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