Document ID: EPA-HQ-OPP-2012-0108-0010
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2012-06-26T04:00Z

SEQ CHAPTER \h \r 1 		

			

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

Table of Contents

I.	OVERVIEW

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

	

	B.	Use Profile
………………………………………………………………
……………………....4										7									

									

	  

	

II.	SCIENCE ASSESSMENT

	A.	Product Characterization
………………………………………………………………
………5								14								

        

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

											

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

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

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

	F. 	Insect Resistance Management 
……………………………………………………………..7
3															

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

		

III.     	REGULATORY POSITION FOR 5307 CORN
……………………………99

	

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
Agrobacterium-mediated 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 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 plant-incorporated
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; Agrisure™ Viptera™ 3222; Agrisure™
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 

	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).   



1. Transformation System

Based on amino acid sequence similarity 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 (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 gene and the C-terminus (a portion of Domain III and Variable
Region 6) of the cry1Ab gene (Hofte and Whiteley, 1989) (see Figure 1). 

Figure 1. Schematic Illustrating the Origin of the Amino Acid Residues
Present in eCry3.1Ab 

		

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
N-terminus, 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-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

Pollenc	0.10b

		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.

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
post-translational 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 bacterial- derived
proteins, except that the microbial-derived version contains one
additional methionine and six histidine residues at the N-terminus.

CLASSIFICATION:  ACCEPTABLE	477347-03

Characterization of Test Substance ECRY3.1AB-0208 and Certificate of
Analysis a

	ECRY3.1AB-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 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	477347-04

Bioactivity of Test Substance ECRY3.1AB-0208 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 were addressed in MRID
No. 484425-09	477347-07

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 non-coding 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 PMI-0105 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 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. N-terminal
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	484425-02

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 microbially-produced 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 number functional element southern blot analysis
Southern blot analyses of 5307 maize demonstrated that (1) 5307 maize
contains a single copy of ecry3.1Ab, pmi, the CMP promoter 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	484425-04

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 T-DNA 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 size of 30 amino acids: Assessment of
amino acid sequence to known or putative allergens. 	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
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	484425-07

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 non-target 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 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	484425-08

 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 dry-weight 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.

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	484425-09

[This report supersedes MRID No. 47734707]

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

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 kernel.	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 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).

EnviroLogix™ QuickStix™ Kit for eCry3.1Ab Corn Bulk Grain and the
QuickStix™ Kit for eCry3.1Ab for leaf and seed are suitable analytical
methods for the qualitative detection of eCry3.1Ab protein residues. 

EnviroLogix™ also obtained USDA GIPSA performance certification of the
QuickStix™ 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	477347-17

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 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 prepared- The data in this report
were used to support the results reported in MRID No:  484425-01.
488026-01

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 prepared- The 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 prepared- The 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

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 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. 

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. 

Source of the trait.  Bacillus thuringiensis is not considered to be a
source of allergenic proteins. 

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.

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.  

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. 

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

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-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  HYPERLINK "http://www.epa.gov/pesticides/cumulative"
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. 

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) 

ogix™ QuickStix™ Kit for eCry3.1Ab Corn Bulk Grain (Cat. No. AS TBD
BG) and EnviroLogix™ QuickStix™ Kit for eCry3.1Ab Corn Leaf and Seed
(Cat. No. AS TBD LS). Lastly, Syngenta has submitted documentation that
the EnviroLogix™ QuickStix™ Kit for eCry3.1Ab Corn Bulk Grain (Cat.
No. AS TBD BG) received USDA GIPSA certification of the test 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 eCry3.1Ab protein as contained in test
substance ECRY3.1AB-0208 and event 5307 maize under simulated mammalian
gastric conditions b	The eCry3.1Ab protein from either microbial or
plant sources 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.	477347-11

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 database accession number ADC30135): Assessment of
amino acid sequence similarity to known or putative allergens	No
significant sequence similarities in the overall amino acid 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	484425-18

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.  

 that was common to PMI and a food allergen α-parvalbumin 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
putative protein allergens.

CLASSIFICATION:  ACCEPTABLE	484425-20

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 conditions	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 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.	484425-23

[This report supersedes 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 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.	484425-24

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.  

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.	484425-25

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 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 × DAS-59122-7 × 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 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
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. 

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	484429-01	

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 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-transgenic, near-isogenic, 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

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

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 lepidopteran-active 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 delta-endotoxins (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 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
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. 

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

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 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	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



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.

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) Plant-Incorporated 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.

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 cost.  

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

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

Testing methods which utilize the tiered approach were last published by
the EPA as Harmonized OPPTS Testing Guidelines, Series 850 and 885 (EPA
712-C-96-280, February 1996). 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).
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. A less than 50%
mortality effect at the MHD is taken to indicate minimal risk.  However,
greater than 50% mortality does not necessarily indicate the existence
of unacceptable risk in the field, but it does trigger the need to
collect additional dose-response information and a refinement of the
exposure estimation before deciding if the risk is acceptable or
unacceptable. Where potential hazards are detected in Tier I testing
(i.e. mortality is greater than  50%), additional information at lower
test doses is required which can serve to confirm whether any effect
might still be detected at more realistic field [1X EEC] concentrations
and routes of exposure.  

When screening tests indicate a need for additional data, the 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  (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 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 non-target
organisms directly in the field. This approach, with an emphasis on
testing invertebrates found in corn fields, was also recommended by the
August 2002 SAP and was supported by several public comments. Based on
these recommendations, the Agency has required field studies on long
term invertebrate population/community and 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-analysis of the data
collected from 42 field studies indicated that non-target invertebrates
are generally more abundant in Bt cotton and Bt maize fields than in
non-transgenic fields managed with insecticides (Marvier et al., 2007). 
In addition, a comprehensive review of short and long term field studies
on the effects of invertebrate populations in Bt corn and cotton fields
indicated that no unreasonable adverse effects are taking place as a
result of wide scale Bt crop cultivation (Sanvido et al. 2007).  Another
review of field tests published to date concluded that the 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.   

 

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 non-target
testing be conducted at a test dose 10X the EEC whenever possible, the
test dose margin can be less than 10X where uncertainty in the system is
low or where high concentrations of test material are not possible to
achieve due to test organism feeding habits.  BPPD may also allow for
testing at lower doses in cases where many species are tested or tests
are very sensitive, although the concentration used must exceed the EEC.
 For the purposes of the non-target organism studies submitted in
support of 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. 

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	Microbially-produced 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 invertebrate testing, Gammarus fasicatus 	885.4240

 	Lyophilized Event 5307 corn leaves, V6-V8 stage	No adverse effects on
survival or reproduction of Gammarus fasicatus exposed to Event 5307
corn leaf material for 5 days.  

Classification:  Acceptable	48442533

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

	Microbially-produced 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
Microbially-produced 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
Microbially-produced 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

	Microbially-produced 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

	Microbially-produced 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

	Microbially-produced 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

Earthworm toxicity, 

Eisenia fetida	850.6200	Microbially-produced 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	Microbially-produced 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 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 treatment-related 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.

	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 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% (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-observed-effect 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
soil-deposited 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 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.

	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. 

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 anti-lepidopteran 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 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.  

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 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 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.    

 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. 

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.  

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 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.

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.

Event 5307 (coleopteran active) Environmental Risk Assessment Summary

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.

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. 

Bioassays for Interaction Between Cry1Ab and Cry1F

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.

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 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.

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 coleopteran-active
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 coleopteran-active 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 CPB-inactive
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.

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. 

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 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.    

 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 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 single-trait 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.

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 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. 

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. 

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
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 δ-endotoxin.  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);  HYPERLINK "http://www.nap.edu"
http://www.nap.edu .

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

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:  HYPERLINK
"http://www.epa.gov/scipoly/sap/2000/october/octoberfinal.pdf"
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.   HYPERLINK
"http://www.epa.gov/scipoly/sap/2002/august/august2002final.pdf"
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.   HYPERLINK
"http://www.epa.gov/scipoly/sap/meetings/2004/june/final1a.pdf"
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 Vector pNOV1300) in Event
MIR162 Maize (OECD Unique Identifier: SYN-IR162-4),  HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_00
6599.pdf"
http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006
599.pdf . 

USEPA, 2010a. Biopesticides Registration Action Document – Cry1Ab and
Cry1F Bacillus thuringiensis (Bt) Corn Plant-Incorporated Protectants, 
HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/cry1f-cry1ab-brad.pdf"
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 SYN-IR604-8,  HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/mcry3a-brad.pdf"
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 T-DNA) in Event DAS-59122-7
Corn (OECD Unique Identifier: DAS-59122-7),  HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/cry3435ab1-brad.pdf"
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 ™ 3100 [EPA Reg.
No. 67979-RL, OECD Unique ID. SYN-BTØ11-1 x SYN-IR162-4 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 SYN-IR162-4 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.

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

BPPD   SEQ CHAPTER \h \r 1 has 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 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).

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 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 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.

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. 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).

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.

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 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.  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 (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 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 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 2nd toxin

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
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. 

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.

	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 ½ 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:

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).

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 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.

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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.

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 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.

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).  HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/cry3bb1-brad.pdf"
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);

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 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   HYPERLINK  \l "Benefits"  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, 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 plant-incorporated
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 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 fifteen-year 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 determined
on a case-by-case basis, as will novel refuge configurations that may
present unique durability profiles. 

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was established for mCry3A in maize [40 CFR § 174.505].

 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].

 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.  

 OPPTS Testing Guidelines, Series 850 and 885 website: 

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

 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.

 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.   

 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.

 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.

 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. ( HYPERLINK "http://delphi.nceas.ucsb.edu/btcrops/"
http://delphi.nceas.ucsb.edu/btcrops/ ).

 The use of BPPD in this chapter  refers to the BPPD IRM Team consisting
of Alan Reynolds and Jeannette Martinez

Bacillus thuringiensis eCry3.1Ab Corn	

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