synergism Studies 

The synergism studies discussed in the risk assessment of the Cry proteins expressed in the Bt11 x

MIR604 x TC1507 x 5307 hybrid above (MRIDs 48442908 and 48442909) are also applicable to the 

environmental risk assessment of Bt11 x MIR162 x MIR604 x TC1507 x 5307 hybrid corn because the 

Vip3Aa20 protein was also included in these studies. These studies showed no increase in toxicity to 

sensitive insect species as a result of the combination of the Cry1Ab, Vip3Aa20, mCry3A, Cry1F, and 

eCry3.1Ab proteins. The results also support the functional equivalence of the combined PIP Bt11 x 
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MIR604 x MIR162 x TC1507 x 5307 corn product with its respective parental corn events Bt11, 

MIR604, MIR162, TC1507 and 5307 corn, respectively. Therefore, increased toxicity is not expected in 

nontarget organisms as a result of the combination of these proteins in Bt11 x MIR162 x MIR604 x

TC1507 x 5307 hybrid corn. 

c. Protein Expression in Bt11 x MIR162 x MIR604 x TC1507 x 5307 Corn Plants

The expression of Cry1Ab, Vip3Aa20, mCry3A, Cry1F, and eCry3.1Ab individually in single-trait 

Bt11, MIR162, MIR604, TC1507, and 5307 corn plants, respectively, was compared with the expression 

of each of these proteins in the combined trait Bt11 x MIR604 x TC1507 x 5307 corn plants (MRID 

48442901). This study is also discussed in the environmental risk assessment submitted by Syngenta for 

Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn (MRID 48442904). The data show that Cry1Ab is 

expressed at levels that are significantly higher in R1 leaves and in R1 whole plants. EPA communicated 

concerns for these increased levels of expression in Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn 

plants in a letter to Syngenta dated March 15, 2012, and asked Syngenta to provide further information 

to justify equivalence of protein expression between the single and combined trait plants. Syngenta 

provided a response (MRID 48802801), which discussed the relevance of these increased levels of 

expression to margins of exposure for nontarget organisms and overall exposure during the life of the 

plant. 

In the letter, EPA also raised concern over the presence of mCry3A protein in pollen of the Bt11 x

MIR162 x MIR604 x TC1507 x 5307 stack. As discussed above, in the original risk assessment for this 

protein, mCry3A was not detectable in MIR604 corn pollen, and tests for effects of mCry3A in aquatic 

invertebrates were not required due to the expected lack of exposure. MRID 48802801 also contains 

Syngenta’s response for these concerns, which are identical to their justification and conclusion 

discussed above for Bt11 x MIR604 x TC1507 x 5307 corn.

The comparative protein expression study for Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn and 

Syngenta’s response to the Agency’s concerns is currently under review, and conclusions of its 

biochemical equivalence to its respective parental events are to be provided in the product 

characterization memo for Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn (USEPA 2012b). Provided 

that the expression study and supplemental information are found to be acceptable, a conclusion can be 

made that significant increased exposure of nontarget organisms to the Cry1Ab, Vip3Aa20, mCry3A, 

Cry1F, and eCry3.1Ab proteins is not expected as a result of cultivation of Bt11 x MIR162 x MIR604 x

TC1507 x 5307 corn plants. 

d. Risk Assessment Conclusions for Cry1Ab, Vip3Aa20, mCry3A, Cry1F, and

 eCry3.1Ab as Expressed in Bt11 x MIR162 x MIR604 x TC1507 x 5307 Corn

EPA has concluded that the individual proteins expressed in Bt11 x MIR162 x MIR604 x TC1507 x

5307 corn will have no adverse effect on nontarget organisms, do not persist in the soil, and pose no risk 

of gene flow and development of weediness in wild relatives. Synergism studies submitted to EPA also 

show that increased toxicity is not expected from the combination of these proteins. While data are 
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currently under review to show that these proteins are not expressed in Bt11 x MIR162 x MIR604 x

TC1507 x 5307 hybrid corn at levels that significantly increase nontarget exposure above levels of 

concern, a significant increase in nontarget exposure is not anticipated. Therefore, a conclusion is made 

that cultivation of Bt11 x MIR604 x TC1507 x 5307 corn will not result in adverse effects to nontarget 

organisms or the environment, and additionally will have No Effect, direct or indirect, on Federally

listed threatened and endangered species or their designated Critical Habitats. This conclusion is 

dependent on a finding that the expression study for Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn 

and its associated supplemental information are found to be acceptable and to support the conclusion of 

no significant increase in exposure. 

IV. References

Carstens, K.L., Anderson, J.A., Bachman, P., DeShrijver, A., Dively, G. Federici, B. Hamer, M., 

Gielkens, M., Jensen, P., Lamp, W., Raushen, S., Ridley, G., Romeis, J., Waggoner, A. 2011. 

Genetically modified crops and aquatic ecosystems: considerations for environmental risk 

assessment and non-target organism testing. Transgenic Res., published online 26 November 

2011.

Colby, S.R. 1967. Calculating synergistic and antagonistic responses of herbicide combinations. Weeds 

15:20-22.10.

Crocker, D., Hart, A., Gurney, J., McCoy, C. 2002. Project PN0908: Methods for estimating daily food 

intake of wild birds and mammals. Central Science Laboratory. Unpublished report to the (UK) 

Department for Environment, Food and Rural Affairs. Included as an appendix to US EPA 

MRID 46155615.

Jensen, P.D., Dively, G.P., Swan, C.M., Lamp, W.O. 2010. Exposure and non-target effects of 

transgenic Bt corn debris in streams. Environ. Entomol. 39(2):707-714.

Lee, M.K., Walters, F.S. , Hart, H., Palekar, N., Chen, J-S. 2003. The mode of action of the 

Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab δendotoxin. App. Environ. Micro. 69(8): 4648-4657.

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.

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

and Adequacy of Regulation is available from the National Academy Press, 2101 Constitution 

Avenue, N.W., Lockbox 285, Washington, DC 20055; (800) 624-6242 or (202) 334-3313 (in the 

Washington metropolitan area); http://www.nap.edu.

Romeis, J., Meissle, M., Bigler, F. 2006. Transgenic Crops expressing Bacillus 
Bacillus thuringiensis eCry3.1Ab Corn

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thuringiensis toxins and biological control. Nature Biotechnology 24: 63-71. 

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

N. A., Pokelsek, J., Stephen, M.L. 2007. Toxins in transgenic crop byproducts may affect 

headwater stream ecosystems. PNAS: 104(41): 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.

Saxena, D. and Stotzky, G. (2001) Bacillus thuringiensis (Bt) toxin released from root exudates and 

biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and 

fungi in soil. Soil Biol. Biochem. 33: 1225–1230. 

Swan, C.M., Jensen, P.D. , Dively, G.P., Lamp, W.O.. 2009. Processing of transgenic crop residues in 

stream ecosystems. J. App. Ecol. 46:1304-1313.

United States Environmental Protection Agency (USEPA) (1998). “Guidelines for Ecological Risk 

Assessment.” EPA 630/R-95-002F. Washington, DC, USA. [Federal Register, May 14, 1998. 

63(93): 26846-26924.]

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. Available at the EPA 

website: http://www.epa.gov/scipoly/sap/1999/index.htm#december

USEPA. 2001. SAP Report No. 2000-07. Sets of scientific issues being considered by the 

Environmental Protection Agency regarding: Bt plant-pesticides risk and benefit assessments. 

Dated March 12, 2001. Web site: http://www.epa.gov/scipoly/sap/2000/october/octoberfinal.pdf

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

Environmental Protection Agency regarding: Corn rootworm plant-incorporated protectant 

nontarget insect and insect resistance management issues. Dated November 6, 2002. 

http://www.epa.gov/scipoly/sap/2002/august/august2002final.pdf

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

ecological risk, and insect resistance management for Bt cotton products. Dated August 19, 2004. 

http://www.epa.gov/scipoly/sap/meetings/2004/june/final1a.pdf

USEPA, 2009. Biopesticides Registration Action Document – Bacillus thuringiensis Vip3Aa20 

Insecticidal Protein and the Genetic Material Necessary for Its Production (via Elements of 
Bacillus thuringiensis eCry3.1Ab Corn

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Vector pNOV1300) in Event MIR162 Maize (OECD Unique Identifier: SYN-IR162-4), 

http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006599.pdf. 

USEPA, 2010a. Biopesticides Registration Action Document – Cry1Ab and Cry1F Bacillus 

thuringiensis (Bt) Corn Plant-Incorporated Protectants, 

http://www.epa.gov/oppbppd1/biopesticides/pips/cry1f-cry1ab-brad.pdf.

USEPA, 2010b. Biopesticides Registration Action Document – Modified Cry3A Protein and the Genetic 

Material Necessary for its Production (Via Elements of PZM26) in Event MIR604 Corn SYNIR604-8, http://www.epa.gov/oppbppd1/biopesticides/pips/mcry3a-brad.pdf. 

USEPA, 2010c. Biopesticides Registration Action Document – Bacillus thuringiensis Cry34Ab1 and 

Cry35Ab1 Proteins and the Genetic Material Necessary for Their Production (PHP17662 TDNA) in Event DAS-59122-7 Corn (OECD Unique Identifier: DAS-59122-7),

http://www.epa.gov/oppbppd1/biopesticides/pips/cry3435ab1-brad.pdf. 

USEPA, 2011. Review of Molecular Characterization data, Test Substance Characterizations, Protein 

Expression Analyses and Synergism Study for Combination PIP Bt11 x MIR162 x TC1507 

corn, Agrisure TM 3100 [EPA Reg. No. 67979-RL, OECD Unique ID. SYN-BTØ11-1 x SYNIR162-4 x DAS-Ø15Ø7-1] in support for Sec. 3 Registration, submitted by Syngenta Seeds, Inc. 

– Field Crops- NAFTA. Memorandum from A. Waggoner, through J. Kough, to J. Kausch. 

Dated March 25, 2011. US Environmental Protection Agency, Washington, D.C.

USEPA, 2012a. Review of Molecular Characterization Data, Test Substance Characterizations and 

Protein Expression Analyses for Combination PIP Bt 11 x MIR604, TC1507 x 5307 corn [EPA 

Reg. No. 67979-EU; OECD Unique ID. SYN-BTØ11-1 x SYN-IR-604-5 x DAS-Ø15Ø7-1 x 

SYN-05037-1] in support for FIFRA Section 3 Registration, submitted by Syngenta Seeds, Inc. –

Field Crops- NAFTA. Memorandum from J. Facey, through J. Kough, to M. Mendelsohn. US 

Environmental Protection Agency, Washington, D.C.

USEPA, 2012b. Review of Molecular Characterization Data, Test Substance Characterizations, Protein 

Expression Analyses and Synergism Study for Combination PIP Bt11 x MIR162 x MIR604 x 

TC1507 x 5307 corn [EPA Reg. No. 67979-EG, OECD Unique ID. SYN-BTØ11-1 x SYNIR162-4 x SYN-IR-604-5 x DAS-Ø15Ø7-1 x SYN-05307-1] in support for Sec. 3 Registration, 

submitted by Syngenta Seeds, Inc. – Field Crops- NAFTA. Memorandum from A. Balen, 

through J. Kough, to M. Mendelsohn. US Environmental Protection Agency, Washington, D.C.

Wolt, J.D. and Peterson, R.K.D. 2010. Prospective formulation of environmental risk assessments: 

Probabilistic screening for Cry1A(b) maize risk to aquatic insects. Ecotoxicol. Environ. Safety

73: 1182-1188.
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Yu, C. Mullins, M.A., Warren, G.W., Koziel, M.G. and Estruch, J.J. (1997). The Bacillus

thuringiensis Vegetative Insecticidal Protein Vip3A Lyses Midgut Epithelium Cells of 

Susceptible Insects. Applied and Environmental Microbiology 63 (2): 532-53.

F. Insect Resistance Management (IRM)

Background

BPPDjhas reviewed the Insect Resistance Management (IRM) plan and supporting data submitted by

Syngenta for two proposed commercial Bt corn products containing the new active ingredient 

eCry3.1Ab (event 5307). The two products (EPA Reg No. 67979-EU and 67979-EG) are “stacked 

pyramids” consisting of eCry3.1Ab and previously-registered Bt corn toxins for control of lepidopteran 

stalk borers and corn rootworm. Separate IRM volumes were submitted for each proposed product: 

Bt11 x MIR604 x TC1507 x 5307 (MRID# 484430-08) and Bt11 x MIR162 x MIR604 x TC1507 x 

5307 (MRID# 484429-10). 

Conclusions and Recommendations

1) Syngenta has submitted adequate data and modeling to demonstrate that the 5307 pyramided products 

(Bt11 x MIR604 x TC1507 x 5307 and Bt11 x MIR162 x MIR604 x TC1507 x 5307) will have high 

durability with a 5% refuge in the Corn Belt (relative to single toxin products deployed with 20% 

refuge). There should be strong durability for both of the target pest complexes: lepidopteran stalk/ear 

borers (European corn borer, Southwestern corn borer, and corn earworm) and corn rootworm.

2) For southern regions in which cotton is also grown, Syngenta proposed a 20% refuge for Bt11 x 

MIR162 x MIR604 x TC1507 x 5307 (67979-EG) but a 50% refuge for Bt11 x MIR604 x TC1507 x 

5307 (67979-EU), presumably because the latter product has one fewer lepidopteran-active toxin. 

Larger refuge sizes in this region are driven by concerns with corn earworm (a corn and cotton pest). 

BPPD notes that a 20% refuge is compatible with the requirements for other similar Bt corn pyramids 

(including two Syngenta products). Syngenta may be able to support a 20% refuge for the Bt11 x 

MIR604 x TC1507 x 5307 pyramid with a relatively simple model (as was done to support the other 

registered pyramids).

3) Syngenta did not propose a seed blend with initial product registration for either of the two 5307 

pyramids. BPPD notes that additional information and modeling may be necessary to support such a 

refuge strategy.

4) Syngenta provided data to demonstrate that the risk of cross resistance between eCry3.1Ab and 

mCry3A (Syngenta’s other registered rootworm toxin) should be low. The company did not, however, 

provide an assessment of the cross resistance potential between eCry3.1Ab and other rootworm toxins 

 

j The use of BPPD in this chapter refers to the BPPD IRM Team consisting of Alan Reynolds and Jeannette Martinez
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not registered by Syngenta (i.e., Cry3Bb1 and Cry34/35Ab1). BPPD recognizes that eCry3.1Ab (an 

engineered toxin) should have significant structural differences with Cry3Bb1 and Cry34/35Ab1, and 

therefore, a low likelihood of cross resistance. But no information was provided in the submission to 

verify this assumption. Accordingly, BPPD recommends that Syngenta provide additional data or 

analysis to address the cross resistance potential of eCry3.1Ab with Cry3Bb1 and Cry35/35Ab1. As 

part of this effort, BPPD also recommends that Syngenta work to develop a eCry3.1Ab-resistant colony

(similar to the work done for mCry3A -- see Meihls et al. 2011). 

5) Existing programs for resistance monitoring (lepidopteran pests), remedial action, and compliance 

assurance should be compatible with the 5307 pyramids. Additional data and information, however, are 

recommended for corn rootworm resistance monitoring (see #6 below). BPPD recommends that 

Syngenta update and submit grower education materials (e.g., technology use guides) to include the 

5307 product line and the appropriate refuge requirements. In addition, Syngenta should submit annual 

IRM reports for resistance monitoring, compliance, and sales.

6) BPPD recommends that Syngenta submit the following additional data for corn rootworm resistance 

monitoring with the eCry3.1Ab toxin:

 Baseline susceptibility data;

 A proposal for a plant-based diagnostic assay including an investigation into the feasibility of 

using the “Sublethal Seedling Assay” (Nowatzki et al. 2008);

 A monitoring plan for northern corn rootworm (Diabrotica barberi);

 Corn rootworm damage guidelines (for unexpected pest damage investigations of eCry3.1Ab 

corn). 

Background

Syngenta Seed, Inc. has proposed to register three Bt corn products containing the new active ingredient 

event 5307, which expresses the eCry3A.1Ab toxin. Applications were submitted for registration of 

Event 5307 alone as a breeding intermediate and in two stacked/pyramided end products (referred to as 

the “5307” pyramids in this review). The proposed products are as follows:

 5307 (breeding intermediate); EPA Reg. No. 67979-EE

 Bt11 x MIR604 x TC1507 x 5307; EPA Reg. No. 67979-EU

 Bt11 x MIR162 x MIR604 x TC1507 x 5307; EPA Reg. No. 67979-EG

The eCry3A.1Ab toxin in event 5307 was engineered from “variable regions” of mCry3A and Cry1Ab 

(Walters et al. 2010). It is active against the corn rootworm (CRW) complex, including western corn 

rootworm (WCRW, Diabrotica virgifera virgifera) and northern corn rootworm (NCRW, Diabrotica 

barberi). MIR604 (mCry3A) is also targeted against CRW and has been previously registered in a 

number of stacked and pyramided products (see BRAD -- BPPD 2006).
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Bt11 (Cry1Ab) and TC1507 (Cry1F) x TC1507 are effective against European corn borer (ECB, 

Ostrinia nubilalis), the major lepidopteran target pest in the Corn Belt, as well as corn earworm (CEW, 

Helicoverpa zea), southwestern corn borer (SWCB, Diatraea grandiosella), and fall armyworm (FAW, 

Spodoptera frugiperda). MIR162 (Vip3Aa20) is also targeted against the lepidopteran pests CEW, 

SWCB, and FAW but does not provide efficacy against ECB. All three of the lepidopteran toxins have 

been previously registered and deployed in stacked/pyramided products (BRADs: BPPD 2001 and 

2009a). Syngenta has indicated that a number of other secondary lepidopteran pests will be targeted 

including black cutworm (Agrotis ipsilon), dingy cutworm (Feltia jaculifera), lesser corn stalk borer 

(Elasmopalpus lignosellus), southern corn stalk borer (Diatrea crambidoides), common stalk borer 

(Papaipema nebris), sugarcane borer (Diatrea saccharalis), and western bean cutworm (WBCW, Richia 

albicosta). 

Because 5307 (EPA Reg. No. 67979-EE) is intended for breeding purposes only (and not for 

commercial corn production), a specific IRM plan is not required for the product (and it is not addressed 

further in this review). Although no refuge is typically planted with breeding corn, such registrations 

have been limited in terms of overall acreage (at county and national levels) to mitigate potential 

resistance.

Summary of Syngenta’s IRM Submissions for 5307 Pyramided Products 

Two separate IRM plans were submitted for the stacked/pyramided registrations containing the new 

active ingredient 5307, though much of the same data was shared between the two applications. Each of 

these listed studies listed is reviewed in this memorandum and the assessment of the shared data is 

applicable to both products.

MRID# 484429-10: Insect Resistance Management Plan for Bt11 x MIR162 x MIR604 x 

TC1507 x 5307 Maize

MRID# 484430-08: Insect Resistance Management Plan for Bt11 x MIR604 x 5307 x TC1507

Maize

The major components of Syngenta’s IRM plan included dose determinations, investigation of cross 

resistance, and simulation modeling. These sections and BPPD’s review of the overall IRM plan are 

described below.

1. Dose Considerations

Lepidopteran Toxins

Because all of the lepidopteran-active toxins in the two proposed products have been previously 

registered, Syngenta did not conduct new dose studies for ECB, SWCB, or CEW. Rather, data (MRID# 

480506-06) developed for a separate pyramided product containing Cry1Ab, Cry1F, and Vip3Aa20 

were cited. In conducting these studies, Syngenta employed the methodology prescribed by the 1998 
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Science Advisory Panel (SAP 1998), using techniques “1” and “4” for each of the insects. Specifically, 

the two SAP procedures are as follows:

1) Serial dilution bioassay with artificial diet containing lyophilized tissues of Bt plants using 

tissues from non-Bt plants as controls;

4) Survey large numbers of commercial plants (using a controlled infestation with a susceptible 

laboratory strain) in the field to make sure that the cultivar is at the LD99.9 or higher to assure 

that 95% of heterozygotes would be killed.

Syngenta reported the results of these tests in their IRM submission for the registration of Bt11 x 

MIR162 x TC1507 corn (EPA Reg. No. 67979-15). Based on those data, Syngenta concluded that Bt11 

x MIR162 x TC1507 corn has high dose expression for ECB and SWCB. This conclusion was 

supported by the data obtained from the Bt11 x MIR162 x TC1507 pyramid and also from the single 

toxin components (Bt11 and TC1507) which showed similar high dose activity. MIR162 has no activity

against ECB, but showed high activity for SWCB in one test (technique “4”) but not the other (technique 

“1”). Against CEW, Bt11 x MIR162 x TC1507 expresses an “effective” high dose as a pyramid, though 

the three single toxin events were shown to have less than high dose expression by themselves. 

Effective high dose indicates that the combined activity of the toxin in the pyramid produces complete 

mortality (though the single toxin constituents may allow some survival). 

In the current submission, Syngenta concluded that these Cry1Ab and Cry1F dose results should also be 

valid for Bt11 x MIR162 x MIR604 x TC1507 x 5307 and Bt11 x MIR604 x 5307 x TC1507 since field 

expression data show comparable toxin levels between the pyramided and constituent single toxin 

products. The lepidopteran dose data were fully described in MRID# 480506-06 (reviewed in BPPD 

2011).

BPPD Review - Lepidopteran Dose Considerations

Data for lepidopteran dose expression were previously reviewed for the registration of Bt11 x MIR162 x 

TC1507 corn (see review in BPPD 2011). An excerpt of the main conclusions in BPPD’s review is as 

follows:

BPPD generally agrees with Syngenta’s characterization of the dose profile for Bt11 x MIR162 x 

TC1507 corn -- i.e., high dose activity against the major target pests (with CEW considered an 

“effective” high dose). Dose testing was not reported for FAW in this submission for Cry1Ab or 

Cry1F. The results obtained for the other toxin combinations in these studies, including the 

single trait (Bt11, MIR162, TC1507) and dual toxin (Bt11 x MIR162, Bt11 x TC1507) 

constituent products, support previously-determined dose conclusions for MIR162 (summarized 

in BPPD 2009a). 

BPPD questions, however, whether the sample sizes tested in SAP method #1 were adequate for 

a definitive “high dose” conclusion. Syngenta tested only 20 - 24 insects per treatment (with 
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100% mortality on Bt). It is unclear if these small scale tests are sufficient to conclude that each 

toxin will produce 99.99% mortality to the larger ECB and SWCB populations. 

BPPD notes that no new dose data were developed specifically for the either of the “5307” pyramided 

products. Syngenta’s dose conclusions for these products are largely supported by a reference to field 

expression data showing comparable levels of protein in the pyramided products to the single toxin 

events. These data were submitted as part of the “5307” applications, but were not summarized in the 

IRM volume. Field efficacy trials (another means to verify dose performance in pyramided/stacked 

products) against the major target pests were also not submitted or referenced in the report. However, 

provided that the protein expression levels are comparable (as described by Syngenta), BPPD agrees 

with the dose characterization of the “5307” pyramided products as having a high dose for ECB and 

SWCB and an “effective” high dose for CEW. 

Rootworm Toxins (MRID# 484429-10, Appendix A)

Dose studies for the CRW-active toxins eCry3.1Ab and mCry3A were conducted by Dr. Bruce Hibbard 

(USDA-ARS, Columbia, MO). CRW larval mortality to single toxin and pyramided events was 

assessed from artificially-infested plots in Central Missouri over a three year period. Selection intensity

(survival relative to the control) was calculated to provide an indication of the effectiveness (dose) of 

each toxin and the pyramid.

Within each test field (1-3 locations with 3-5 replicates were tested per year) plots were established of 

eCry3A.1Ab (designated event SYN101 in the report), mCry3A (MIR604), SYN101 x MIR604, and a 

non-Bt isoline. During the seasons prior to testing, soybeans were planted to reduce the possibility of 

wild CRW infesting the plots. Insecticides were not used in the test fields (including seed treatments) to 

avoid confounding factors, though the fields were otherwise managed with corn agronomic practices 

typical of the region. In the Bt test plots, each plant was verified for Bt expression using gene strip tests. 

Buffers (vegetation-free areas) were established outside each test plot to prevent CRW larval movement 

between plots. Prior to testing, plots were thinned to 16 plants per row (3.05 m row length). 

WCRW eggs (obtained from wild populations) were artificially infested in each plot when the corn 

plants reached the V2-V3 growth stage. The infestation rate was 1,380 eggs per 30.5 cm (row foot) for 

2007-2008 tests, but was reduced to 875 eggs/foot in 2009 testing to reduce potential density dependent 

mortality in the control plots (based on work by Hibbard et al. 2010). Test plots were fitted with screen 

tents to collect emerging adult CRW approximately 5-6 weeks after infestation. After initial beetle 

emergence, all but four plants in each plot were cut off at 1 m and stripped of leaves (presumably for 

easier beetle collection). Beetles were collected 2-3 times per week for the duration of the study.

Survival of WCRW was assessed in terms of “selection intensity,” or the percentage of beetles emerging 

from the Bt treatments relative to the non-Bt control plots. In addition to WCRW, any northern corn 

rootworm (NCRW) and southern corn rootworm (SCRW) beetles (presumably from wild populations) 

were also collected from the field tents.
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The results from the trials showed that both the single toxin (SYN101 and MIR604) and pyramided 

(MIR604 x SYN101) treatments significantly reduced WCRW and SCRW populations relative to 

control plots. NCRW beetle emergence was also reduced by the Bt treatments, though in most of the 

treatments sites no beetles were collected in the non-Bt tents. The mCry3A and eCry3.1Ab toxins 

appeared to have a greater impact on WCRW emergence than SCRW. In terms of selection intensity, 

single trait SYN101 reduced beetle survival by 99.79%, while pyramided MIR604 x SYN101 lowered 

emergence by 99.91% over the five test locations/years. MIR604 alone resulted in an average selection 

intensity of 97.84%. There appeared to be little difference in beetle counts or selection intensity

between males and females. The study authors did not report selection intensity for SCRW (though the 

average beetle counts were provided), but noted selection intensities for NCRW of 97.13% (MIR604), 

98.85% (SYN101) and 100% (MIR604 x SYN101). Based on the results, Syngenta concluded that the 

mCry3A x eCry3.1Ab pyramid expresses a “highly effective dose” for WCRW and NCRW. Data from 

the dose investigations are summarized in Table 1 below.

To determine whether each toxin was acting independently, the researchers conducted a statistical 

analysis of beetle emergence. Had the two toxins acted independently, 0.0982 beetles would have been 

expected to emerge from the MIR604 x SYN101 plots (based on the survival results from the single 

toxin plots). In the experiments, 0.8 beetles emerged (averaged across all five locations), a statistically

higher amount than the 0.0982 expected. 

Table 7. Mortality (Selection Intensity) of CRW to eCry3.1Ab and mCry3A in Single Toxin and 

Pyramided Toxin Deployments (compiled from data provided in Tables 3 and 4 of MRID# 484429-10, 

Appendix A) 

Treatment

SCRW NCRW 1 WCRW

Beetle Ct.2

(avg.)

Sel. Int. 

(%)

Beetle Ct.2

(avg.)

Sel. Int. 

(%)

Beetle Ct.2

(avg.)

Sel. Int. 

(%)

MIR604 13.1 b NR 0.2 b 97.13 19.0 b 97.84

SYN101 13.6 b NR 0.1 b 98.85 1.9 c 99.79

MIR604 x 

SYN101 10.6 b NR 0.0 b 100 1.9 c 99.91

Isoline 36.8 a NR 7.9 a 0 879.0 a 0

1 NCRW beetles were collected at only two of the five test sites.

2 Total beetle counts for both sexes are reported. For each treatment, mean values with separate letters are statistically 

different by Fisher’s Least Square Difference test.

BPPD Review - Corn Rootworm Dose Considerations

Syngenta’s dose studies for CRW follow the methodology employed for other rootworm-active PIP 

toxins including Cry34/35, mCry3A, and Cry3Bb1 (see methodology and data published in Storer et al. 
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2006; Hibbard et al. 2010a, b; and data summarized in BPPD 2009b). BPPD agrees with Syngenta that 

the events containing eCry3.1Ab have high activity against WCRW, although it is unlikely that the toxin 

is expressed at a “high dose” level (as defined by the 1998 SAP).

None of the other registered CRW toxins (Cry34/35, mCry3A, and Cry3Bb1) is known to express a true 

high dose for CRW, even in pyramided toxin deployments. Still, eCry3.1Ab greatly reduced WCRW 

survival (>99%) and may be among the most efficacious toxins for rootworm registered to date. The 

study authors compared the eCry3.1Ab results with data from other toxins and noted that selection 

intensity for Cry34/35 was 96.47% (Storer et al. 2006) and 94.88% for mCry3A (Hibbard et al. 2010b). 

Data analyzed for a separate product (SmartStax) showed selection intensity ranges of 96.17 - 99.96%

for Cry3Bb1, 94.20 - 99.18% for Cry34/35, and 98.22 - 99.97% for Cry3Bb1 x Cry34/35 pyramid

(reviewed in BPPD 2009b).

Fewer conclusions can be drawn for either NCRW or SCRW. Although the researchers reported high 

reductions in NCRW emergence (see Table 1), the data were based on only two locations (no beetles 

were collected in the other locations). No artificial infestations were performed, so it is assumed that the 

NCRW observed in the two locations were from wild populations. Even in the plots where NCRW were 

found, overall beetle numbers were low (7.9 avg. beetles/non-Bt field). Similarly, SCRW collections 

presumably arose from feral populations, though beetles were fairly numerous (36.8 avg. beetles/non-Bt 

field). Although not reported, the selection intensity appeared to be lower with SCRW than with either 

WCRW or NCRW (e.g., 71.2% for the pyramid). However, because infestation was not controlled, any

conclusions regarding the efficacy of eCry3.1Ab against either NCRW or SCRW are limited. 

2. Cross Resistance

Lepidopteran Toxins

Syngenta’s conclusions regarding potential cross resistance between Cry1F, Cry1Ab, and Vip3A were 

based upon data and rationales that were previously submitted for Bt11 x MIR162 x TC1507 (EPA Reg. 

No. 67979-15), a lepidopteran-active pyramid containing the same toxins as the proposed products. 

These cross resistance data were assessed in BPPD’s review of the Bt11 x MIR162 x TC1507 

submission (MRID# 480506-07 and -08; reviewed in BPPD 2011). Previously, the cross resistance 

potential for Vip3A was assessed for the initial registration of MIR162 (see discussion in BPPD 2009).

For ECB, Syngenta cited a number of published studies to support the lack of cross resistance between 

Cry1Ab and Cry1F. Vip3A (MIR162) is not active against ECB and was therefore not considered in the 

cross resistance assessment. Syngenta noted the following points for ECB:

 Resistance is most likely to result from a decrease in Bt (Cry) toxin binding with epithelial cells 

in the insect midgut. Other mechanisms (e.g. metabolic adaptations) would likely be too weak to 

overcome the high levels of mortality causes by PIP toxins in the field.

 Midgut binding assays have shown that Cry1Ab and Cry1F do not strongly compete for the same 

binding sites in ECB (Hua et al. 2001).
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 Studies with Cry1Ab and Cry1F-resistant ECB colonies did not show high levels of cross 

resistance to the reciprocal toxin (Pereira et al. 2008; Siqueira et al. 2004)

Fewer data are available for CEW and the three toxins. Syngenta cited work with Cry1F and Cry1Ac 

(developed for Dow AgroScience’s WideStrike cotton product, see Storer 2002), based on the rationale 

that Cry1Ab and Cry1Ac share binding sites and likely would have similar patterns of cross resistance. 

Work with resistant colonies has been limited since stable field-relevant resistance has not been 

established. However, Syngenta noted that a Cry1Ab-resistant laboratory CEW strain was still 

susceptible to Vip3A (Konasale et al. 2008). [BPPD note: After review, BPPD believes Syngenta 

intended to reference Anilkumar et al. 2008, a study in which Cry1Ac-resistant CEW were tested.]

Syngenta referenced previously-submitted resistant colony studies for FAW (MRID# 480506-07; 

reviewed in BPPD 2011). The company concluded that these data showed incomplete cross resistance 

between Cry1F and Cry1Ab, but no cross resistance with Cry1F and Vip3A.

Overall, Syngenta concluded that: 1) Based on midgut binding characterizations and studies with 

resistant colonies, Cry1F likely has “little or no potential cross resistance” with Vip3A in CEW or FAW; 

2) Similarly, there is low potential for “field relevant” cross resistance between Cry1F and Cry1Ab in 

ECB or FAW; 3) More limited information on CEW (midgut binding studies with Cry1F and Cry1Ac) 

suggests there is “potential for moderate levels of cross resistance between Cry1F and 

Cry1Ab.....however, this risk is highly mitigated by the presence of Vip3A.”

BPPD Review - Lepidopteran Cross Resistance Potential

BPPD generally agrees with the overall conclusion that there is low potential for cross resistance 

between the toxins among the lepidopteran pests, despite the cursory information provided by Syngenta 

in its previous submission (MRID# 480506-07 and -08; see review in BPPD 2011). No new data were 

provided with the two 5307 pyramid applications. 

Cry1F and Cry1Ab may have some risk for cross resistance. Both are part of the Cry1 family of Bt 

toxins that share some structural similarities and several studies have found cross resistance in various 

insects between different toxins in the group (e.g., Denholf et al. 1993; Granero et al. 1993; Gould et al. 

1995, Bolin et al. 1999; others). Nevertheless, the weight of evidence from midgut receptor binding 

characterizations and resistant colony work suggests that among the major corn pests (ECB and FAW), 

the potential for cross resistance between Cry1F and CryAb1 should be low. There are other factors that 

may reduce the probability for cross resistance among the targeted pests. As discussed in BPPD (2011), 

recent research (Pereira et al. 2010) suggests that resistance to Cry1F may be the result of a unique 

process different from identified resistance mechanisms in other Bt cry toxins. Also, an analysis of 

separate data by BPPD (2010a) concluded that there is low cross resistance potential between Cry1Ab 

and Cry1F for the major target pests of Bt corn.
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Fewer data and/or information regarding CEW were available or cited by Syngenta. Previous binding 

work (Storer 2002) was conducted with Cry1F and Cry1Ac (data developed by Dow AgroSciences); 

Syngenta believes that Cry1Ac and Cry1Ab can be expected to have similar binding patterns. BPPD 

cannot confirm this hypothesis with the information cited by Syngenta but notes that a separate analysis 

(BPPD 2010b) concluded that it is likely that Cry1Ab and Cry1F will have a low cross resistance 

potential in CEW.

The presence of Vip3A in one of the proposed 5307 pyramids (Bt11 x MIR162 x MIR604 x TC1507 x 

5307) will likely help mitigate the potential risk of Cry1F - Cry1Ab cross resistance for CEW and FAW. 

For example, a Cry1Ac-resistant CEW colony was tested and found to be cross resistant to Cry1Ab, but 

not Vip3A (Anilkumar et al. 2008). Similarly, Cry1F-resistant FAW were even more susceptible to 

Vip3A than an unselected colony (MRID# 480506-07; reviewed in BPPD 2011). On the other hand, 

ECB may have a greater concern for cross resistance in a Bt11 x MIR162 x TC1507 lepidopteran 

pyramid because Vip3A is not active against the insect. Cry1Ab and Cry1F, therefore, must maintain 

separate pathways of toxicity to realize the benefits of the pyramid.

Syngenta has not addressed cross resistance potential with SWCB in this or previous submissions. It is 

commonly assumed that SWCB behaves similar to ECB and that conclusions made for ECB can be 

applied to this insect as well. However, the 2010 SAP noted key differences between the insects, 

particularly in response to Bt toxins (SAP 2011). To illustrate, Vip3A has at least some activity against 

SWCB while having no activity against ECB (BPPD 2009a). Therefore, it is unclear to BPPD whether 

the assumption that SWCB will also be at low risk for cross resistance with Cry1Ab, Cry1F, and/or 

Vip3A will have validity for a lepidopteran pyramid containing the toxins.

While the likelihood of cross resistance may be low in terms of modified midgut receptors, it is possible 

that other mechanisms of cross resistance (e.g., behavioral modifications or expression of cry-proteases) 

could arise with pyramided Bt corn. The 2010 SAP concluded that the prediction of low cross resistance 

potential between Cry1F and the Cry1A toxins for ECB and SWCB remains uncertain and outlined a 

number of epistatic mechanisms that could lead to increased survival on pyramided Bt corn (SAP 2011). 

For these reasons, cross resistance should be considered in predictive modeling to evaluate the durability

of a Cry1F/Cry1Ab/Vip3A pyramid (particularly for worst case scenarios). 

Corn Rootworm Toxins

Syngenta tested the cross resistance potential between eCry3.1Ab and mCry3A by investigating midgut 

binding patterns in WCRW. Potential interactions of the toxins with WCRW binding sites were 

investigated in qualitative and quantitative competitive binding assays.

The qualitative binding studies were published in Walters et al. (2010). Brush border membrane 

vesicles preparations were made from first instar WCRW larvae and exposed to activated 

(chymotrypsin-processed) mCry3A or eCry3.1Ab. Heterologous and homologous competition 

experiments were performed for each of the toxins. The resulting western blots showed that both 

unlabeled mCry3A and eCry3.1Ab competed with their respective labeled homologues. On the other 
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hand, in the heterologous competitions unlabeled eCry3.1Ab did not inhibit labeled mCry3A binding, 

while unlabeled mCry3A failed to diminish the binding of labeled eCry3.1Ab. Based on these 

competition studies, the study authors concluded that mCry3A and eCry3.1Ab recognize different 

binding sites in WCRW. This novel activity is likely due to the exchange of Cry1Ab and Cry3A 

variable regions resulted in the engineered toxin (eCry3.1Ab).

A second set of experiments was conducted to quantify the level of competition among mCry3A and 

eCry3.1Ab for binding sites in WCRW. The studies were performed by Drs. Juan Jurat-Fuentes and 

Siva Rama Krishna Jakka of the University of Tennessee (Appendix C of MRID# 484429-10). Both 

toxins were chymotrypsin-activated and radiolabeled with I125 for detection by photographic plates. In 

initial testing, both toxins were shown to specifically bind to the BBMV preparations, which suggested 

limited binding sites in WCRW. The level of specific binding was higher for eCry3.1Ab (70-80%) than 

for mCry3A (50%). Three separate binding competition assays were conducted: 1) labeled mCry3A 

with unlabeled eCry3.1Ab and mCry3A; 2) labeled eCry3.1Ab with unlabeled mCry3A and eCry3.1Ab, 

and 3) labeled mCry3A with unlabeled mCry3A (non-activated) and eCry3.1Ab (activated). 

Results from the assays showed little or no competition between mCry3A and eCry3.1Ab for BBMV 

binding sites. In the first competition, labeled mCry3A binding was reduced slightly (to ~ 80%) when 

exposed to the higher concentrations of unlabeled eCry3.1Ab. Homologous competition with unlabeled 

mCry3A resulted in a significantly greater reduction in labeled mCry3A binding (to ~ 40-60% at the two 

highest concentrations). The authors suggested that the low level of competition observed could be 

explained if there are multiple binding sites that recognize mCry3A with one that is shared with 

eCry3.1Ab. However, the shared binding site would have low affinity for eCry3A.1Ab since 

competition was observed at only at the peak concentrations tested. No competition was observed in the 

second assay: eCry3.1Ab binding was not reduced by even the highest concentrations of unlabeled 

mCry3A. These results suggest that the specific binding site(s) for eCry3.1Ab are not recognized by 

mCry3A. Competition experiments with non-activated protoxins showed similar patterns as were 

observed in the first competition, indicating that both chymotrypsin-activated and non-activated 

mCry3A and eCry3.1Ab toxins recognize the same binding sites. 

Considering the results of both studies, Syngenta concluded that eCry3.1Ab and mCry3A maintain 

independent binding sites in WCRW and that separate genetic mutations would be needed to develop 

resistance to both toxins. As such, the toxins are viable in a pyramided product for resistance 

management of CRW.

BPPD Review - Corn Rootworm Cross Resistance Potential

Syngenta’s cross resistance analysis was based on the assumption that Bt resistance will develop through 

an alteration of the toxin binding receptors in the target, a mechanism that has been documented for a 

number of pests (Ferre et al. 1991; Tabashnik 1994; Lee et al. 1995; Ferre & Van Rie 2002). Syngenta 

showed through an analysis of WCRW midgut binding activity and competitive binding assays that 

eCry3.1Ab and mCry3A largely recognize unique binding sites. As such, cross resistance between the 

two toxins is not expected according to the traditional Bt resistance model of binding site modification. 
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This is perhaps not surprising, considering that eCry3.1Ab is an engineered toxin with different 

structural elements (transferred from Cry1Ab) than mCry3A (Walters et al. 2010).

Syngenta did not consider alternate resistance mechanisms or cross resistance scenarios in addition to 

toxin - receptor binding. It has been suggested that resistance in CRW to Bt may evolve differently than 

lepidopteran pests on which the toxin-receptor mechanism of resistance is based. Resistance to Bt in 

CRW may involve multiple loci exerting small changes in susceptibility as opposed to a single locus 

creating high level resistance (as is assumed for lepidoptera) (SAP 2011). Several selection experiments 

with CRW have suggested that resistance may be incomplete (Meihls et al. 2008, 2011) or could occur 

at low levels (Lefko et al. 2008). The SAP also noted the potential for epistatic processes in which a 

single genetic change (e.g., protease expression or regulation of cadherin levels) could confer broader 

resistance to Bt toxins. BPPD notes, however, that specific cases of epistasis (or broader mechanisms of 

cross resistance) have not yet been documented in CRW or other Bt corn pests. In addition, one study of 

a Cry3Bb1-resistant colony showed no evidence of cross resistance to Cry34/35Ab1 (Gassmann et al. 

2011).

Syngenta’s assessment of cross resistance was limited to the novel eCry3.1Ab and existing mCry3A 

proteins, both of which are proprietary to the company. No analysis of the other registered CRW toxins, 

Cry3Bb1 and Cry34/35Ab1, was included in the submission. Customarily, the registrant of a new PIP 

active ingredient will analyze cross resistance for all existing toxins in the landscape and not just the 

toxin(s) in the proposed product or pyramid (e.g., refer to the cross resistance discussion for mCry3A in 

BPPD 2006). BPPD recognizes that because eCry3.1Ab is an engineered toxin, it is likely to have 

significant structural differences with Cry3Bb1 and Cry34/35Ab1 and perhaps different midgut binding 

patterns in CRW. But no information was provided in the submission to verify this assumption; 

therefore, BPPD recommends that Syngenta provide additional data or analysis to address the cross 

resistance potential of eCry3.1Ab with Cry3Bb1 and Cry35/35Ab1.

In addition to midgut binding analysis (as submitted by Syngenta), tests with resistant colonies can be 

useful to assess cross resistance, provided such colonies are available. Resistant colonies have been 

selected with Cry3Bb1 (Meihls et al. 2008), Cry34/35Ab1 (Lefko et al. 2008), and mCry3A (Meihls et 

al. 2011), though it is unknown whether such colonies could be accessed for future testing. Should they

be available, BPPD recommends testing with the resistant colonies to improve the cross resistance 

analysis for mCry3A. BPPD is unaware of any selection experiments that have been conducted for the 

relatively new eCry3A.1Ab protein, though it is recommended that selection experiments be initiated 

with this protein as well.

3. Simulation Modeling

Lepidoptera

Since the lepidopteran-active toxins (Cry1Ab, Cry1F, and Vip3Aa20) in Syngenta’s proposed 5307 

products have been previously registered, the applicant cited existing modeling for CEW, ECB, and 

SWCB. For CEW, Syngenta cited simulations conducted for the initial registration of Vip3Aa20 
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(MIR162). These simulations, described in BPPD (2009a), were used to support deployment of a 20% 

refuge in cotton-growing regions, where the risk of resistance in CEW may be high. Syngenta did not 

conduct additional simulations for CEW because the company is proposing to maintain the existing 20% 

refuge standard for the new Cry1Ab/Cry1F/Vip3Aa20 pyramid.

Modeling for ECB and SWCB was previously developed for the registration of Syngenta’s Bt11 x 

MIR162 x 1507 pyramid (EPA Reg. No. 67979-15). The model (detailed in MRID# 480506-06) was 

deterministic for two loci (Cry1Ab and Cry1F) and was similar in design to the single locus simulation 

created by Caprio (1998). Vip3A was not included in the simulations since it is not active against ECB. 

Results from the modeling showed that resistance did not develop (within 100 years) to the 

Cry1Ab/Cry1F pyramid with either a 5% or 20% refuge. Syngenta asserted that the model included a 

high degree of conservatism including assumptions that resistance carries no fitness costs, resistance is 

complete, no natural refuge in the system, and that single trait versions of Cry1Ab and Cry1F are 

present. The company concluded that the simulations support the use of Cry1Ab, Cry1F, and Vip3Aa20 

in a pyramid with a 5% refuge.

BPPD Review - Lepidopteran Modeling

Syngenta’s ECB/SWCB models were assessed for the registration of Bt11 x MIR162 x 1507 (see full 

review in BPPD 2011). BPPD generally agreed with Syngenta that, under the modeling conditions 

investigated, Bt11 x TC1507 x MIR162 was highly durable relative to the corresponding single trait 

PIPs (i.e., resistance did not evolve within the time frame of the model). Furthermore, BPPD agreed that 

some of the parameter choices were conservative for the model, including assumptions of complete 

resistance, no fitness costs for resistance, and no natural refuge. Also, the exclusion of MIR162 

(Vip3Aa20) from the modeling adds conservatism for SWCB, which has susceptibility to the toxin.

Despite these conclusions, BPPD’s analysis was limited by the cursory description provided for the 

model (Appendix F, MRID# 480506-06), which lacked a number of critical details in the methodology

description. BPPD had additional questions regarding some of the parameter assumptions and was 

unable to find descriptions of others. The scope of the modeling appeared to be limited, as no alternate 

scenarios (i.e., worst cases hypotheses) or sensitivity analysis were conducted (or reported in the 

submission). For these reasons, it was difficult for BPPD to assess the relative durability of refuge 

options for Bt11 x TC1507 x MIR162 based solely on the limited modeling submitted by Syngenta. 

BPPD recommended that Syngenta provide an improved modeling analysis to address the concerns so 

that the expected durability of Bt11 x TC1507 x MIR162 could be confirmed (BPPD 2011). A revised 

modeling report was required as a term of registration, which was subsequently submitted to EPA 

(MRID# 485509-02). As of the date of this memorandum, this submission has not yet been formally 

reviewed by BPPD.

Syngenta’s CEW model was submitted and reviewed for the registration of MIR162 (Vip3Aa20). The 

model was designed by Dr. Michael Caprio (Mississippi State University) and included several 

scenarios in which Vip3Aa20 x Cry1Ab corn (with either 20% or 50% refuge) was planted in a 

landscape containing Bt cotton (Vip3Aa19 x Cry1Ab). Simulations with 20% or 50% corn refuge did 
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not evolve resistance during the 25-year model timeframe. BPPD’s assessment of the model concluded 

that it, coupled with other evidence, provided sufficient support to warrant the use of a 20% refuge for 

the MIR162 pyramid in southern cotton-growing regions (BPPD 2009a). Syngenta is proposing to 

employ the same 20% refuge strategy in cotton regions with the 5307 pyramids (containing Cry1Ab, 

Cry1F, and/or Vip3Aa20) and did not conduct any new CEW modeling for these new products. BPPD 

agrees that the modeling originally submitted for MIR162 is adequate to support Syngenta’s proposed 

lepidopteran refuge strategy for the 5307 applications.

Corn Rootworm

Syngenta partnered with Dr. Michael Caprio (Mississippi State University) to conduct simulations for 

CRW. Dr. Caprio (in collaboration with U.S EPA’s Office of Research development) had previously 

designed a stochastic model that was used for an analysis of a “seed blend” IRM strategy (Caprio and 

Glaser 2010). For Syngenta’s simulations, Caprio modeled the time to resistance for a MIR604 

(mCry3A) x 5307 (eCry3.1Ab) pyramid with a 5% block or seed blend refuge. Single trait events (MIR 

604 and 5307) were also modeled as a mosaic with 20% block refuges. 

Caprio’s model employed a landscape structure with four 36 ha fields, with each of the fields including a 

20 x 20 grid of patches (each 30m x 30m). The grid allowed for structured refuges to be placed within 

the Bt fields so that adult dispersal could be modeled. Fields were populated at the start of each year in 

the model run with the PIP crop and refuges. For the single toxin mosaic simulations, each of two 

toxins was randomly assigned to two of the fields. Refuges were located at one side of the Bt field 

(randomly selected for each field). Bt fields had a 30% probability of not having a refuge each year of 

the simulation (corresponding with a non-compliance rate of 30%). No partial non-compliance was 

incorporated and Caprio noted that there was a small probability (0.81%) in any given year of there 

being no refuge in any of the four fields (an unlikely scenario in the real world). If resistance to one of 

the two toxins developed, in subsequent years all fields were planted exclusively with the second toxin. 

Dual toxin simulations for the pyramid (with 5% structured refuge or seed blend) were modeled using 

the same landscape features, though no single toxin PIPs were included (all four fields were planted with 

the same dual gene PIP). 

Adult dispersal was modeled in a routine that included both population and individual parameters. For 

each dispersal routine, a certain proportion of the total population was determined to move at least one 

patch (randomly drawn from binomial distribution). Movement on an individual basis included vector 

(direction) and distance, both of which were randomly generated (dispersal distance was based on data 

developed by Nowatski et al. 2003). A second routine covered “aging” in which individuals aged 

sequentially by one day in the simulations. This feature allowed for the modeling of a developmental 

delay for insects exposed to PIP toxins, which was accomplished by holding back 12% of such 

individuals from progressing through life stages each day (resulting in an average overall delay of 7 days 

for the population).

Larval movement included a base movement rate (the same for both Bt and non-Bt plants) and 

asymmetrical movement away from Bt plants. Base larval movement was sampled for each simulation 
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from a distribution that ranged from 1 to 10% with 5% being the most likely value. Asymmetrical 

movement incorporated the genotype of the insect and mortality on Bt, such that susceptible genotypes 

(more likely to be killed on Bt) had greater movement rates on Bt plants. The frequency (ratio) of 

asymmetrical movement was determined for each simulation from a probability distribution (0.1 to 1.0, 

0.5 most likely). 

Many of the other parameters were populated using uncertainty distributions, as was done for larval 

movement. Values for toxin mortality (used to derive genotype fitness) were provided by Syngenta, 

while other biological parameters were carried over from previous modeling (Caprio and Glaser 2010). 

Information on the parameter assumptions and model features is detailed in Table 2 below.

Table 8. Parameter Values and Assumptions for Syngenta’s (Caprio) Stochastic Model to Evaluate the 

Durability of 5307 x MIR604 (taken from data provided in Table 1 and other information in Appendix D 

of MRID# 484429-10)

Parameter Value or Assumption Notes

Min Most Likely Max

Initial R-allele freq. 0.001 0.005 0.02

Same values for both toxins 

but drawn independently for 

each locus

Mortality due to 

toxin 1 0.95 0.9808 0.9997 Toxin not specified; assumed to be eCry3.1Ab

Mortality due to 

toxin 2

0.833 0.95 0.9906 Toxin not specified; assumed 

to be mCry3A

Overall mortality 0.99165 0.99904 0.9997

Base larval 

movement 0.01 0.05 0.1 Total movement for the lifetime of the larvae

Asymmetrical 

movement ratio 0.1 0.5 1.0

Insect aging

One day per simulated day; 12% on Bt 

plants have one day developmental 

delay

Cross resistance Not reported BPPD assumes to be zero 

Non compliance 

(structured refuges) 30% (no partial non-compliance)

Model duration 4440 days (31.7 years)

Definition of 

resistance

R allele > 0.5

Model continued to run if 

resistance developed to one 

toxin - all subsequent fields 

were assumed to be planted to 

2

nd toxin
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Several hundred simulations were conducted for each toxin deployment scenario with each simulation 

run for the equivalent of 4440 days (31.7 years). Resistance did not evolve to either toxin in the 

pyramid seed blend scenario (510 total simulations). Similarly, resistance did not evolve to the pyramid 

in the structured refuge scenario (535 simulations) though in three simulations resistance evolved to the 

second toxin. Resistance to both toxins evolved only in simulations with the single toxin mosaic 

scenario. The average time to resistance was 26.7 years for toxin 1, 28.7 years for toxin 2, and 29.9 

years for both toxins; however, resistance did not evolve to both toxins in 62.7% of the simulations (311 

total). Caprio also presented a risk profile for the single toxin modeling with levels of risk ranging from 

0% to 50%. For a 10% level of risk, the time to resistance would be ≥ 25.3 years for both toxins. This 

means that 90% of the simulations evolved resistance after 25.3 years, while in the other 10% of 

simulations resistance developed more quickly (the researcher essentially assumes a 10% chance of 

being “wrong” about the estimated durability). As the level of risk drops, the resistance values also drop 

(more outlying simulations are included); at the 0% level of risk the time to resistance was 17.4 years for 

both toxins (i.e., none of the simulations had resistance develop earlier than 17.4 years). Results from 

the modeling are summarized in Table 3.

Caprio (and Syngenta) concluded that the pyramid of mCry3A and eCry3.1Ab with 5% refuge (seed 

blend or block refuge) is more durable (“less risky”) than a mosaic of single toxin products with a 20% 

refuge. Given that resistance did evolve to toxin 2 in three simulations of the pyramid/structured refuge 

scenario, Caprio also concluded that the seed blend (in which resistance never evolved) was somewhat 

more durable than the structured refuge. This was likely due to the non-compliance rate (30%) that was 

assumed for structured refuges.

Table 9. Results from Syngenta’s modeling (taken from data provided in Table 2 and other information 

in Appendix D of MRID# 484429-10)

Scenario Simulations

Mean Time to Resistance (years)

Toxin 1 Toxin 2 Both Toxins

Single toxin mosaic: 50% 

toxin 1, 50% toxin 2 311 28.67 26.73 29.92

Pyramid with 5% structured 

refuge 535 No resistance * No resistance

Pyramid with 5% seed blend 

refuge 510 No resistance evolved to either toxin

* Resistance evolved in three simulations to toxin 2. The time to resistance was not reported for these 

simulations.

BPPD Review - Corn Rootworm Modeling

Syngenta’s modeling largely produced expected results, in that pyramided deployment of toxins were 

relatively more durable than single toxin PIP despite having lower percent refuges. This output is 
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consistent with other models that have analyzed pyramids as a resistance management approach for Bt 

crops (Roush 1998, Caprio 1998, Zhao et al. 2003, Gould et al. 2006, Onstad and Meinke 2010). 

Caprio’s stochastic model differs from other models by using a probabilistic approach for assigning 

parameter in which values are drawn from a distribution (with a most likely value) for each simulation. 

This allows the model to incorporate variability and uncertainty associated with biological parameters 

(e.g., resistance allele frequencies, dose mortality, and movement). Many simulations are run for each 

scenario from which a “risk profile” of estimated durability can be drawn for different levels of 

uncertainty (selected by the risk assessor). As the criterion for certainty in the durability estimate is 

raised, the time to resistance decreases. BPPD has previously reviewed and utilized Caprio’s model in 

evaluations of other products (see BPPD 2010a) and has found it to be a rigorous risk assessment tool.

While BPPD agrees with Syngenta regarding the overall conclusions, the modeling was limited in scope 

and the handling of some parameters. Three general scenarios were run that included landscapes of 

either single toxin or pyramided toxin PIPs. The single toxin simulations included a mosaic of both 

traits (eCry3.1Ab and mCry3a) but both pyramid scenarios (seed blend and block refuge) were run with 

no single trait PIPs in the landscape. This was a realistic choice in the case of eCry3.1Ab since no 

products (single trait or pyramid) have been previously approved with the toxin. In the case of mCry3A, 

however, single toxin PIPs expressing the trait have been registered since 2006 and would likely be 

grown along with 5307 pyramided products should they be registered. Single trait products can act as a 

“stepping stone” to resistance for pyramids, particularly if non-compliance with single trait refuge 

requirements is high. It is uncertain how the durability of the toxins would have been affected in a 

simulation including both single traits and pyramids. BPPD expects that resistance would have evolved 

to the single trait mCry3A (as occurred in the single toxin mosaic simulations), though it is unknown 

whether resistance would have evolved to the pyramid within the modeled time frame. One possible 

way to address this issue would be to increase the resistance allele frequency for mCry3A to account for 

previous selection to the single toxin. It is unclear, however, whether such an approach was considered 

in Syngenta’s simulations. 

The model duration (31.7 years) may have also limited the analysis between the 5% pyramid block and 

5% pyramid seed blend refuges. For the vast majority of simulations (including the single toxin 

mosaics), resistance did not evolve during the model time period. In only three simulations (out of over 

1,000 total) was resistance observed in the pyramid toxin scenarios. Because of this, it is difficult to 

make any relative comparisons between the pyramid refuge options (i.e., blocks vs. blends); in essence, 

the only conclusion that can be drawn is that pyramided deployments are superior to a mosaic of single 

toxin PIPs. The study author (Caprio) suggested that blended refuge may be superior to block refuge for 

pyramids, though this was based on the three cases of resistance to one of the toxins that arose during 

the block refuge simulations. BPPD suspects, however, that this result is not statistically relevant given 

that only 0.56% of the simulations (3 of 535) showed resistance. Expanding the time horizon of the 

model could have helped to tease out differences between block refuge and seed blends and perhaps 

further flesh out the role non-compliance plays on block refuge durability. 
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Though not explicitly stated in the report, cross resistance was presumably assumed to be negligible and 

not included in the modeling. Data submitted by Syngenta (and reviewed earlier in this memorandum) 

support the assumption of little cross resistance potential through the traditional toxin-midgut binding 

site model. BPPD cannot, however, entirely eliminate the possibility of cross resistance through means 

other than toxin-receptor interactions. The 2011 SAP identified epistasis, in which a single genetic 

change (e.g., protease expression or regulation of cadherin levels) exerts a broad effect, as a potential 

mechanism that could result in resistance to multiple toxins. Because of this, Syngenta could have 

assumed a low level of cross resistance as a worst case scenario in the modeling (it is unclear if the 

model assumed epistasis as part of genotype survival). This would likely have reduced the durability of 

both traits in all scenarios, though the relative impact of cross resistance/epistasis on durability between 

seed blend and block refuges is unknown.

Other potential parameters and considerations, such as mating (random/non-random), pre-ovipositional 

dispersal, oviposition, and density-dependent effects, were not discussed in the report. These parameters 

can be important for relative evaluations of block and seed blend refuges, though Syngenta’s application 

proposes only block refuge for the 5307 pyramids. BPPD assumes that these parameters were addressed 

in the same manner as Caprio’s previous modeling (Caprio and Glaser 2010), though this cannot be 

confirmed.

Despite the limitations in the model and lack of clarity in the report, BPPD concludes that Syngenta’s 

modeling demonstrates high durability of the mCry3A x eCry3.1Ab pyramid with a 5% block refuge. A 

number of other published and unpublished models have also shown that multi-toxin PIPs with high 

efficacy and low cross resistance have superior durability to single toxin PIPs. On the other hand, 

additional information may be need to fully assess the durability of a 5% seed blend, including more 

complete descriptions of how parameters such as genotypic fitness/movement, assumptions regarding

heterozygote survival, and oviposition were addressed in the model. BPPD notes, however, that 

Syngenta has not proposed a seed blend strategy for 5307 corn at the time of their application. 

4. Proposed IRM Plan

For Bt11 x MIR604 x TC1507 x 5307 (EPA Reg. No. 67979-EU) and Bt11 x MIR162 x MIR604 x 

TC1507 x 5307 (67979-EG), Syngenta has proposed similar IRM strategies. The plans are based on 

reduced (5%) refuges for pyramided Bt corn and established paradigms for stewardship (i.e., resistance 

monitoring, remedial action, and compliance).

Refuge

Separate refuge requirements were proposed for the Corn Belt (i.e., non-cotton growing regions) and 

cotton-growing regions. Larger refuges have been required in cotton regions because of CEW, which 

can have up to two generations on corn before moving onto cotton during the same growing season. In 

the Corn Belt, resistance management is primarily geared towards stalk borers (ECB and SWCB) and 

CRW.
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Corn Belt Refuge (non-cotton growing regions)

For both Bt11 x MIR604 x TC1507 x 5307 and Bt11 x MIR162 x MIR604 x TC1507 x 5307, 

Syngenta proposes a 5% structured refuge. The refuge can be planted as either a “common 

refuge” for both ECB/SWCB and CRW or as two separate (5%) refuges for each pest complex.

The common refuge must be planted adjacent to or within the Bt field. In-field refuges must be 

at least four strips wide and if crop rotation is used, it must be employed for both the refuge and 

Bt fields. Insecticide treatments can be made in the refuge for corn rootworm larvae with soil 

insecticides. Non-rootworm late season pests can be treated with foliar (non-Bt) insecticides in 

the refuge provided adult CRW are not present and economic thresholds are reached. If adult 

CRW are present, both the refuge and Bt field must be treated in the same manner. Economic 

thresholds are to be determined by local or regional professionals. Syngenta also proposed that 

“pests on the [Bt] maize acres can be treated as needed without having to treat the common 

refuge.” 

With the separate refuge option, discrete refuges (each 5%) must be planted for lepidoptera and 

rootworm. The lepidopteran refuge must be planted within 1⁄2 mile of the Bt field and in-field 

strips must be at least four rows wide. Insecticides can be used in the refuge for 1) corn 

rootworm larvae (soil insecticides), or 2) corn borers (non-Bt foliar applications) provided 

economic thresholds are reached. The corn rootworm refuge has essentially the same restrictions 

as the common refuge in terms of deployment (adjacent to or within the Bt field), in-field strip 

width (at least four rows), and insecticide use for rootworm and other pests. 

Cotton-Growing Regions

Syngenta proposed different refuge requirements for each product in cotton regions, though both 

strategies maintain the common and separate refuge options. For Bt11 x MIR162 x MIR604 x 

TC1507 x 5307, Syngenta proposed a 20% lepidopteran refuge while for Bt11 x MIR604 x 

TC1507 x 5307, a 50% lepidopteran refuge would be required. 

The common refuge (20% for Bt11 x MIR162 x MIR604 x TC1507 x 5307 and 50% for Bt11 x 

MIR604 x TC1507 x 5307) must be planted with the same deployment and insecticide use 

restrictions as the common refuge for the Corn Belt. For the separate refuges, both products 

must have a 20% corn rootworm refuge. Each product must also have a lepidopteran refuge: 

20% for Bt11 x MIR162 x MIR604 x TC1507 x 5307 and 50% for Bt11 x MIR604 x TC1507 x 

5307. The separate refuges must also be planted with the same restrictions as the separate 

refuges for the Corn Belt region.

Syngenta concluded that their proposal for the 5% refuge is supported by a number of factors, including 

submitted data reviewed in this memorandum. Specifically, Syngenta cited the following justification:
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 EPA has set a precedent of lower refuge for pyramided products for both lepidoptera and corn 

rootworm (e.g., MON 89034, SmartStax);

 Two of the toxins in the pyramids, Bt11 (Cry1Ab) and TC1507 (Cry1F), are expressed at high 

dose levels for ECB and SWCB;

 For CEW, the Bt11 x TC1507 x MIR162 pyramid offers three modes of action and a functional 

high dose;

 For CRW, MIR604 (mCry3A) x 5307 (eCry3.1Ab) offers two effective modes of action;

 Cross resistance potential for both lepidoptera and corn rootworm among the toxin groups in the 

pyramids is low;

 Simulation modeling demonstrates the durability of the Bt11 x MIR162 x MIR604 x TC1507 x 

5307 pyramid relative to single toxin deployments.

BPPD Review - Refuge

As discussed in the modeling section, BPPD concludes that there is adequate scientific support to 

demonstrate that the 5307 pyramids will have high durability with a 5% structured refuge (relative to 

single trait PIPs with 20% refuge). Syngenta has not proposed a seed blend for any of the 5307 

products; BPPD notes that additional information and modeling may be necessary to support such a 

refuge strategy.

Syngenta’s proposed language for the refuge requirement includes the following statement: “Pests on 

the Bt11 x MIR162 x MIR604 x TC1507 x 5307 [or Bt11 x MIR604 x TC1507 x 5307] maize acres can 

be treated as needed without having to treat the common refuge.” This language has not been 

specifically included in the registration terms and conditions of similar Bt corn products, but the 

registrations do not prohibit the practice. Treatment of Bt acres should not increase the risk of resistance 

since most pest insects present would presumably be resistant to the toxin(s) (or otherwise have avoided 

lethal exposure to the toxin). Therefore, differential insecticide usage in which the PIP field is treated 

but the refuge is not would not be expected to create a selection gradient disfavoring susceptible insects 

(as would be the case if only the refuge was treated).

For cotton-growing regions, Syngenta proposed a 50% lepidopteran refuge for Bt11 x MIR604 x 

TC1507 x 5307, while requesting a 20% refuge for Bt11 x MIR162 x MIR604 x TC1507 x 5307. BPPD 

assumes the difference is due to the fact that the latter product has three active toxins against lepidoptera 

(CEW in particular) while the former has two. CEW has been the primary target pest of concern in 

cotton areas (given its annual life cycle on corn and cotton) and larger refuges (50%) were required for 

single toxin corn PIPs in the region. However, EPA has approved a number of two toxin pyramids with 

a 20% refuge in cotton regions, including two Syngenta products (EPA Reg. Nos. 67979-12 and 67979-

15) and another registrant’s product containing Cry1Ab and Cry1F (29964-7). Syngenta may be able to 

support a 20% refuge for the Bt11 x MIR604 x TC1507 x 5307 pyramid with a relatively simple model 

(as was done to support the other registered pyramids).
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Syngenta’s refuge proposal did not specifically define the cotton-growing region that would require 20% 

(or 50%) refuges. Based on the paradigm established for previously-registered Bt corn PIPs, this region 

should be defined as follows: Alabama, Arkansas, Georgia, Florida, Louisiana, Mississippi, North 

Carolina, South Carolina, Oklahoma (only the counties of Beckham, Caddo, Comanche, Custer, Greer, 

Harmon, Jackson, Kay, Kiowa, Tillman, Washita), Tennessee (only the counties of Carroll, Chester, 

Crockett, Dyer, Fayette, Franklin, Gibson, Hardeman, Hardin, Haywood, Lake, Lauderdale, Lincoln, 

Madison, Obion, Rutherford, Shelby, and Tipton), Texas (except the counties of Carson, Dallam, 

Hansford, Hartley, Hutchinson, Lipscomb, Moore, Ochiltree, Roberts, and Sherman), Virginia (only the 

counties of Dinwiddie, Franklin City, Greensville, Isle of Wight, Northampton, Southampton, Suffolk 

City, Surrey, Sussex), and Missouri (only the counties of Dunkin, New Madrid, Pemiscot, Scott, 

Stoddard).

Stewardship (Resistance Monitoring, Remedial Action, Compliance Assurance)

Syngenta did not address resistance monitoring, remedial action, and refuge compliance for the 5307 

pyramids other than to indicate that existing programs would be utilized for the new products. 

The existing stewardship framework developed for the lepidopteran toxins (Cry1Ab, Cry1F, and 

Vip3Aa20) has been coordinated by the Agricultural Biotechnology Stewardship Technical Committee 

(ABSTC) for over a decade. BPPD agrees that no additional information is needed for the inclusion of 

the 5307 products into the ABSTC programs for resistance monitoring, remedial action, and compliance

assurance.

Corn rootworm resistance monitoring has not been implemented by ABSTC and each registrant has 

developed a toxin-specific plan (though there are common structural elements between plans). 

Syngenta’s CRW monitoring and remedial action plans were developed for the MIR604 product 

registrations (67979-5, -8; amended September, 2010). The general structure should be applicable to the 

5307 registrations, however, additional information is recommended for the eCry3.1Ab toxin. Baseline 

susceptibility data for eCry3.1Ab were not addressed or submitted with the IRM volume. Also, a 

proposal for a diagnostic assay (to distinguish potentially resistant rootworm from susceptibles) was not 

included in the submission. BPPD recommends that Syngenta develop both baseline susceptibility data 

and a (plant-based) diagnostic assay for eCry3.1Ab as a term or condition of registration. 

Resistance monitoring for CRW has been challenged by the “non-high dose” nature of the Bt toxins. 

CRW do not adapt well to artificial diets and monitoring assays based on toxin-diet incorporation have 

been variable and may be unreliable for detecting shifts in susceptibility. As part of the 2010 

extensions of Bt corn registrations, BPPD notes that Syngenta was required to pursue the use of the 

“Sublethal Seedling Assay” (Nowatzki et al. 2008) as a potential diagnostic assay for mCry3A. This 

technique involves assessing the rate of larval development on corn seedlings and was shown to be more 

sensitive to changes in susceptibility to Cry34/35 (a separately registered CRW toxin). It is 

recommended that Syngenta also investigate the feasibility of this approach for eCry3.1Ab. In addition 

to the Sublethal Seedling Assay, the 2010 extension of mCry3A required Syngenta to: 1) develop a 

resistance monitoring plan for northern corn rootworm (Diabrotica barberi) and 2) establish corn 
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rootworm damage guidelines (for unexpected pest damage investigations). BPPD recommends that 

these requirements also be applied to the eCry3.1Ab registrations.

Syngenta’s submission indicated that they plan to submit the usual annual reports required for Bt corn 

(i.e., resistance monitoring, compliance, and sales) for the 5307 products. BPPD notes that Syngenta’s 

grower education materials (e.g., technology use guides) will need to be updated to include the 5307 

product line and refuge requirements. BPPD recommends that these materials be submitted to the 

Agency prior to or concurrent with the first commercial growing season of the products.

References

Anilkumar, K.J., A. Rodrigo-Simón, J. Ferré, M. Pusztai-Carey, S. Sivasupramaniam, and W. J. Moar, 

2008. Production and Characterization of Bacillus thuringiensis Cry1Ac-Resistant Cotton Bollworm 

Helicoverpa zea (Boddie). Appl. Environ. Microbiol. 74: 462-469.

BPPD, 2001. Biopesticide Registration Action Document (BRAD): Cry1Ab and Cry1F Bacillus 

thuringiensis (Bt) Corn Plant Incorporated Protectants (updated September, 2010). Available at 

http://www.epa.gov/oppbppd1/biopesticides/pips/cry1f-cry1ab-brad.pdf

BPPD, 2005. Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 Proteins and the Genetic Material 

Necessary for their Production in Event DAS-59122-7 Corn Biopesticide Registration Action Document 

(BRAD). Available at 

http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006490.pdf.

BPPD, 2006. Biopesticide Registration Action Document (BRAD): Modified Cry3A Protein and the 

Genetic Material Necessary for its Production (via elements of pZM26) in Event MIR604 Corn SYNIR604-8 (updated September, 2010). Available at 

http://www.epa.gov/oppbppd1/biopesticides/pips/mcry3a-brad.pdf.

BPPD, 2008. Biopesticide Registration Action Document (BRAD): Bacillus thuringiensis Cry1A.105 

and Cry2Ab2 Insecticidal Proteins and the Genetic Material Necessary for Its Production in Corn

(updated September, 2010). Available at http://www.epa.gov/oppbppd1/biopesticides/pips/mon-89034-

brad.pdf

BPPD, 2009a. Biopesticide Registration Action Document (BRAD): Bacillus thuringiensis Vip3Aa20 

Insecticidal Protein and the Genetic Material Necessary for Its Production (via Elements of Vector 

pNOV1300) in Event MIR162 Maize (OECD Unique Identifier: SYN-IR162-4) (updated March, 2009). 

Available at http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006599.pdf

BPPD, 2009b. Review of Additional Insect Resistance Management (IRM) information for SmartStax 

(MON 89034 x TC1507 x MON 88017 x DAS 59122-7) Bt corn. A. Reynolds memorandum to M. 

Mendelsohn, dated July 1, 2009.
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BPPD, 2010a. Science Assessment of Pioneer’s modeling addenda (MRIDs 478710-01, 478879-01, and 

479108-01) in support of their application of registration for Optimum AcreMax1 Insect Protection. J. 

Martinez memorandum to M. Mendelsohn, dated February 4, 2010.

BPPD, 2010b. Review of Insect Resistance Management (IRM) considerations with a proposed reduced 

refuge for Bt corn event 1507 x MON 810 in the Corn Belt. A. Reynolds memorandum to A. Sibold, 

dated October 20, 2010.

BPPD, 2010c. EPA PRELIMINARY RISK ASSESSMENT of Monsanto’s and Dow’s 5% Seed 

Mixture Request for a Section (3) Full Commercial Registration of SmartStax Corn. J. Martinez 

memorandum to M. Mendelsohn, dated October 29, 2010.

BPPD, 2011. Review of the Insect Resistance Management (IRM) plan and supporting data for Bt corn 

event Bt11 x MIR162 x TC1507. A. Reynolds memorandum to J. Kausch, dated March 2, 2011.

Caprio, M., 1998. Evaluating resistance management strategies for multiple toxins in the presence of 

external refuges. J. Econ. Entomol. 91: 1021-1031.

Caprio, MA and JA Glaser. 2010. “Simulation model evaluation of pest resistance development to 

refuge in the bag concepts related to Pioneer Submission”. Report generated for OPP/BPPD on January

22, 2010.

Denholf, P., S. Jansens, M. Peferoen, D. Degheele, and J. Van Rie, 1993. Two different

Bacillus thuringiensis delta-endotoxin receptors in the midgut brush border membrane of the European 

Corn Borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae). Appl. Environ. Microbiol. 59: 1828-

1837.

Ferré, J., M. D. Read, J. Van Rie, S. Jansens, and M. Peferoen, 1991. Resistance to the Bacillus

thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut 

membrane receptor. Proc. Natl. Acad. Sci. USA. 88: 5119-5123.

Ferré, J. and J. Van Rie, 2002. Biochemistry and genetics of insect resistance to Bacillus thuringiensis. 

Annu. Rev. Entomol. 47: 501-533.

Gassmann, A.J., J.L. Petzold-Maxwell, R.S. Keweshan, and M.W. Dunbar, 2011. Field-evolved 

resistance to Bt maize by western corn rootworm. PLoS ONE. 6(7): 1-7.

Gould, F., A. Anderson, A. Reynolds, L. Bumgarner, and W. Moar, 1995. Selection and genetic 

analysis of a Heliothis virescens (Lepidoptera: Noctuidae) strain with high levels of resistance to 

Bacillus thuringiensis toxins. J. Econ. Entomol. 88: 1545-59.
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Gould, F., M. B. Cohen, J. S. Bentur, G. C. Kennedy, and J. Van Duyn, 2006. Impact of small fitness 

costs on pest adaptation to crop varieties with multiple toxins: a heuristic model. J. Econ. Entomol. 99:

2091-2099.

Granero, F., V. Ballester, and J. Ferre, 1996. Bacillus thuringiensis crystal proteins CRY1Ab and 

CRY1Fa share a high affinity binding site in Plutella xylostella (L.). Biochem. Biophys. Res. Comm. 

224: 779-783.

Hibbard B.E., L.N. Meihls, M.R. Ellersieck, and D.W. Onstad, 2010a. Density-dependent and densityindependent mortality of the Western corn rootworm: impact on dose calculations of rootworm-resistant 

Bt corn. J. Econ. Entomol. 103: 77-84.

Hibbard, B.E., T.L. Clark, M.R. Ellersieck, L.N. Meihls, A.A. El Khishen, V. Kaster, H. York-Steiner, 

and R. Kurtz, 2010b. Mortality of western corn rootworm larvae on MIR604 transgenic maize roots: 

Field survivorship has no significant impact on survivorship of F1 progeny on MIR604. J. Econ. 

Entomol. 103: 2187-2196.

Hua, G., L. Masson, J.L. Jurat-Fuentes, G. Schwab, and M.J. Adang, 2001. Binding Analyses of 

Bacillus thuringiensis Cry {delta}-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia 

nubilalis. Appl. Environ. Microbiol. 67: 872-879.

Lee, M.K., F. Rajamohan, F. Gould, and D.H. Dean, 1995. Resistance to Bacillus thuringiensis Cry1A 

δ-endotoxins in a laboratory-selected Heliothis virescens strain is related to receptor alteration. Appl. 

Environ. Microbiol. 61: 3836-3842.

Lefko, S.A., T.M. Nowatzki, S.D. Thomposon, R.R. Binning, M.A. Pascual, M.L. Peters, E.J. Simbro, 

and B.F. Stanley, 2008. Characterizing laboratory colonies of western corn rootworm (Coleoptera: 

Chrysomelidae) selected for survival on maize containing event DAS-59122. J. Appl. Entomol. 132: 

189-204

Meihls L.N., M.L. Higdon, B.D. Siegfried, T.A. Spencer, N.J. Miller, T.W. Sappington, M.R. Ellersieck, 

and B.E. Hibbard, 2008. Increased survival of western corn rootworm on transgenic corn within three 

generations of on-plant greenhouse selection. Proceed. Nat. Acad. Sci. 105: 19177-19182.

Meihls L.N., M.L. Higdon, M.R. Ellersieck, and B.E. Hibbard, 2011. Selection for resistance to 

mCry3A-expressing transgenic corn in western corn rootworm. J. Econ. Entomol. 104: 1045-1054.

Nowatzki, T.M., S.A. Lefko, R.R. Binning, S.D. Thompson, T.A. Spencer, and B.D. Siegfried, 2008. 

Validation of a novel resistance monitoring technique for corn rootworm (Coleoptera: Chrysomelida) 

and event DAS-59122-7 maize. J. Appl. Entomol. 132: 177-188.

Onstad, D. and L. Meinke, 2010. Modeling Evolution of Diabrotica virgifera virgifera (Coleoptera: 

Chrysomelidae) to Transgenic Corn with Two Insecticidal Traits. J. Econ Entomol. 103: 849-860.
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Pereira E.J., H.A. Siqueira, M. Zhuang, N.P. Storer, B.D. Siegfried, 2010. Measurements of Cry1F 

binding and activity of luminal gut proteases in susceptible and Cry1F resistant Ostrinia nubilalis larvae 

(Lepidoptera: Crambidae). J. Invertebr. Pathol. 103: 1-7.

Roush, R.T., 1998. Two toxin strategies for management of insecticidal transgenic crops: will 

pyramiding succeed where pesticide mixtures have not? Phil. Trans. R. Soc. Lond. 353: 1777-1786.

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 on February 9-10, 1998. Report dated, April 28, 1998. (Docket Number: OPPTS-00231). 

Scientific Advisory Panel (SAP), 2011. Transmittal of the meeting minutes of the FIFRA Scientific 

Advisory meeting held on December 8-9, 2011 to address scientific issues associated with insect 

resistance management for SmartStax refuge-in-a-bag, a plant-incorporated protectant (PIP) corn seed 

blend. Report dated, March 3, 2011. (Docket Number: EPA-HQ-OPP-2010-0772).

Siqueira, H.A.A., D. Moellenbeck, T. Spencer, and B.D. Siegfried, 2004. Cross-resistance of CrylAbselected Ostrinia nubilalis (Lepidoptera: Crambidae) to Bacillus thuringiensis d-endotoxins. J. Econ. 

Entomol. 97: 1049-1057.

Storer, N., 2002. Product durability plan for cotton expressing Cry1F and Cry1Ac insecticidal crystal 

proteins from Bacillus thuringiensis. Unpublished report submitted to EPA by Dow Agrosciences. 

MRID# 458084-15. 

Storer, N., 2003. A spatially explicit model simulation western corn rootworm (Coleoptera: 

Chrysomelidae) adaptation to insect-resistance maize. J. Econ. Entomol. 96: 1530-1547.

Storer, N., J.M. Babcock, and J.M. Edwards, 2006. Field measures of western corn rootworm 

(Coleoptera: Chrysomelidae) mortality caused by Cry34/35Ab1 proteins expressed in maize event 

59122 and implications for trait durability. J. Econ. Entomol. 99: 1381-1387.

Tabashnik, B. E., 1994. Evolution of resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 39: 47-

79.

Walters, F., C. deFontes, H. Hart, G. Warren, and J. Chen, 2010. Lepidopteran-active variable-region 

sequence imparts coleopteran activity in eCry3.1Ab, an engineered Bacillus thuringiensis hybrid 

insecticidal protein. Appl. Environ. Microbiol. 76: 3082-3088.

Zhao, J., J. Cao, Y. Li, H. Collins, R. Roush, E. Earle, and A. Shelton, 2003. Transgenic plants 

expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nature Biotechnology. 

21: 1493-1497.
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G. Benefits and Public Interest Finding for Initial Registrations of Event 5307 Corn

The criteria for determining whether registration of a pesticide chemical is in the public interest are set

forth in a Federal Register Notice dated March 5, 1986 volume 51, No. 43 (OPP-32500; FRL-2977-2)

titled Conditional Registration of New Pesticides. There is a presumption that registration of a pesticide 

chemical is in the public interest if one of the following criteria is met: i) the use is for a minor crop; (ii) 

the use is a replacement for another pesticide that is of continuing concern to the Agency; (iii) the use is 

one for which an emergency exemption under FIFRA Section 18 has been granted for lack of an 

alternative pest control method, or (iv) the use is against a pest of public health significance. Further, 

EPA may determine that such a registration is in the public interest on the basis of the following criteria: 

i) there is a need for the new chemical that is not being met by currently registered pesticides; ii) the new 

pesticide is comparatively less risky to health or the environment than currently registered pesticides; or 

iii) the benefits (including economic benefits) from the use of the new active ingredient exceed those of 

alternative registered pesticides and other available nonchemical techniques.

Syngenta has provided data to support its claim that 5307 CRW-protected corn is in the public interest

(MRID# 484425-41). EPA’s analysis supports the following conclusions:

1. Results of efficacy trials conducted from 2006 to 2009 indicate that 5307 corn

provides effective control of key rootworm pests of field corn, including western corn rootworm 

(Diabrotica virgifera virgifera), northern corn rootoworm (Diabrotica barberi), and Mexican corn 

rootworm (Diabrotica virgifera zeae). Efficacy was measured in terms of root damage (i.e., crop 

protection) and adult emergence (i.e., population reduction). 

2. The eCry3.1Ab protein expressed in 5307 is a novel mode of action for corn rootworm control. The 

protein is a chimera engineered from the mCry3A and Cry1Ab Bt proteins. It has unique biochemical 

properties and low cross resistance potential with other registered Bt PIPs targeting CRW (refer to the 

Insect Resistance Management chapter in this BRAD document). As such, 5307 is expected to provide

insect resistance management (IRM) benefits for this and other CRW-protected corn products (detailed 

in #3 below).

3. CRW is a highly adaptive insect and has been able to overcome a number of cultural and chemical 

control regimes (Meinke et al. 1998, Spencer et al. 2005). Recent reports have suggested that CRW may

be developing resistance to Cry3Bb1, a separately-registered Bt toxin (Gassmann et al. 2011, Gassmann 

2012). Bt toxins for CRW control may have a higher risk of resistance than other PIPs because they are 

not expressed at “high dose” levels for the insect (Tabashnik and Gould 2012). On the other hand, the 

availability of multiple toxins for CRW with unique modes of action (i.e., a toxin mosaic) may enhance 

IRM by reducing the selection pressure on any one toxin. The 5307 (eCry3.1Ab) protein would 

represent the fourth Bt toxin for CRW and would further expand the existing toxin mosaic. 

Furthermore, it is expected that 5307 will be included with other Bt proteins to provide multiple control 

mechanisms in a single product (known as a toxin “pyramid”). Simulation models predict that toxin 
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pyramids with unique modes of action are more durable (less susceptible to resistance) than single toxin 

PIPs (Roush 1998, Zhao et al. 2003, Onstad et al. 2010). Syngenta has proposed two such pyramids 

with 5307 and a second CRW toxin, MIR604 (mCry3A): a) Bt11 x MIR604 x TC1507 x 5307 and b) 

Bt11 x MIR162 x MIR604 x TC1507 x 5307.

4. If 5307 corn is registered, it will be the fourth CRW-protected Bt corn product on the market.

The availability of multiple CRW-protected corn products will increase grower choice and price 

competition, likely resulting in lower seed prices for consumers and higher adoption rates.

5. Data provided by Syngenta from a three-year study showed that 5307 corn provided significantly

greater crop yield (bushels/acre) than non-Bt corn with or without insecticide use. Syngenta reported a 

yield benefit of 63 bushels/acre compared to non-Bt corn without insecticide and 25 bushels/acre 

relative to non-Bt corn treated with Force 3G insecticide. Yield from 5307 was comparable (statistically 

equivalent) to MIR604 corn (a separate CRW-protected PIP registered by Syngenta).

6. Registration of 5307 corn is expected to result in further reduction of chemical insecticide use by 

growers. This is of special importance since many pesticides registered for CRW-control are highly 

toxic to humans and the environment, while eCry3.1Ab-expressing corn poses no foreseeable human 

health or environmental risks (refer to the Human Health Assessment and Environmental Assessment 

chapters in this BRAD document). Syngenta’s submission provided a summary of the conventional 

insecticides used for CRW control; many of these products are “restricted use” due to high mammalian 

toxicity and bear environmental hazards statements warning of dangers to birds, mammals, fish, aquatic 

invertebrates, and bees. EPA previously conducted an assessment of these insecticides and their 

associated mammalian and environmental toxicities for Cry3Bb1, a previously-registered CRW PIP (see 

U.S. EPA 2010). This analysis also demonstrated the high toxicity and environmental concerns of many 

CRW insecticides used in field corn.

REFERENCES

Gassmann A.J., J.L. Petzold-Maxwell, R.S. Keweshan, and M.W. Dunbar, 2011. Field-evolved 

resistance to Bt maize by western corn rootworm. PLOS one, Vol. 6 (7): 1-7.

Gassmann, A.J., 2012. Field-evolved resistance to Bt maize by western corn rootworm: Predictions 

from the laboratory and effects in the field. J. Invertebr. Pathol. In press.

Meinke, L.J., B.D. Siegfried, R.J. Wright, and L.D. Chandler, 1998. Adult susceptibility of Nebraska 

western corn rootworm (Coleoptera: Chrysomelidae) populations to selected insecticides. J. Econ 

Entomol. 91: 594-600.

Onstad, D. and L. Meinke, 2010. Modeling Evolution of Diabrotica virgifera virgifera (Coleoptera: 

Chrysomelidae) to Transgenic Corn with Two Insecticidal Traits. J. Econ Entomol. 103: 849-860.
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Roush, R.T., 1998. Two toxin strategies for management of insecticidal transgenic crops: will 

pyramiding succeed where pesticide mixtures have not? Phil. Trans. R. Soc. Lond. 353: 1777-1786.

Spencer, J.L., E. Levine, S.A. Isard, and T.R. Mabry, 2005. Movement, dispersal and behaviour of 

western corn rootworm adults in rotated maize and soybean fields. In: Vidal, S. et al. (Eds.), Western 

Corn Rootworm: Ecology and Management, CABI Publishing, Cambridge, pp. 121-144.

Tabashnik, B.E. and F. Gould, 2012. Delaying corn rootworm resistance to Bt corn. J. Econ Entomol. 

105: 767-776.

U.S. EPA. 2010. Biopesticide Registration Action Document for Bacillus thuringiensis Cry3Bb1 Protein 

and the Genetic Material Necessary for Its Production (Vector PV-ZMIR13L) in MON 863 Corn 

(OECD Unique Identifier: MON-ØØ863-5). http://www.epa.gov/oppbppd1/biopesticides/pips/cry3bb1-

brad.pdf

Zhao, J., J. Cao, Y. Li, H. Collins, R. Roush, E. Earle, and A. Shelton, 2003. Transgenic plants 

expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nature Biotechnology. 

21

III. REGULATORY POSITION FOR EVENT 5307 CORN 

Pursuant to section 3(c)(7)(C) of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the 

Environmental Protection Agency (EPA) may conditionally register a new pesticide active ingredient for 

a period of time reasonably sufficient for the generation and submission of required data that are lacking 

because insufficient time has elapsed since the imposition of the data requirement for those data to be 

developed. EPA may grant such conditional registration only if EPA determines that (1) the use of the 

pesticide product during the period of the conditional registration will not cause any unreasonable 

adverse effect on the environment, and (2) the registration and use of the pesticide during the conditional 

registration is in the public interest. EPA determines that all of these criteria have been fulfilled. 

The first criterion under FIFRA section 3(c)(7)(C), as mentioned above, has been met because 

insufficient time has elapsed since the imposition of the requirements for the following data:

1) Additional data or analysis to address the cross resistance potential of eCry3.1Ab with Cry3Bb1 and 

Cry35/35Ab1. As part of this effort, BPPD also recommends that Syngenta work to develop a 

eCry3.1Ab-resistant colony (similar to the work done for mCry3A -- see Meihls et al. 2011). 

2) Additional data for corn rootworm resistance monitoring with the eCry3.1Ab toxin:

 Baseline susceptibility data;

 A proposal for a plant-based diagnostic assay including an investigation into the feasibility of 

using the “Sublethal Seedling Assay” (Nowatzki et al. 2008);

 A monitoring plan for northern corn rootworm (Diabrotica barberi);
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 Corn rootworm damage guidelines (for unexpected pest damage investigations of eCry3.1Ab 

corn). 

The applicants submitted and/or cited data sufficient for EPA to determine that conditional registration 

of the Bacillus thuringiensis eCry3.1Ab insecticidal protein and the genetic material necessary for its

production (via elements of vector PSYN12274) in 5307 Corn (SYN-537-1) under FIFRA section 

3(c)(7)(C) will not result in unreasonable adverse effects to the environment, as discussed above. The 

applicants submitted and/or cited satisfactory data pertaining to the proposed use. The human health

effects and non-target organism effects data are considered sufficient for the period of the conditional 

registration. These data demonstrate that no foreseeable human health hazards or environmental effects 

are likely to arise from the use of the products and, furthermore, that the risk of resistance developing to 

the eCry3.1Ab proteins during the conditional registrations is not expected to be significant. 

Registration of the Bacillus thuringiensis eCry3.1Ab insecticidal protein and the genetic material 

necessary for its production (via elements of vector PSYN12274) in 5307 Corn (SYN-537-1) is in 

the public interest for the following reasons:

1. Results of efficacy trials conducted from 2006 to 2009 indicate that 5307 corn

provides effective control of key rootworm pests of field corn, including western corn rootworm 

(Diabrotica virgifera virgifera), northern corn rootoworm (Diabrotica barberi), and Mexican corn 

rootworm (Diabrotica virgifera zeae). Efficacy was measured in terms of root damage (i.e., crop 

protection) and adult emergence (i.e., population reduction). 

2. The eCry3.1Ab protein expressed in 5307 is a novel mode of action for corn rootworm control. The 

protein is a chimera engineered from the mCry3A and Cry1Ab Bt proteins. It has unique biochemical 

properties and low cross resistance potential with other registered Bt PIPs targeting CRW (refer to the 

Insect Resistance Management chapter in this BRAD document). As such, 5307 is expected to provide

insect resistance management (IRM) benefits for this and other CRW-protected corn products (detailed 

in #3 below).

3. CRW is a highly adaptive insect and has been able to overcome a number of cultural and chemical 

control regimes (Meinke et al. 1998, Spencer et al. 2005). Recent reports have suggested that CRW may

be developing resistance to Cry3Bb1, a separately-registered Bt toxin (Gassmann et al. 2011, Gassmann 

2012). Bt toxins for CRW control may have a higher risk of resistance than other PIPs because they are 

not expressed at “high dose” levels for the insect (Tabashnik and Gould 2012). On the other hand, the 

availability of multiple toxins for CRW with unique modes of action (i.e., a toxin mosaic) may enhance 

IRM by reducing the selection pressure on any one toxin. The 5307 (eCry3.1Ab) protein would 

represent the fourth Bt toxin for CRW and would further expand the existing toxin mosaic. 

Furthermore, it is expected that 5307 will be included with other Bt proteins to provide multiple control 

mechanisms in a single product (known as a toxin “pyramid”). Simulation models predict that toxin 

pyramids with unique modes of action are more durable (less susceptible to resistance) than single toxin 

PIPs (Roush 1998, Zhao et al. 2003, Onstad et al. 2010). Syngenta has proposed two such pyramids 
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with 5307 and a second CRW toxin, MIR604 (mCry3A): a) Bt11 x MIR604 x TC1507 x 5307 and b) 

Bt11 x MIR162 x MIR604 x TC1507 x 5307.

4. If 5307 corn is registered, it will be the fourth CRW-protected Bt corn product on the market.

The availability of multiple CRW-protected corn products will increase grower choice and price 

competition, likely resulting in lower seed prices for consumers and higher adoption rates.

5. Data provided by Syngenta from a three-year study showed that 5307 corn provided significantly

greater crop yield (bushels/acre) than non-Bt corn with or without insecticide use. Syngenta reported a 

yield benefit of 63 bushels/acre compared to non-Bt corn without insecticide and 25 bushels/acre 

relative to non-Bt corn treated with Force 3G insecticide. Yield from 5307 was comparable (statistically

equivalent) to MIR604 corn (a separate CRW-protected PIP registered by Syngenta).

6. Registration of 5307 corn is expected to result in further reduction of chemical insecticide use by 

growers. This is of special importance since many pesticides registered for CRW-control are highly 

toxic to humans and the environment, while eCry3.1Ab-expressing corn poses no foreseeable human 

health or environmental risks (refer to the Human Health Assessment and Environmental Assessment 

chapters in this BRAD document). Syngenta’s submission provided a summary of the conventional 

insecticides used for CRW control; many of these products are “restricted use” due to high mammalian 

toxicity and bear environmental hazards statements warning of dangers to birds, mammals, fish, aquatic 

invertebrates, and bees. EPA previously conducted an assessment of these insecticides and their 

associated mammalian and environmental toxicities for Cry3Bb1, a previously-registered CRW PIP (see 

U.S. EPA 2010). This analysis also demonstrated the high toxicity and environmental concerns of many

CRW insecticides used in field corn.

In view of these minimal risks and the clear benefits related to the Bacillus thuringiensis eCry3.1Ab 

insecticidal protein and the genetic material necessary for its production (via elements of vector 

PSYN12274) in 5307 Corn (SYN-537-1), EPA believes that the use of the products during the 

limited period of the conditional registrations will not cause any unreasonable adverse effects.

Although the data with respect to this particular new active ingredient are satisfactory, they are not 

sufficient to support an unconditional registration under FIFRA section 3(c)(5). Additional data are 

necessary to evaluate the risk posed by the continued use of these products. Consequently, EPA is

imposing the data requirements specified earlier in this chapter.

EPA has determined, as explained in section II(G) of this Biopesticides Registration Action Document 

(BRAD), that the third criterion for a FIFRA section 3(c)(7)(C) conditional registration has been 

fulfilled because use of the Bacillus thuringiensis eCry3.1Ab insecticidal protein and the genetic 

material necessary for its production (via elements of vector PSYN12274) in 5307 Corn (SYN-537-

1) under these registrations is in the public interest. 

The submitted data, in support of these registrations under FIFRA section 3(c)(7)(C), have been 

reviewed and determined to be adequate. Studies mentioned above are included in the terms, conditions, 
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and limitations of these registrations. These registrations will not cause unreasonable adverse effects to 

man or the environment and are in the public interest. 

The expiration date of the registrations is proposed as August 1, 2016.

C. Period of Registration 

In the 2001 Bt Corn reassessment, EPA determined that it was appropriate to amend the then-existing 

registrations to extend the period of registration of those products to an expiration date of October 15, 

2008. All of the products being assessed at that time were efficacious against lepidopteran pests. EPA 

based this action on the finding that use of Cry1Ab or Cry1F expressed in corn will not significantly

increase the risk of unreasonable adverse effects on the environment “for the limited time period of 7 

additional years (to October 15, 2008).” These registrations were later amended to extend the period of 

registration to an expiration date of September 30, 2010. EPA subsequently granted time-limited 

registrations to products efficacious against coleopteran corn rootworm pests. For example, EPA 

registered Cry3Bb1 on February 24, 2003, to May 1, 2004, and extended that registration twice, to 

February 24, 2008, and September 30, 2010.

As set forth elsewhere in this document, EPA’s primary concern for the Bt protected transgenic corn 

products is the possibility that target pests will develop resistance to one or more of the plantincorporated protectant (PIP) toxins. Development of resistance to a Bt toxin would be a grave adverse 

effect, and, for over 15 years, EPA has imposed stringent requirements intended to countermand the 

potential development of resistance. Registrants similarly have been busily developing various products, 

product mixes (i.e., so-called “pyramids” and “stacks”), and resistance strategies, to maximize 

agronomic benefits and address resistance management issues. The result has been a vast array of 

product combinations and, occurring over the past couple of years, a re-emergence of varying refuge 

requirements for different products.

As discussed in the 2001 Bt PIP BRAD (at IID13), the earliest Bt corn registrations did not include 

mandatory refuge requirements. There was a lack of scientific consensus as to what the appropriate 

refuge requirement should be, and, it was assumed that the limited market penetration of these early 

crops would be so low as to guarantee that adequate natural refuges would be available from 

neighboring non-Bt corn fields. From 1995 to 1997, Bt corn registrations included voluntary refuge 

requirements of 0% to 20% in the Corn Belt. In 1999, the Agricultural Biotechnology Stewardship 

Technical Committee (ABSTC), in conjunction with the National Corn Growers Association, proposed 

uniform insect resistance management (IRM) requirements for Bt corn registrations. With some 

modifications, this proposal, put in place for the 2000 growing season, formed the baseline IRM 

requirements for almost all Bt corn registrations for the better part of a decade: farmers were required to 

plant a 20% refuge that could be treated for insects, or a 50% treated refuge in cotton-growing areas; all 

refuges to be planted within one-half mile of the Bt corn field. 

These uniform requirements brought certainty and consistency to the market after the initial period 

where many Bt corn products had different refuge requirements. Recently, however, as product 
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developers have begun to conceive of products with different combinations of “pyramided” products 

(i.e., products containing two or more toxins efficacious against the same pest) and “stacked” products 

(i.e., products combining toxins efficacious against different pests), the refuge requirements have begun 

to vary. For example, certain products require a 20% external refuge; some products permit a 5% 

external refuge; one product incorporates a 10% seed blend refuge; we have applications in process for 

products that propose to incorporate a 5% seed blend refuge; and other permutations are possible.

Given the profusion of various toxin combinations and refuge options, we can no longer proceed on the 

basis that, as concerns insect resistance management, all products are equal. It was a relatively simple 

proposition when the default requirement of a 20% sprayed refuge applied to almost all of the Bt corn 

crops in the market. Under those circumstances, the relative durability of products against the 

development of resistance was functionally equivalent, and, as a consequence, imposing functionally

equivalent registration periods was appropriate. That is now no longer the case.

As part of our continually evolving regulatory approach to the continually evolving product mix 

wrought by developers, we think it appropriate to revise our regulatory requirements in scientifically

defensible ways to reflect the comparative level of risks posed by the products that we regulate. Here, 

for example, where we’ve determined that a particular product, or category of products, likely will pose 

less risk of insect resistance developing to a particular PIP protein, we think it appropriate to grant that 

particular product, or category of products, a registration for a period greater than that granted a 

corresponding product that poses a greater risk of insect resistance developing. This approach is 

reflective of complementary principles: first, to ensure that we apply our limited resources to the 

products that pose greater risk of adverse effects to the environment; and, second, to conserve the 

resources that registrants and applicants must expend in amending the registrations of products that pose 

less risk of adverse effects to the environment. 

The scheme that we are following includes registration periods of five, eight, and twelve years; a fifteenyear registration period will also be available, if adequately supported by our science assessment. In this 

scheme, (i) a product with a single PIP toxin, and a 20% external refuge, qualifies for a five-year 

registration; (ii) a product with pyramided PIP toxins (i.e., two or more toxins with distinct, non-cross 

reacting modes of action), that are non-high dose (the definition for a high dose product remains 

unchanged), with either a seed blend or external refuge, qualifies for an eight-year registration; (iii) a 

product with pyramided PIP toxins (i.e., two or more toxins with distinct, non-cross reacting modes of 

action), that are high-dose, with either a seed blend or external refuge, qualifies for a twelve-year 

registration; (iv) a product with pyramided PIP toxins (i.e., two or more toxins with distinct non-cross 

reacting modes of actions), with either a seed blend or external refuge, that has been determined by 

EPA’s science assessment to be 150% as durable as the baseline single toxin product with a 20% 

external refuge, would qualify for a fifteen-year registration. Products determined by EPA’s science 

assessment to be less than 100% as durable as the baseline single toxin product with a 20% external 

refuge would not qualify for a five-year registration and the registration period for such products will be 

determined on a case-by-case basis consistent with the level of risk they pose. Similarly, instances where 

other risk issues may arise, or where novel resistance concerns may be present, would also be 
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determined on a case-by-case basis, as will novel refuge configurations that may present unique 

durability profiles.