SEQ CHAPTER \h \r 1

BIOPESTICIDES REGISTRATION ACTION DOCUMENT

Bacillus thuringiensis modified Cry1Ab (SYN-IR67B-1) and Vip3Aa19
(SYN-IR102-7) insecticidal proteins and the genetic material necessary
for their production in COT102 X COT67B cotton

	U.S. Environmental Protection Agency

	Office of Pesticide Programs

	Biopesticides and Pollution Prevention Division

						Table of Contents

I.
Overview................................................................
...........................................................	.  3

	A.  Executive Summary

	B.  Use Profile

	C.  Regulatory History

II.	Science Assessment
........................................................................
..............................  10

	A. Product Characterization
........................................................................
...........................10

              B. Human Health
Assessment..............................................................
..................................	 25

	C. Environmental Assessment
........................................................................
......................	 43

	D. Insect Resistance Management
........................................................................
................	 85

	E. Benefits and Public Interest
Finding...........................................................
....................	106

III.     Terms and Conditions of the
Registration............................................................
120

IV.     Regulatory Position for COT102 X COT67B
Cotton...……..............	132

Regulatory Action Team

Product Characterization and Human Health

John Kough, Ph.D.

Annabel Waggoner, B.S.

Rebbeca Edelstein, Ph.D.

Sharlene Matten, Ph.D.

Chris Wozniak, Ph.D.

Environmental Fate and Effects

Zigfridas Vaituzis, Ph.D.

Annabel Waggoner, B.S.

Tessa Milofsky, M.S.

Mika Hunter, B.S.

Insect Resistance Management

Jeannette Martinez, M.S.

Benefit Assessment

Jeannette Martinez, M.S.

Biopesticides Registration Action Document Team Leader

Alan Reynolds, M.S.

Office of General Council

Angela Huskey, Esq.

Chris Kaczmarek, Esq.

Keith Matthews, Esq.I.  Overview

A.  Executive Summary

EPA has conditionally registered a new pesticide product containing
Syngenta Seeds Inc.’s new active ingredients, Bacillus thuringiensis
Vip3Aa19 (OECD Unique Identifier SYN-IR102-7) and modified Cry1Ab (OECD
Unique Identifier SYN-IR67B-1) insecticidal proteins and the genetic
material necessary for their production in COT102 X COT67B cotton.
Syngenta has trademarked this product as VipCot -- the trademark name of
VipCot will be used in this document to describe COT102 X COT67B cotton.
 The Agency has determined that the use of this pesticide is in the
public interest and that it will not cause any unreasonable adverse
effects on the environment during the time of conditional registration.

The new cotton plant-incorporated protectant, VipCot, produces its own
insecticidal proteins within the cotton plant.  These proteins were
derived from Bacillus thuringiensis (Bt), a naturally occurring soil
bacterium. The modified Cry1Ab and Vip3Aa19 proteins used in this
product control lepidopteran pests of cotton.

On June 26, 2008, tolerance exemptions under 40 CFR Part 174 were
approved for Bacillus thuringiensis modified Cry1Ab protein as
identified under OECD Unique Identifier SYN-IR67B-1 in cotton (40 CFR
174.529) and Vip3Aa proteins in corn and cotton (40 CFR 174.501).  The
exemption for Vip3Aa is inclusive of the Vip3Aa19 protein and its use in
cotton.

Benefits

Results of efficacy trials conducted in 2005 and 2006 show that VipCot
cotton and its single event cotton isolines provide good protection
against three major cotton pests:  tobacco budworm (Heliothis
virescens), cotton bollworm (Helicoverpa zea), and pink bollworm
(Pectinophora gossypiella).  The Vip3Aa19 protein expressed in VipCot
cotton has not been previously registered and provides a unique mode of
action.  When coupled with modified Cry1Ab in VipCot, the proteins have
the potential to provide benefits for insect resistance management
including:  high-dose (for both proteins expressed together) against the
major target pests, lack of cross-resistance (Vip3Aa19), and the
potential to delay development of resistance in other cotton varieties
expressing Cry toxins.   As an additional registered Bt cotton product,
VipCot will likely result in direct and indirect human and environmental
health benefits by providing growers with an additional choice of Bt
cotton option and the potential to increase grower choice and price
competition, resulting in lower seed prices for consumers and higher
adoption rates.  Registration of VipCot may also result in further
reduction of chemical insecticide use by growers.

Public Interest Finding

To grant a conditional registration under Section 3(c)(7)(C) of FIFRA,
EPA must determine that such conditional registration will, inter alia,
be in the public interest.  EPA determines whether conditional
registration of a pesticide is in the public interest in accordance with
the criteria set forth at 51 Fed. Reg. 7628 (Conditional Registration of
New Pesticides, March 5 1986).  On the basis of analysis utilizing these
criteria, EPA concludes that the use of VipCot protected cotton will be
in the public interest, because it results in direct and indirect human
and environmental health benefits by providing growers with an
additional Bt cotton product which has the potential to extend the
useful life of Bt cotton technology generally due to a novel mode of
action (Vip3Aa19) and low likelihood of cross-resistance with other Bt
Cry proteins.

Product Characterization

VipCot (COT102 x COT67B) was developed by conventional breeding of
COT102 (Vip3Aa19) plants with COT67B (modified Cry1Ab) plants.

Event COT102 cotton, which was developed by Agrobacterium-mediated
transformation of cotton using elements of a vector referred to as both
pNOV3001 and pCOT1, expresses the insecticidal protein, Vip3Aa19 as well
as a selectable marker, hygromycin B phosphotransferase (APH4).  The
Vip3Aa19 protein is intended to control several lepidopteran pests of
cotton including Heliothis virescens (tobacco budworm, TBW), Helicoverpa
zea (cotton bollworm, CBW), Spodoptera frugiperda (fall armyworm),
Spodoptera exigua (beet armyworm), and Trichoplusia ni (cabbage looper).
 Vip3A is a vegetative (i.e., produced during the vegetative stage of
bacterial growth) insecticidal protein from Bacillus thuringiensis (Bt),
a gram positive bacterium commonly found in soil.

Event COT67B cotton, which was developed by Agrobacterium-mediated
transformation of cotton using elements of vectors pNOV4641 and
pNOV1914, expresses the insecticidal protein, modified Cry1Ab.  This
protein contains an additional 26 amino acid sequence at the C-terminus
(termed the ‘Geiser motif’).  The modified Cry1Ab protein is
intended to control several lepidopteran pests of cotton including
Heliothis virescens (tobacco budworm), Helicoverpa zea (cotton
bollworm), Pectinophora gossypiella (pink bollworm), Spodoptera
frugiperda (fall armyworm), Spodoptera exigua (beet armyworm), and
Trichoplusia ni (cabbage looper).

DNA characterization (i.e., Southern blot analysis) was used to confirm
the integrity of the COT102 and COT67B inserts in the stacked product
COT102 x COT67B.  Samples from COT102 x COT67B cotton gave the same
results as those observed for the individual events, indicating that the
molecular characterization data provided for the individual events are
also applicable to COT102 x COT67B.

Protein expression data, together with data indicating that there is no
evidence of either a synergistic or antagonistic interaction between
Vip3Aa19 and modified Cry1Ab in cotton bollworm or tobacco budworm,
demonstrate that data on the individual events and individual proteins
can be used to support the safety of the COT102 x COT67B (VipCot)
combined product.

Human Health Assessment

There is a reasonable certainty that no harm will result from aggregate
exposure to the U.S. population, including infants and children, to the
modified Cry1Ab and Vip3Aa19 proteins. This includes all anticipated
dietary exposures and all other exposures for which there is reliable
information. The Agency has arrived at this conclusion because no
toxicity to mammals has been observed, nor any indication of
allergenicity potential for the plant-incorporated protectant.

Syngenta submitted four acute oral toxicity studies conducted on mice,
which all indicated that Vip3Aa is non-toxic to humans.  Three of the
studies were conducted with microbially-produced Vip3Aa proteins with
slight variations in amino acid sequence (1-2 amino acid differences),
and one study was conduced with transgenic corn leaf tissue as the test
material.  No treatment-related adverse effects were observed in any of
the studies.  The oral LD50 for mice (males, females, and combined) was
greater than 3675 mg Vip3Aa/kg body weight (the highest dose tested).
For modified Cry1Ab, an acute oral toxicity study in mice indicated that
the protein is non-toxic to humans.  Groups of five male and five female
mice were given 0 or 1830 mg/kg bodyweight microbially-produced modified
Cry1Ab by oral gavage as a single dose.  There were no effects on
clinical condition, body weight, food consumption, clinical pathology,
organ weight, or macroscopic or microscopic pathology that were
attributed to the test substance.

Since Vip3Aa and modified Cry1Ab are proteins, allergenic potential was
also considered. Currently, no definitive tests for determining the
allergenic potential of novel proteins exist.  Therefore, EPA uses a
weight-of-evidence approach where the following factors are considered:
source of the trait; amino acid sequence comparison with known
allergens; and biochemical properties of the protein, including in vitro
digestibility in simulated gastric fluid (SGF) and glycosylation.  This
approach is consistent with the approach outlined in the Annex to the
Codex Alimentarius “Guideline for the Conduct of Food Safety
Assessment of Foods Derived from Recombinant-DNA Plants.”  The
allergenicity assessment for Vip3Aa and modified Cry1Ab is as follows:

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
Vip3Aa19 and modified Cry1Ab with known allergens showed no significant
sequence identity over 80 amino acids or identity at the level of eight
contiguous amino acid residues.

Digestibility.  The Vip3Aa and modified Cry1Ab proteins were digested
rapidly in simulated gastric fluid containing pepsin.

Glycosylation.  Vip3Aa and modified Cry1Ab (expressed in cotton) were
shown not to be glycosylated.

Conclusion.  Considering all of the available information, EPA has
concluded that the potential for Vip3Aa and modified Cry1Ab to be food
allergens is minimal.

Environmental Assessment

The Agency concludes that for the VipCot cotton breeding stack (COT102 x
COT67B, containing modified Cry1Ab and Vip3Aa19) no unreasonable adverse
effects will result to the environment or any federally-listed
threatened or endangered species from commercial cultivation of COT102 x
COT67B cotton.  This conclusion is based on prior assessments conducted
on Vip3Aa and Cry1Ab proteins individually.  Furthermore, the Agency has
determined that Events COT102, COT67B, and VipCot cotton will have No
Effect (NE) on endangered and/or threatened species listed by the US
Fish and Wildlife Service (USFWS) and the National Marine Fisheries
Services (NMFS), including mammals, birds, terrestrial and aquatic
plants, and invertebrate species. Therefore, no consultation with the
USFWS is required under the Endangered Species Act.

The Agency believes that cultivation of VipCot cotton may result in
fewer adverse impacts to non-target organisms than result from the use
of chemical pesticides. Under normal circumstances, Bt cotton requires
substantially fewer applications of chemical pesticides. This should
result in fewer adverse impacts to non-target organisms because
application of nonspecific conventional chemical pesticides is known to
have an adverse effect on non-target beneficial organisms found living
in the complex environment of an agricultural field.  Many of these
beneficial organisms are important integrated pest management controls
(IPM) for secondary pests such as aphids and leafhoppers. Therefore, the
overall result of cultivation of VipCot cotton, expressing Vip3Aa19 and
modified Cry1Ab proteins, is that the number of chemical insecticide
applications for non-target pest control will be reduced for management
of multiple pest problems.

Insect Resistance Management

In order to reduce the possibility of the target pests developing
resistance to Vip3Aa19 and modified Cry1Ab (as expressed in VipCot
cotton), EPA is requiring Syngenta Seeds, Inc. to ensure that a portion
of the planted acreage of this product be set aside where non-Bt cotton
will be grown to serve as a “refuge.”  Under the established refuge
strategy for Bt cotton, growers can choose from three structured refuge
options:

Option 1:   95:5 external structured, unsprayed refuge; 150 ft wide,
within ½ mile of edge of field.

Option 2:   80:20 external sprayed refuge; within 1 linear mile,
preferably ½ mile, of edge of field.

Option 3:   95:5 embedded refuge; contiguous or within 1 mile2 of field
and 150 ft wide.

In addition to the refuge options above, growers of VipCot may
participate in a community refuge plan in which multiple growers
contribute to the overall required refuge acres by planting 20%
external, sprayed or 5% external, unsprayed refuge.

BPPD has concluded that based on the modeling, dose, and efficacy
studies, the requested refuge options 1-3 and community refuge plan are
acceptable for VipCot cotton.  Syngenta will also be required to develop
and conduct a resistance monitoring program for Vip3Aa19 and modified
Cry1Ab with the major target pests (cotton bollworm, tobacco budworm,
and pink bollworm).  Additional requirements for remedial action (in the
event of resistance), grower education, compliance assurance, and annual
reported will also be implemented for VipCot as terms of registration.

B.  Use Profile

Pesticide Name:  Bacillus thuringiensis Vip3Aa19 (OECD Unique Identifier
SYN-IR102-7) and modified Cry1Ab (OECD Unique Identifier SYN-IR67B-1)
insecticidal proteins and the genetic material necessary for their
production in COT102 X COT67B cotton

Trade and Other Names: VipCot Cotton; COT102 X COT67B Cotton

OPP Chemical Code: 006499 (Vip3Aa19) and 006529 (modified Cry1Ab)

Basic Manufacturers:  Syngenta Seeds, Inc.

Type of Pesticide:  Plant-Incorporated Protectant

Uses:  Cotton

Target Pest(s):  tobacco budworm, cotton bollworm, pink bollworm

C.  Regulatory History

Syngenta Seeds, Inc. was issued an Experimental Use Permit (EUP) for
VipCot Bt cotton containing Vip3Aa19 (Event COT102) and modified Cry1Ab
(Event COT67B) on April 26, 2007 (EPA Reg. No. 67979-EUP-7).  These
proteins were selected to provide protection of cotton from feeding
damage caused by major lepidopteran pests including tobacco budworm,
cotton bollworm, and pink bollworm.  On April 26, 2007, EPA established
a temporary exemption from the requirement of a tolerance for Vip3Aa19
(72 FR 26300, amended 72 CFR 40752; 40 CFR 174.501) in the food and feed
commodities of cotton.  For the purpose of the EUP, modified Cry1Ab was
determined to be covered under the permanent tolerance exemption for
Cry1Ab in all crops (40 CFR 174.511).   Both the EUP and temporary
tolerance exemption were originally set to expire on May 1, 2008.
However, Syngenta was granted an extension of both the EUP and temporary
tolerance exemption (72 FR 68744) on November 27, 2007 which expire on
May 1, 2009.

A separate EUP (EPA Reg. No. 67979-EUP-5) was previously issued to
Syngenta for two Bt cotton events (Event COT202 and COT203) containing
Vip3A.  These two events were not part of the more recent VipCot EUP and
have not been proposed for commercial registration.  This EUP expired on
March 31, 2006.

On December 14, 2006, Syngenta submitted an application (EPA Reg. No.
67979-O) to register VipCot (Event COT 102 x Event COT67B) under Section
3 of the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA).  On
April 5, 2007, Syngenta submitted a second application for a seed
increase registration (EPA Reg. No. 67979-RR).  This application was
subsequently withdrawn by the registrant on January 7, 2008.

On September 6, 2007, Syngenta submitted a petition to EPA under the
Federal Food, Drug, and Cosmetic Act (FFDCA), as amended by the Food
Quality Protection Act of 1996 (FQPA), requesting a permanent tolerance
exemption for Vip3Aa in all plants (PP 7F7254).  A separate petition was
submitted to request a permanent exemption for modified Cry1Ab in all
plants on November 9, 2007 (PP 7F7290).  After review of the supporting
data, EPA determined that the permanent tolerance exemptions would be
limited to corn and cotton (Vip3Aa) and cotton (modified Cry1Ab).

On June 26, 2008 (73 FR 45620 and 73 FR 40760), the Agency established
permanent exemptions from the requirement of a tolerance for residues of
the Bacillus thuringiensis Vip3Aa proteins in corn and cotton (40 CFR
174.501) and modified Cry1Ab protein as identified under OECD Unique
Identifier SYN-IR67B-1 in cotton (40 CFR 174.529) when used as
plant–incorporated protectants.

On June 26, 2008, a conditional registration was issued for VipCot Bt
Cotton (EPA Reg. No. 67979-9).

II. Science Assessment

The classifications that are found for each data submission are assigned
by 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 CFR Part 158.  Both “ACCEPTABLE” and “SUPPLEMENTAL” studies
may be used in the risk assessment process as appropriate.  An
“UNACCEPTABLE” rating indicates that new data need to be submitted.

	II.A.  Product Characterization

II.A.1. Event COT102 Cotton (OECD Unique Identifier: SYN-IR102-7)
Expressing Vip3Aa19

Event COT102 cotton, which was developed by Agrobacterium-mediated
transformation of cotton using elements of a vector referred to as both
pNOV3001 and pCOT1, expresses the insecticidal protein, Vip3Aa19 as well
as a selectable marker, hygromycin B phosphotransferase (APH4).  The
Vip3Aa19 protein is intended to control several lepidopteran pests of
cotton including, but not limited to, Helicoverpa zea (cotton
bollworm/corn earworm), Heliothis virescens (tobacco budworm),
Spodoptera frugiperda (fall armyworm), Spodoptera exigua (beet
armyworm), and Trichoplusia ni (cabbage looper).  Vip3A is a vegetative
(i.e., produced during the vegetative stage of bacterial growth)
insecticidal protein from Bacillus thuringiensis (Bt), a gram positive
bacterium commonly found in soil.

Transformation System:

COT102 cotton was produced by Agrobacterium tumefaciens-mediated
transformation of hypocotyls of Gossypium hirsutum L. cultivar Coker 312
with plasmid pNOV3001 (also referred to as pCOT1).  Plasmid pNOV3001
(pCOT1) contains T-DNA with the vip3Aa19 and aph4 expression cassettes.
The vip3Aa19 expression cassette contains the vip3Aa19 coding sequence
under the regulation of the Act2 promoter and intron (derived from
Arabidopsis thaliana), and NOS terminator (derived from Agrobacterium
tumefaciens).  The aph4 expression cassette contains the aph4 coding
sequence under the regulation of the Ubq3 promoter and intron (derived
from Arabidopsis thaliana) and the NOS terminator (derived from
Agrobacterium tumefaciens).  The vip3Aa19 gene encodes a protein that
differs from the Vip3Aa1 protein from Bacillus thuringiensis strain AB88
by one amino acid at position 284 (The vip3Aa1 gene encodes lysine at
position 284, and the vip3Aa19 gene encodes glutamine).  Vip3Aa19
confers resistance to several lepidopteran pests.  The aph4 gene encodes
hygromycin B phosphotransferase (APH4), an enzyme that catalyzes the
phosphorylation of hygromycin and some related aminoglycosides.
Expression of APH4 allows growth in the presence of hygromycin and was
used as a selectable marker, enabling selection of transformed cells.

Characterization of the DNA Inserted in the Plant and Inheritance and
Stability:

Characterization of the DNA isolated from event COT102 cotton using
restriction enzyme digests and Southern blot analysis as well as DNA
sequencing indicates that the DNA was inserted in the cotton genome at a
single locus, and the insert contains one copy each of the vip3Aa19 and
aph4 expression cassettes.  There were no other detectable elements
other than those associated with the respective cassettes.  No backbone
sequences from plasmid pNOV3001 (pCOT1) were detected in the cotton
genome.  Southern blot analysis and protein expression data also
demonstrated the stability of the insert over multiple generations.

Protein Characterization:

The insecticidal protein produced in event COT102 cotton, designated as
Vip3Aa19, is a variant of the naturally occurring Vip3Aa1 protein
isolated from Bacillus thuringiensis strain AB88, differing from the
Vip3Aa1 protein by one amino acid (Vip3Aa19 contains a glutamine at
position 284, while Vip3Aa1 contains a lysine).  Both proteins are 789
amino acids in length and have a molecular weight of approximately 89
kDa.  Syngenta has also developed a transgenic corn variety, MIR162,
that produces another variant, designated as Vip3Aa20, differing from
the naturally occurring Vip3Aa1 protein by two amino acids; at position
284, Vip3Aa20 has the same amino acid substitution as Vip3Aa19 (i.e.,
K284Q), and in addition, at position 129, Vip3Aa20 contains an
isoleucine, while Vip3Aa1 contains a methionine (M129I).

The following techniques were used to characterize and compare the
plant-produced and the E. coli-produced Vip3Aa proteins: sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot
analysis, densitometry, mass spectrometry, glycosylation analysis,
N-terminal amino acid sequencing, and insecticidal activity assays.
Glycoslyation analysis indicated that the proteins are not glycoslyated.
 These analyses demonstrated the structural and functional similarity
between the plant-produced Vip3Aa19 and the E. coli-produced Vip3Aa19,
Vip3Aa20, and Vip3Aa1 proteins and justified the use of E. coli-produced
proteins in toxicity studies.

Analytical Detection Methods:

Syngenta has provided a validation study for SeedChek Vip3A/FLCry1Ab, a
lateral flow test kit that detects both Vip3A and Cry1Ab.  The SeedChek
Vip3A/FLCry1Ab lateral flow test kit was tested for the qualitative
detection of modified Cry1Ab and Vip3A proteins in cotton seed and
cotton leaf.  The study showed that the SeedChek kit is able to detect
Vip3A and Cry1Ab in both cotton seed and cotton leaf.  No unexpected
cross reactivity with other transgenic varieties or nontransgenic
controls was observed.  An independent lab validation of this method is
still needed.

Protein Expresson:

Expression level data were provided for Vip3Aa19 and APH4 in different
plant tissues and at different growth stages in COT102.

Table 1. Mean Expression Levels of Vip3Aa19 and APH4 from COT102 Plant
Tissues

Tissue Type	Vip3Aa19

(µg/g dry weight + standard deviation)	APH4

(µg/g dry weight + standard deviation)

Leaves*	44 + 10 - 277 + 41 	< 0.42 – 8.2 + 1.4

Squares	116 + 22	2.2 + 0.4

Flowers	162	1.68

Pollen	3.47	64.3

Bolls	19 + 4	< 0.39

Whole Plants	25 + 4	< 0.37

Seed	7 + 2	1.4 + 0.3

Roots	16 + 2	0.53 + 0.11

*Ranges reflect means at different growth stages for leaves

The data submitted for product characterization for event COT102 cotton
are summarized in Table 2 below.

Table 2. Product Characterization Data Submitted for Event COT102 Cotton
(reviewed in Edelstein 2008 unless otherwise noted)

Study Type/Title

Summary

MRID #

Expression Levels/ Quantitation of VIP3A and APH4 Protein in Cotton
Tissues and Whole Plants Derived from Transformation Event COT102

Transgenic cotton plants (COT102) and a non-transgenic isoline (Coker
312) were grown concurrently in 2001 in Camilla, GA; Maricopa, AZ; and
Idalou, TX.  Ten whole transgenic plants (including roots) and two
control plants were harvested approximately 2, 4, 9, 13, 15, and 22 week
post-emergence (stages:  four-leaf, squaring, first white bloom, peak
bloom, first open boll, pre-harvest, respectively). Tissue extracts were
analyzed for VIP3A and APH4 by ELISA.  VIP3A protein was detected in
COT102 whole plants, leaves, roots, squares, and bolls at all six
developmental stages examined. VIP3A levels varied in all plant tissues,
generally declined with time, but stayed constant in the roots. The
highest levels were found in leaves at the squaring stage (mean of 8.56
to 10.78 μg VIP3A/g fresh tissue).  Low VIP3A levels were found in seed
(mean of 2.51 to 3.23 μg VIP3A/g) and in pollen (1.09 μg VIP3A/g).
VIP3A was not detected in cotton fiber or nectar.  The protein marker,
APH4, was detected in COT102 plants at low, non-quantifiable levels at
some developmental stages in leaves, roots, bolls, squares, and whole
plants and at quantifiable levels in pollen (2.25 μg APH4/g air-dried
pollen).  APH4 was not detected in cotton fiber or nectar.  Geographic
location appeared not to have a significant effect on VIP3A levels, but
no statistical analysis was done.  APH4 levels appears to be similar
across locations, but the lack of data points in many instances and the
detectable levels falling below the level of quantitation (LOQ) do not
allow for any definitive conclusions to be made.  The estimated amount
of VIP3A/acre cotton varied considerably among the developmental stages
with the greatest mean level found at the peak bloom stage (105.80 g
VIP3A/acre based on whole plant VIP3A levels).

Classification:  ACCEPTABLE

45835801

Characterization of Inserted DNA/Molecular Characterization and Genetic
Stability of Event COT102b

Southern blot analysis and DNA sequencing suggest that event COT102 has
one transgene insertion site with a single copy of intact vip3A(a) and
aph4 expression cassettes (containing one copy of the vip3A(a) gene,
aph4 gene, actin-2 promoter, and ubq3 promoter). DNA sequence alignment
revealed an exact sequence match between the pCOT-1 vector and event
COT102, and showed the lack of Agrobacterium sequence beyond the T-DNA
borders. VIP3 protein expression measurement (by ELISA) of five
generations of COT102 seedlings (F1, BC1F2, BC2F1, BC2F2, and BC3F1)
showed that the vip3A(a) gene was stable across generations and
segregated in a Mendelian fashion, consistent with a single transgene
insertion site.  MRID 458358-02 provided very scant experimental
details. Insufficient experimental methods details were provided for the
Southern blots, DNA cloning and sequencing, PCR analysis, and protein
detection and segregation analysis by ELISA, precluding confirmation of
their appropriateness by an independent reviewer. Sample Southern blots
demonstrating the integration copy number and lack of rearrangements
through appropriate restriction analyses must be provided in order to
assess the results of this study. Further information is required
regarding the number of plants utilized in the segregation and
heritability analysis.

Classification: SUPPLEMENTAL, upgradeable to acceptable pending
submission of additional methods details and correction/clarification of
typographic errors in Figure 1, Figure 2, and/or the text of MRID
458358-02.

Superseded by MRID 47017603

45835802

Characteristics of Bacillus thuringiensis VIP3A Protein and VIP3A Cotton
Plants Derived from Event COT102b

The Bacillus thuringiensis (Bt) VIP3A insect control protein as
expressed in transgenic cotton seed confers protection against the
bollworm complex and other lepidopteran cotton pests.  The seeds are
derived from transgenic cotton event COT102, which contains the
insecticidal gene via plasmid vector pCOT1.  The product active
ingredient is (0.0015 % dry weight Bacillus thuringiensis VIP3A Protein
and the genetic material necessary for its production (pCOT1 in cotton).
 The product also contains (0.0001% dry weight marker protein and the
genetic material necessary for its production (pCOT1 in cotton).  VIP3A
protein in transgenic cotton plants derived from Event COT102, is
produced by a synthetic vip3A(a) gene, which encodes a polypeptide of
789 amino acids.  The VIP3A toxin is proteolytically activated to a
toxin core in the lepidopteran larval midgut and forms pores in the gut
membranes of sensitive species.  Several formulated microbial Bt
products containing VIP3A-like proteins and the genetic components in
plasmid pCOT1, as well as its expression analysis, are described in MRID
457665-01.

Classification: ACCEPTABLE.  The wide certified limits of the active
ingredient need to be explained, although they are within the bounds
covered by the acute oral toxicity studies submitted for review.

45766501

Characterization of the active ingredient/Characterization of VIP3A
Protein Produced in COT102-Derived Cotton and Comparison with VIP3A
Protein Expressed in Both Maize (Corn) Derived from Event PACHA and
Recombinant Escherichia colib

VIP3A protein produced in cotton plants derived from transgenic cotton
event “COT102" was characterized for its biochemical and functional
similarity with VIP3A expressed in recombinant Escherichia coli and
“Pacha” derived transgenic maize plants. Samples of purified VIP3A
protein from E. coli and maize were dissolved in buffer for analysis by
SDS-PAGE and Western blotting.  VIP3A from cotton leaves was extracted
following published procedures and prepared for SDS-PAGE and Western
blotting.  VIP3A proteins from all three sources were determined to have
the predicted molecular weight of ca. 89,000 and cross-reacted
immunologically with the same anti-VIP3A antibody.   No evidence of any
post-translational modification of VIP3A was observed in any of the
three Vip3A protein sources. Peptides representing ca. 85% (673/789) of
the complete VIP3A amino acid sequence were identified by mass spectral
analysis of cotton produced VIP3A protein. Amino acid sequences
corresponded identically to the predicted amino acid sequence of the
VIP3A protein. Comparisons of bioactivity of E. coli-expressed and
cotton-expressed VIP3A protein in larvae of four lepidopteran species
demonstrated comparable activities, with the exception of the tobacco
budworm bioassays (TBW).  A 35% difference in mortality was noted in TBW
assays comparing these two sources of test substance. In the absence of
an in-depth statistical analysis, it is not possible to assign a
particular factor as the causal agent in delimiting this result. Given
that both test substances contain other constituents, it is difficult to
assess the reason for this observation. TBW is considered as one of the
least sensitive species of lepidopteran insects evaluated. A similar
rank order of species sensitivity was found for both test solutions; FAW
was the most sensitive to VIP3A, while CBW and TBW were the least
sensitive.  These data indicate that VIP3A proteins from recombinant E.
coli, Pacha-derived maize and event COT102-derived cotton are
substantially equivalent.

Classification:  ACCEPTABLE

45835812

Expression Level/ Analysis of Processed COT102 Cottonseed Products for
Yield and Presence of Gossypol and Vip3A Proteinb

Processing transgenic COT102 and control Coker 312 cotton seeds resulted
in similar yields for the hulls, lint, kernels, refined oil, and
de-fatted meal.  Analysis of the refined oil and de-fatted meal
(non-toasted and toasted) by ELISA detected VIP3A protein in COT102 meal
but not in oil, and not in meal or oil from control seeds.  Analysis of
both COT102 and Coker 312 de-fatted meal for the plant toxin gossypol
detected free gossypol (HPLC method) and total gossypol (free +
protein-bound; spectrophotometric method).  Refined oil had >100-fold
lower levels of total gossypol than meal.  MRID 45835803 provided
inadequate and/or conflicting details for some experimental methods and
results.

Classification: ACCEPTABLE. Submission of additional methods details and
correction and/or clarification of the MRID 458358-03 text as listed
under “Deficiencies” is, however, recommended to ensure adequate
recording in the official record.

The additional information was subsequently determined to be unnecessary
because no adverse effects were observed in the nontarget studies.

45835803

The mode of action of the Bacillus thuringiensis vegetative insecticidal
protein Vip3A differs from that of Cry1Ab delta-endotoxin 	This
publication (Lee et al., 2003), which examined the differences in the
mechanism of insecticidal activity of Cry1Ab and Vip3A, was submitted by
the registrant to provide additional product characterization data,
specifically Vip3A’s mode of action. The submitted publication
examined differences in the mechanism of insecticidal activity of Cry1Ab
and Vip3A proteins.  Ligand blotting showed that activated Cry1Ab and
Vip3A-G (Vip3A proteolytically cleaved with lepidopteran gut juice)
bound different receptor molecules in midgut of Tobacco hornworm
(Manducta sexta, Linnaeus) and that Vip3A-G did not bind Cry1A
receptors.  Voltage clamping assays showed that Vip3A-G formed distinct
pores in dissected midgut from M. sexta but not in the monarch butterfly
(Danaus plexippus, Linnaeus).  Cry1Ab and Vip3A both formed
voltage-independent and cation-selective stable ion channels in planar
lipid bilayers, but their primary conductance state and cation
specificity differed.

Classification: ACCEPTABLE

	46880801

Characterization of Test Substance/Re-Characterization of Vip3A Protein
Test Substance (Vip3A-0204)

The purpose of this study was to re-characterize the microbially
produced test substance, VIP3A-0204.  The purity, integrity, and
bioactivity of the test substance were determined and compared with
previous analyses after being stored ca. 15 months under desiccation at
-20 (C.  Total protein in VIP3A-0204 was quantified
spectrophotometrically, and the purity was determined using SDS-PAGE
followed by densitometric analysis. The integrity of the Vip3Aa19
protein in test substance VIP3A-0204 was determined using Western blot
analysis, and bioactivity was assessed in insect feeding assays using
freshly hatched first-instar S. frugiperda (fall army worm) larvae.

This re-characterization study demonstrated that VIP3A-0204 largely
retained its insecticidal activity (LC50 of 34 ng Vip3A/cm2 diet surface
vs. 45.1 initially) after storage for 15 months.  The purity of test
substance VIP3A-0204 was determined to be ca. 92% Vip3Aa19 by weight.
Western blot analysis revealed a dominant immunoreactive band
corresponding to the predicted molecular weight of Vip3Aa19 of ca. 89
kDa.  These results are similar to those obtained in previous analyses,
demonstrating that the test substance is stable when stored desiccated
at -20 (C for approximately 15 months.

Classification: ACCEPTABLE

47017602

Characterization of the inserted DNA/ Additional Molecular
Characterization of Event COT102 Cotton by Southern Analysis

Molecular analysis of event COT 102 was performed using restriction
enzyme digestion and Southern blot analysis to determine the number of
inserts, copy number of functional elements, and the presence or absence
of plasmid backbone sequences.  This study also assessed the inheritance
and stability of the insert.  Data from the Southern analyses
demonstrated that the BC4F1 generation of COT102 cotton: (1) contains a
single intact insert; (2) contains a single copy of the vip3Aa19 gene
and the aph4 gene; (3) contains a single copy of the Act2 promoter; (4)
contains a single copy of the Ubq3 promoter; (5) does not contain any
detectable backbone sequences from the transformation plasmid pCOT1; and
(6) the insert is stably integrated into the cotton genome.  These
results are consistent with results from previous molecular analysis
studies on event COT 102.

Classification: ACCEPTABLE

47017603

Inheritance and Stability/ Stability of Vip3Aa19 and APH4 Protein
Expression Across Multiple Generations of Event COT102 Cotton	The
purpose of this study was to use ELISA to analyze the levels of
expression of the Vip3Aa19 and hygromycin B phosphotransferase (APH4)
proteins in leaves (collected at the 1st white bloom stage) of three
generations (F1, BC1F1, and BC4F1) of Event COT102 cotton.  The levels
of Vip3Aa19 protein measured were comparable (ca. 60 µg/g dry weight)
in all three generations analyzed.  APH4 protein was detectable in all
three generations analyzed, but the concentrations were below the limit
of quantification (LOQ).  The consistency of Vip3Aa19 and APH4 protein
concentrations demonstrate the stability of transgenic protein
expression across multiple generations of COT102 cotton at the 1st white
bloom stage.

Classification: ACCEPTABLE

47017609

II.A.2. Event COT67B Cotton (OECD Unique Identifier: SYN-IR67B-1)
Expressing Modified Cry1Ab

Event COT67B cotton, which was developed by Agrobacterium-mediated
transformation of cotton using elements of vectors pNOV4641 and
pNOV1914, expresses the insecticidal protein, modified Cry1Ab.  This
protein contains an additional 26 amino acid sequence at the C-terminus
(termed the ‘Geiser motif’).  The modified Cry1Ab protein is
intended to control several lepidopteran pests of cotton including, but
not limited to, Helicoverpa zea (cotton bollworm/corn earworm),
Heliothis virescens (tobacco budworm), Spodoptera frugiperda (fall
armyworm), Spodoptera exigua (beet armyworm), and Trichoplusia ni
(cabbage looper).

Transformation System:

COT67B cotton was produced by Agrobacterium tumefaciens-mediated
cotransformation of Gossypium hirsutum L. cultivar Coker 312 using
transformation vectors pNOV4641 and pNOV1914, each carrying one T-DNA.
Plasmid pNOV4641 contains a full-length cry1Ab gene that encodes a
full-length Cry1Ab protein that is identical to the Cry1Ab protein
produced by Bacillus thuringiensis subsp. kurstaki strain HD-1, except
that it contains an additional 26 amino acids, which Syngenta describes
as the ‘Geiser motif,’ in the C-terminal portion of the protein.
The cry1Ab gene is under the regulation of the Act2 promoter and intron
(derived from Arabidopsis thaliana) and NOS terminator (derived from
Agrobacterium tumefaciens).  Plasmid pNOV1914 contains a hygromycin B
phosphotransferase gene (aph4) derived from Escherichia coli that
confers resistance to the antibiotic hygromycin B and was used as a
selectable marker.  The two-T-DNA system enabled Syngenta to separate
the two inserts by traditional breeding.  COT67B cotton contains only
the T-DNA from plasmid pNOV4641 encoding the modified Cry1Ab protein;
the T-DNA from pNOV1914 containing the aph4 gene is absent.

Characterization of the DNA Inserted in the Plant and Inheritance and
Stability:

Characterization of the DNA isolated from event COT67B cotton using
restriction enzyme digests and Southern blot analysis as well as DNA
sequencing indicates that the DNA was inserted in the cotton genome at a
single locus, and the insert contains one copy of the cry1Ab gene.  No
backbone sequences from the transformation plasmid pNOV4641 were found
in COT67B.  The left border and the adjacent 13 bp of the insert along
with 24 bp of the right border (RB) were deleted during the insertion of
the T-DNA.  However, such deletions are common during transformation and
do not affect the functioning of the T-DNA itself.  Additionally, the
analysis showed that COT67B cotton does not contain the selectable
marker gene, hygromycin B phosphotransferase (aph4), the Ubq3 promoter
from the transformation plasmid pNOV1914, or any backbone sequences from
pNOV1914.  Inheritance and stability studies of the cry1Ab gene in
COT67B verified that it is stably integrated into the cotton genome,
segregating in an expected Mendelian fashion of 1:1.

Protein Characterization:

Event COT67B expresses a full-length Cry1Ab protein that is identical to
the Cry1Ab protein produced by Bacillus thuringiensis subsp. kurstaki
strain HD-1, except that it contains an additional 26 amino acids
(described by Syngenta as the ‘Geiser motif’) in the C-terminal
portion of the protein.  Syngenta states that the additional amino acids
have been included because the insertion made fermentation in Bacillus
thuringiensis more efficient, but they have no impact on insecticidal
activity.

The following techniques were used to characterize and compare the
plant-produced and the E. coli-produced modified Cry1Ab proteins: sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western
blot analysis, densitometry, mass spectrometry, glycosylation analysis,
N-terminal amino acid sequencing, and insecticidal activity assays.
Glycoslyation analysis indicated that the proteins are not glycoslyated.
 These analyses demonstrated the structural and functional similarity
between the plant-produced and the E. coli-produced modified Cry1Ab
proteins and justified the use of E. coli-produced protein in toxicity
studies.

Analytical Detection Methods:

Syngenta has provided a validation study for SeedChek Vip3A/FLCry1Ab, a
lateral flow test kit that detects both Vip3A and Cry1Ab.  The SeedChek
Vip3A/FLCry1Ab lateral flow test kit was tested for the qualitative
detection of modified Cry1Ab and Vip3A proteins in cotton seed and
cotton leaf.  The study showed that the SeedChek kit is able to detect
Vip3A and Cry1Ab in both cotton seed and cotton leaf, and no unexpected
cross reactivity with other transgenic varieties or nontransgenic
controls was observed.  An independent lab validation of this method is
still needed.

Protein Expresson:

Expression level data were provided for modified Cry1Ab in different
plant tissues and at different growth stages in event COT67B cotton and
summary results are provided in Table 3 below.  The data were produced
using an ELISA method.

Table 3. Mean Cry1Ab Expression levels in Event COT67B Cotton.

Tissue Type	Cry1Ab

(µg/g dry weight + standard deviation)*

Leaves	65 + 9 – 158 + 40

Squares	93 + 13

Flowers	101

Pollen	12.1

Bolls	47 + 7

Whole Plants	26 + 2

Seed	29 + 5

Roots	17 + 1

*Range reflects means at different growth stages for leaves

Table 4 provides summaries of the product characterization studies and
data provided.

Table 4. Product Characterization Data Submitted for Event COT67B
(reviewed in Edelstein 2008 unless otherwise noted)

Study Type/Title

Summary

MRID #

Characterization of Inserted DNA/ Harper, B. (2006). Molecular
characterization of Event COT67B cotton.

Report No. SSB-125-06.

The purpose of this study was to determine the DNA sequence and
contiguousness of the full length cry1Ab (flcry1Ab) gene present in
Syngenta’s COT67B cotton and its inheritance ratio across generations.
 COT67B cotton plants express a modified full length Cry1Ab Bacillus
thuringiensis protein (FLCry1Ab) that contain an additional 26 amino
acids in the C-terminal portion of the protein described as the
“Geiser motif.” FLCry1Ab confers resistance to certain lepidopteran
insects in cotton. The T-DNA insert (via the pNOV4641 plasmid) in COT67B
cotton was analyzed by Southern blots and DNA sequencing. These analyses
confirmed that there was a single, contiguous copy of the flcry1Ab gene
present in COT67B. No backbone sequences from the transformation plasmid
pNOV4641 were found in COT67B. The left border (LB) and the adjacent 13
bp of the insert along with 24 bp of the right border (RB) were deleted
during the insertion of the T-DNA. However, such deletions are common
during transformation and do not affect the functioning of the T-DNA
itself. Additionally, COT67B cotton did not contain the selectable
marker gene, hygromycin B phosphotransferase (aph4), or the Ubq3
promoter from the transformation plasmid pNOV1914 and was also free of
any backbone sequences from pNOV1914.  Inheritance studies of the
flcry1Ab gene in COT67B verified that it is segregating in an expected
Mendelian fashion of 1:1.

Classification:  ACCEPTABLE

46885901

Expression Levels/ Hill, K. (2006). Quantification of Cry1Ab protein in
Event COT67B cotton tissues and whole plants.

Report No.SSB-022-06d

ol samples were either below the limit of detection (LOD) or below the
limit of quantification (LOQ). The negative control seed from Quitman,
GA was determined to have a low level of Cry1Ab (0.24 μg/g dw) that was
likely due to contamination during processing or extraction.  The
average relative extraction efficiency for the various plant tissues
analyzed varied between 70.7% for whole plants to 78.5% for pollen.  The
absolute amount of Cry1Ab in the cotton tissue samples is unknown and
some Cry1Ab may be unextractable with the methods used.   Extraction
efficiency for the purposes of satisfying the analytical method would
need to use a spike-recovery method. Several deviations from the
protocol were noted by the study authors, but none of these affected the
overall conclusions of the study.

Across all growth stages, mean Cry1Ab concentrations (averaged across
locations) measured in young leaves, old leaves and roots of COT67B
cotton ranged from 87.70 - 323.84, 194.02 - 255.74, and 12.61 - 56.56
μg/g dry weight (dw), respectively. Mean Cry1Ab concentrations measured
in bolls (collected at 1st open boll), whole plants (collected at
pre-harvest), and seed (collected at pre-harvest) averaged 45.24, 42.87,
and 25.17 μg/g dw across locations, respectively. Cry1Ab concentrations
in flowers and pollen collected at the Winnsboro, LA site at peak-bloom
averaged 161.74 and 5.45 μg/g dw, respectively. Cry1Ab concentrations
in nectar taken from the same cotton plants was not detectable (limit of
detection = 0.0002 μg/mL). Cry1Ab concentrations in fiber samples
collected at this site at pre-harvest was <0.02 μg/g dw

The average Cry1Ab protein per acre and per hectare in pre-harvest
COT67B plants collected from 4 sites was determined assuming a planting
density of 50,000 plants/acre (123,500 plants/hectare). The average
Cry1Ab protein concentration ranged from 46 to 183 g/acre (115 to 451
g/hectare).

Classification:  ACCEPTABLE for the purposes of supporting the
Experimental Use Permit.  Statistically-valid trends in the data (e.g.,
expression level differences between tissue types, across developmental
stages, between locations) cannot be made.  For a quantitative analysis,
it is recommended that the expression data submitted to support the
Section 3 registration include an appropriate statistical analysis.

Superseded by MRID 47017607.

46885902

Characterization of test substance/Characterization of Cry1Ab Test
Substance FLCRY1AB-0103 and Certificate of Analysis

The purity, integrity, and bioactivity of E. coli-produced test
substance FLCRY1AB-0103, containing modified full-length Cry1Ab, were
determined initially and after ca. 5 months of being stored under
desiccation at -20 (C.  The purity of test substance FLCRY1AB-0103 was
determined to be ca. 86%, both before and after storage, and Western
blot analysis of the test substance showed a dominant immunoreactive
band corresponding to the predicted molecular weight of ca. 133.5 kDa
before and after storage.  N-terminal sequencing confirmed that the
first 12 amino acids of the test protein corresponded to the predicted
N-terminal sequence of Cry1Ab.  The test substance was insecticidally
active and had a 72-hour LC50 of 3.7 ng Cry1Ab/cm2 diet surface against
first instar larvae of the European corn borer. Re-analysis of
FLCRY1AB-0103 ca. 5 months after the initial analysis demonstrated that
the test substance retained insecticidal activity when stored desiccated
at -20 (C.

Classification: ACCEPTABLE

47017604

Characterization of test substance/Re-Characterization of Cry1Ab Test
Substance FLCRY1AB-0103

The purpose of this study was to re-characterize the purity, integrity,
and bioactivity of microbially produced test substance FLCRY1AB-0103
(containing modified full-length Cry1Ab) after storage at -20 (C for ca.
14 months.  Total protein in test substance FLCRY1AB-0103 was quantified
spectrophotometrically by measuring its absorption at 280 nm (A280
method). The purity of test substance FLCRY1AB-0103 was calculated from
the total sample weight and the total protein as determined by the A280
method in conjunction with densitometry data after electrophoretic
separation. The integrity of the Cry1Ab protein in test substance
FLCRY1AB-0103 was determined using Western blot analysis. Bioactivity of
the Cry1Ab protein in FLCRY1AB-0103 was assessed in insect feeding
assays using freshly hatched first-instar O. nubilalis (European corn
borer) larvae.  The results demonstrated that the test substance
remained intact and retained insecticidal activity during this storage
period.

Classification: ACCEPTABLE

47017605

Characterization of Expressed Substance/ Characterization of the Cry1Ab
Protein Produced in Event COT67B-Derived Cotton Plants and Comparison
with Cry1Ab Protein Produced in Recombinant Escherichia coli

The purpose of this study was to use various biochemical and functional
parameters to demonstrate the biochemical equivalence between the Cry1Ab
protein expressed in transgenic Event COT67B cotton and the Cry1Ab
protein contained in test substance FLCRY1AB-0103 prepared from an E.
coli over-expression system.  Cry1Ab protein was extracted from COT67B
cotton plant tissue and its apparent molecular weight, immunoreactivity,
glycosylation status, and bioactivity were compared to the Cry1Ab
protein from test substance FLCRY1AB-0103. In addition, the microbial-
and plant-derived Cry1Ab proteins were analyzed by peptide mass mapping
and the N-terminal amino acid sequence of Cry1Ab from test substance
FLCRY1AB-0103 was determined.

The Cry1Ab proteins from COT67B and from microbially-derived test
substance FLCRY1AB-0103 both had an apparent molecular weight of ca.
133.5 kDa, and both reacted with anti-Cry1Ab antibodies, as shown by
Western blot analysis. Also, both the protein extract from COT67B and
FLCRY1AB-0103 showed strong insecticidal activity against O. nubilalis
(European corn borer). There was no evidence of post-translational
glycosylation of Cry1Ab protein from COT67B or from microbially-derived
test substance FLCRY1AB-0103. Peptide mass mapping analysis provided
additional evidence of the identity of the insecticidal protein
expressed in COT67B cotton and in test substance FLCRY1AB-0103. Based on
the results of this study it can be concluded that Cry1Ab protein
produced in recombinant E. coli (test substance FLCRY1AB-0103) is a
suitable surrogate for Cry1Ab expressed in COT67B cotton.

Classification: ACCEPTABLE

47017608

Expression levels/Stability of Cry1Ab Protein Expression Across Multiple
Generations of Event COT67B Cotton

The purpose of this study was to use ELISA to analyze the levels of
expression of the modified Cry1Ab protein in leaves (collected at open
boll stage) of the F1, BC1F1, and BC4F1 generations of Event COT67B
cotton. Identical plant tissues from two near-isogenic, nontransgenic
cotton plants (cotton line 2429) from the BC1F1 and BC4F1 generations
were concurrently sampled and analyzed to identify any potential
background effects of the plant matrix on the ELISA.  The levels of
Cry1Ab protein measured in the three generations of COT67B cotton were
comparable (~60 (g/g dry weight). The consistency of the Cry1Ab protein
concentrations demonstrates the stability of transgenic protein
expression across multiple generations of COT67B cotton at the open boll
stage.

Classification: ACCEPTABLE

47017610

Expression Levels/ Analysis for the Presence of Cry1Ab Protein in
Linters, Toasted Cottonseed Oil from Processed Seed of Event COT67B
Cotton Expressing Full-Length Cry1Ab Protein

The purpose of this study was to quantify Cry1Ab protein in linters,
defatted toasted cottonseed meal, and once-refined cottonseed oil
derived from COT67B, and to determine Cry1Ab protein concentrations in
the fuzzy seed used to generate these processed fractions.
Quantification was carried out using an enzyme-linked immunosorbent
assay (ELISA).  The Cry1Ab extraction efficiencies were >69% for fuzzy
seed, linters, and defatted toasted cottonseed meal from COT67B. The
mean concentrations of Cry1Ab protein (corrected for extraction
efficiency) in fuzzy seed, linters, and defatted toasted cottonseed meal
from COT67B were 25.1, 9.6, and 47.5 µg Cry1Ab/g, respectively. Cry1Ab
was not detectable in the once-refined oil from COT67B (limit of
detection = 0.003 µg Cry1Ab/ml). Cry1Ab concentrations in all
cottonseed samples from Coker 312 (negative control) were below the
limit of detection.

Classification: ACCEPTABLE

47017611

II.A.3. COT102 x COT67B Cotton (OECD ID No SYN-IR102-7 x OECD ID No.
SYN-IR67B-1) Expressing Vip3Aa19, APH4, and Modified Cry1Ab

COT102 x COT67B was developed by conventional breeding of COT102 plants
with COT67B plants.

DNA characterization (i.e., Southern blot analysis) was used to confirm
the integrity of the COT102 and COT67B inserts in the stacked product
COT102 x COT67B.  Samples from COT102 x COT67B cotton gave the same
results as those observed for the individual events, indicating that the
molecular characterization data provided for the individual events are
also applicable to COT102 x COT67B.

Analytical Detection Methods:

Syngenta has provided a validation study for SeedChek Vip3A/FLCry1Ab, a
lateral flow test kit that detects both Vip3A and Cry1Ab.  The SeedChek
Vip3A/FLCry1Ab lateral flow test kit was tested for the qualitative
detection of modified Cry1Ab and Vip3A proteins in cotton seed and
cotton leaf.  The study showed that the SeedChek kit is able to detect
Vip3A and Cry1Ab in both cotton seed and cotton leaf, and no unexpected
cross reactivity with other transgenic varieties or nontransgenic
controls was observed.  An independent lab validation of this method is
still needed.

Protein Expresson:

Protein expression levels were provided for Vip3Aa19, APH4, and modified
Cry1Ab in different plant tissues from COT102 x COT67B cotton, and means
are shown below in Table 5.  The protein levels are similar to those
observed in plant tissue from cotton from the individual events.

Table 5. Mean Expression Levels of Vip3Aa19, APH4, and Modified Cry1Ab
from COT102 x COT67B Plant Tissues

Tissue Type	Vip3Aa19

(µg/g dry weight + standard deviation)*	APH4

(µg/g dry weight + standard deviation)*	Cry1Ab

(µg/g dry weight + standard deviation)*

Leaves	55 + 7 – 239 + 46	<0.41 – 6.3 + 1.3	70 + 14 – 185 + 63

Squares	132 + 18	2.1 + 0.5	94 + 10

Flowers	148	1.80	121

Pollen	3.06	74.7	10.7

Bolls	21 + 4	< 0.43	42 + 7

Whole Plants	25 + 7	< 0.40	29 + 7

Seed	7 + 1	1.6 + 0.4	27 + 4

Roots	11 + 3	0.46 + 0.05	20 + 4

*Ranges reflect means at different growth stages for leaves

These data, together with data indicating that there is no evidence of
either a synergistic or antagonistic interaction between Vip3Aa19 and
modified Cry1Ab in cotton bollworm or tobacco budworm (reviewed in the
ecological risk assessment memo for this product), demonstrate that data
on the individual events and individual proteins can be used to support
the safety of the COT102 x COT67B combined product.

Table 6. Product Characterization Data Submitted for COT102 x COT67B
(reviewed in Edelstein 2008 unless otherwise noted)

Study Type/Title

Summary

MRID #

Characterization of Inserted DNA/ Comparative Southern Analysis of
Stacked COT102 x COT67B

Molecular analyses (restriction enzyme digests and Southern blots) were
performed to compare the integrity of the transgenic inserts in the
cotton lines Event COT102 cotton and Event COT67B cotton with the
transgenic inserts in stacked COT102 x COT67B cotton, which was produced
by conventional plant breeding of COT102 and COT67B. The Southern blot
data demonstrated the predicted molecular organization of the vip3Aa19
and aph4 genes from COT102 cotton and the cry1Ab gene from COT67B
cotton. The DNA hybridization patterns from each single event cotton
line were identical to those in stacked COT102 x COT67B cotton,
demonstrating that the integrity of the transgenic inserts was retained
when the component lines were combined into the COT102 x COT67B cotton.

Classification:  ACCEPTABLE

47017606

Expression Levels/ Comparison of Transgenic Protein Expression in Event
COT102, Event COT67B, and Stacked COT102 x COT67B Cotton Lines

The purpose of this study was to use an enzyme-linked immunosorbent
assay (ELISA) to analyze tissues from cotton plants derived from
transformation Event COT102, Event COT67B and from COT102 x COT67B in
order to compare the concentrations of Vip3Aa19, hygromycin B
phosphotransferase (APH4), and Cry1Ab proteins produced in the
transgenic plants.  For the Vip3Aa19 and APH4 proteins, the
concentrations and patterns of expression were generally similar between
the COT102 line and the COT102 x COT67B line. Likewise, for the modified
Cry1Ab protein, the concentrations and patterns of expression were
generally similar between the COT67B line and the COT102 x COT67B line.
Some statistically significant differences were seen in some tissues at
certain sampling stages, but these differences were not consistent by
genotype and/or were not consistent across the growing season.

Classification: ACCEPTABLE

47017607

Analytical Detection Method/Analytical Detection Method for the
Detection of Vip3A and FLCry1Ab Protein in Cotton Tissues Derived from
COT102 x COT67B Cotton (VipCot Cotton)

The SeedChek Vip3A/FLCry1Ab lateral flow test kit was tested for the
qualitative detection of modified Cry1Ab and Vip3A proteins in cotton
seed and cotton leaf.  The study showed that the SeedChek kit is able to
detect Vip3A and Cry1Ab in both cotton seed and cotton leaf, and no
unexpected cross reactivity with other transgenic varieties was
observed.

Classification: ACCEPTABLE

47074101

II.B.  Human Health Assessment

II.B.1.  Human Health Assessment of Vip3Aa

Note:  EPA’s human health assessment was conducted for Vip3Aa
proteins, which include the Vip3Aa19 protein as expressed in cotton.

A.  Mammalian Toxicity and Allergenicity Assessment

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.

Syngenta has submitted acute oral toxicity data demonstrating the lack
of mammalian toxicity at high levels of exposure to Vip3Aa proteins.
These data demonstrate the safety of Vip3Aa at a level well above
maximum possible exposure levels that are reasonably anticipated in the
crops.  Basing this conclusion on acute oral toxicity data without
requiring further toxicity testing and residue data is similar to the
Agency position regarding toxicity testing and the requirement of
residue data for the microbial Bacillus thuringiensis products from
which this plant-incorporated protectant was derived (See 40 CFR Sec.
158.2140)  For microbial products, further toxicity testing (Tiers II &
III) and residue data are triggered by significant adverse acute effects
in studies such as the mouse oral toxicity study, to verify the observed
adverse effects and clarify the source of these effects.

Syngenta submitted four acute oral toxicity studies conducted on mice.
Three of the studies were conducted with microbially-produced Vip3Aa
proteins (Vip3Aa1, Vip3Aa19, and Vip3Aa20) with slight variations in
amino acid sequence (1-2 amino acid differences), and one study was
conducted with transgenic corn leaf tissue expressing Vip3Aa19 as the
test material.  No treatment-related adverse effects were observed in
any of the studies.  The results of these studies showed that the oral
LD50 for mice (males, females, and combined) was greater than 3675 mg/kg
body weight (the highest dose tested) for the tested Vip3Aa proteins.

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 the Vip3Aa19 and Vip3Aa20
proteins, even at relatively high dose levels, they are not considered
toxic.  (This is also true of the Vip3Aa1 protein that was tested.)
Further, amino acid sequence comparisons showed no similarities between
Vip3Aa19 and Vip3Aa20, on the one hand, and known toxic proteins in
protein databases, on the other hand, that would raise a safety concern.

Since Vip3Aa is a protein, allergenic potential was also considered.
Currently, no definitive tests for determining the allergenic potential
of novel proteins exist.  Therefore, EPA uses a weight-of- evidence
approach where the following factors are considered: source of the
trait; amino acid sequence comparison with known allergens; and
biochemical properties of the protein, including in vitro digestibility
in simulated gastric fluid (SGF) and glycosylation.  This approach is
consistent with the approach outlined in the Annex to the Codex
Alimentarius “Guideline for the Conduct of Food Safety Assessment of
Foods Derived from Recombinant-DNA Plants.”  The allergenicity
assessment for Vip3Aa follows:

Source of the trait.  Bacillus thuringiensis, the microorganism from
which Vip3Aa proteins are derived, is not considered to be a source of
allergenic proteins.

Amino acid sequence.  A comparison of the amino acid sequence of
Vip3Aa19 and Vip3Aa20 with known allergens showed no significant
sequence identity over 80 amino acids or identity at the level of eight
contiguous amino acid residues.

Digestibility.  Both Vip3Aa19 and Vip3Aa20 proteins are digested rapidly
in simulated gastric fluid containing pepsin.

Glycosylation.  Both Vip3Aa19 and Vip3Aa20 were shown not to be
glycosylated.

Considering all of the available information on Vip3Aa19 and Vip3Aa20,
EPA concludes that the potential for these specific proteins to be food
allergens is minimal.  Moreover, as further explained below, EPA
believes these data and the other submitted data demonstrating a lack of
mammalian toxicity at high levels of exposure to Vip3Aa19 and Vip3Aa20
can be extrapolated to cover Vip3Aa more generally.

Vip3Aa is the designation assigned to a closely-related group of similar
insecticidal proteins isolated from Bacillus thuringiensis.  The
specific variants referred to throughout this document (i.e., Vip3Aa19
and Vip3Aa20) are isolates of Vip3Aa protein.  All Vip3Aa proteins
(there are 25 known Vip3Aa proteins and there are sequences available
for 19 of these) are highly related.  Indeed, the amino acid sequence of
all the Vip3Aa proteins can only vary up to 5% to be considered a part
of the Vip3Aa group.  With respect to the 19 Vip3Aa proteins for which
sequences are available, they vary by less than 28 amino acids out of
the 789 amino acids that make up the protein.  This level of sequence
similarity makes that group of 19 Vip3Aa protein variants 96% identical
overall.  The sequence identity between any two individual sequences is
even higher.  For example, the sequences of the protein variants tested
by Syngenta (i.e., Vip3Aa19 and Vip3Aa20) are over 99.7% identical.
Finally, as to the few amino acid differences that do exist between the
Vip3Aa variants, these differences do not alter the surrounding
sequence, rarely occur as contiguous amino acids, and are often
substitutions with similar chemical side groups indicating similar
chemical functionality.  Therefore, EPA finds that none of the Vip3Aa
variants would be expected to have significant amino acid sequence
identity -- which is defined as either 35% identity over an 80 amino
acid stretch and, for allergens, at the level of eight contiguous amino
acids -- with a toxin, an anti-nutrient or an allergen.

This conclusion is further supported by EPA’s overall safety
assessment that includes other considerations such as the source of the
trait, digestibility and glycosylation.  As noted above, Bacillus
thuringiensis (from which the Vip3Aa proteins are derived) is not
considered to be a source of allergenic proteins.  Furthermore, since
all the Vip3Aa proteins have extremely homogenous structural
similarities (as explained above), they are highly likely to show
similar biochemical characteristics in terms of digestibility and
glycosylation.  So, as is the case for both Vip3Aa19 and Vip3Aa20, EPA
expects that all Vip3Aa proteins will be rapidly digested under
simulated gastric conditions and will not be glycosylated.  The Vip3Aa
proteins were only shown not to be glycosylated in cotton and corn,
similarly it is unlikely to be glycosylated in any other crops because
in order for a protein to be glycosylated, it needs to contain specific
recognition sites for the enzymes involved in glycosylation, and the
mechanisms of protein glycosylation are similar in different plants
(Lerouge et al., 1998).  Thus, EPA reasonably expects that because the
data on Vip3Aa in cotton and corn demonstrate a lack of protein
glycosylation, it will not be glycosylated in any other plants.

Finally, it is also highly relevant here that microbial pesticide
products, which are distinct from plant-incorporated protectant
pesticide products, containing Bacillus thuringiensis and its components
(which could include microbially-expressed Vip3Aa proteins) are already
exempt from the requirement for a tolerance under 40 CFR part 180.1011.

Accordingly, EPA believes that the foregoing supports EPA’s reasonable
certainty of no harm finding not only for the Vip3Aa19 and Vip3Aa20
protein variants, but also for all other closely-related members of the
Vip3Aa designation as described using the Crickmore classification
system (Crickmore et al., 2007).

B.  Aggregate Exposures

Pursuant to FFDCA section 408(b)(2)(D)(vi), EPA considers available
information concerning aggregate exposures from the pesticide residue in
food and all other non-occupational exposures, including drinking water
from ground water or surface water and exposure through pesticide use in
gardens, lawns, or buildings (residential and other indoor uses).

The Agency has considered available information on the aggregate
exposure levels of consumers (and major identifiable subgroups of
consumers) to the pesticide chemical residue (i.e., the Vip3Aa proteins)
and to other related substances. These considerations include dietary
exposure under the tolerance exemption and all other tolerances or
exemptions in effect for the plant-incorporated protectant’s chemical
residue, and exposure from non-occupational sources. Exposure via the
skin or inhalation is not likely since the plant-incorporated protectant
is contained within plant cells, which essentially eliminates these
exposure routes or reduces these exposure routes to negligible. In
addition, even if exposure can occur through inhalation, the potential
for Vip3Aa to be an allergen is low, as discussed above.  Although the
allergenicity assessment focuses on potential to be a food allergen, the
data also indicate a low potential for Vip3Aa to be an inhalation
allergen. Exposure via residential or lawn use to infants and children
is also not expected because the use sites for Vip3Aa proteins are
agricultural.  Oral exposure, at very low levels, may occur from
ingestion of processed products and, theoretically, drinking water.
However oral toxicity testing showed no adverse effects.

C.  Cumulative Effects

Pursuant to FFDCA section 408(b)(2)(D)(v), EPA has considered available
information on the cumulative effects of such residues and other
substances that have a common mechanism of toxicity. These
considerations included the cumulative effects on infants and children
of such residues and other substances with a common mechanism of
toxicity. Because there is no indication of mammalian toxicity from
exposure to Vip3Aa proteins, we conclude that there are no cumulative
effects for the Vip3Aa proteins.

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

1) Toxicity and Allergenicity Conclusions

The data submitted and cited regarding potential health effects for
Vip3Aa proteins includes the characterization of representative Vip3Aa
proteins, as well as the acute oral toxicity studies, amino acid
sequence comparisons to known allergens and toxins, and in vitro
digestibility of the representative Vip3Aa 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.

Adequate information was submitted to show that the Vip3Aa test
materials derived from microbial cultures were biochemically and
functionally equivalent to the proteins produced by the
plant-incorporated protectant ingredient in the plants.  Microbially
produced proteins were used in the studies so that sufficient material
for testing was available.

The acute oral toxicity data submitted for the representative Vip3Aa
proteins support the prediction that Vip3Aa proteins will be non-toxic
to humans.  As mentioned above, when proteins are toxic, they are known
to act via acute mechanisms and at very low dose levels (Sjoblad et al.,
1992). Since no treatment-related adverse effects were shown to be
caused by the representative Vip3Aa proteins, even at relatively high
dose levels, Vip3Aa proteins are not considered toxic.  Basing this
conclusion on acute oral toxicity data without requiring further
toxicity testing or residue data is similar to the Agency position
regarding toxicity and the requirement of residue data for the microbial
Bacillus thuringiensis products from which this plant-incorporated
protectant was derived (See 40 CFR 158.2140).  For microbial products,
further toxicity testing (Tiers II and III) and residue data are
triggered when significant adverse effects are seen in studies such as
the acute oral toxicity study.  Further studies verify the observed
adverse effects and clarify the source of these effects.

Residue chemistry data were not required for a human health effects
assessment of the subject plant-incorporated protectant ingredients
because of the lack of mammalian toxicity.  However, data submitted
demonstrated low levels of the representative Vip3Aa proteins in corn
and cotton tissues.

Since Vip3Aa are proteins, potential allergenicity is also considered as
part of the toxicity assessment.  Considering all of the available
information, including that (1) Vip3Aa originates from a non-allergenic
source; (2) Vip3Aa19 and Vip3Aa20 have no sequence similarities with
known allergens; (3) Vip3Aa19 and Vip3Aa20 are not glycosylated; (4)
Vip3Aa19 and Vip3Aa20 are rapidly digested in simulated gastric fluid;
and (5) the data developed for Vip3Aa19 and Vip3Aa20 can be extrapolated
to all Vip3Aa proteins due to the extremely high level of structural
similarity that exists between and among Vip3Aa proteins, EPA has
concluded that the potential for Vip3Aa to be an allergen is minimal.

Neither available information concerning the dietary consumption
patterns of consumers (and major identifiable subgroups of consumers
including infants and children) nor safety factors that are generally
recognized as appropriate for the use of animal experimentation data
were evaluated.  The lack of mammalian toxicity at high levels of
exposure to representative Vip3Aa proteins, as well as the minimal
potential to be a food allergen, demonstrates the safety of Vip3Aa at
levels well above possible maximum exposure levels anticipated.

The genetic material necessary for the production of the
plant-incorporated protectant active ingredient include the nucleic
acids (DNA, RNA) that encode these proteins and regulatory regions. The
genetic material (DNA, RNA), necessary for the production of Vip3Aa
proteins has been exempted from the requirement of a tolerance under 40
CFR 174.507 (“Nucleic acids that are part of a plant-incorporated
protectant”).

2) Infants and Children Risk Conclusions

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) also 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 database unless EPA determines that a
different margin of safety will be safe for infants and children.

In this instance, based on all the available information, the Agency
concludes that there is a finding of no toxicity for Vip3Aa proteins.
Thus, there are no threshold effects of concern and, as a result, the
provision requiring an additional margin of safety does not apply.
Further, the considerations of consumption patterns, special
susceptibility, and cumulative effects do not apply.

3) Overall Safety Conclusion

There is a reasonable certainty that no harm will result from aggregate
exposure to the U.S. population, including infants and children, to
Vip3Aa proteins.  This includes all anticipated dietary exposures and
all other exposures for which there is reliable information. The Agency
has arrived at this conclusion because, as discussed above, no toxicity
to mammals has been observed, nor any indication of allergenicity
potential for Vip3Aa proteins.

E.  Other Considerations

1) Endocrine Disruptors

The pesticidal active ingredient is a protein, derived from a source
that is not known to exert an influence on the endocrine system.
Therefore, the Agency is not requiring information on the endocrine
effects of the plant-incorporated protectant at this time.

2) Analytical Method(s)

A validated lateral flow enzyme-linked immunosorbent assay (ELISA)
protocol has been provided to the Agency for detecting Vip3Aa in cotton
as well as a qualitative ELISA method for detecting Vip3Aa in corn.

3) Codex Maximum Residue Level

No Codex maximum residue level exists for the plant-incorporated
protectant Bacillus thuringiensis Vip3Aa proteins and the genetic
material necessary for their production in corn and cotton.

F.  Tolerance Exemptions

The data submitted and reviewed for Vip3Aa support the petition for an
exemption from the requirement of tolerance for Bacillus thuringiensis
Vip3Aa proteins when used as plant–incorporated protectants in or on
the food and feed commodities of corn and cotton.

G.  Supporting Data

The human health studies submitted to support the safety of Vip3Aa are
summarized in Table 7 below.

Table 7. Summary of Vip3Aa Human Health Data (reviewed in Edelstein 2008
unless otherwise noted)

Study Type/Title

Summary

MRID #

Summary of Mammalian Toxicology Data for the VIP3A and APH4 Proteins
Produced by Transgenic VIP3A Cotton Event COT1022

No significant adverse effects were observed in male and female mice
dose by gavage at approximately 3675 mg VIP3A/kg body weight (the
highest dose tested) and the LD50 for pure VIP3A protein was >3675 mg/kg
body weight.  The LD50 for pure APH4 protein in male and female mice was
>774 mg/kg body weight. The allergen database compiled by Syngenta needs
to be better defined or described in order to ascertain the number and
types of allergens searched for homology.

Classification: SUPPLEMENTAL.

Note: this is a summary of multiple studies and is therefore superseded
by the individual studies summarized below, which provide additional
information, including the requested information on the SBI allergen
database.

45766502

Acute Oral Toxicity/ Acute Oral Toxicity of Vip3A Protein in Mice2

Eleven male and 11 female HSD:ICR albino mice were dosed with VIP3A
protein (Lot no. VIP3A-0196 containing ~ 32% by weight VIP3A protein).
The mice were quarantined for 5 days and fasted approximately 16 hours
prior to dosing.  The test material (5050 mg/kg body weight) was dosed
as a 12.5 % w/v suspension in 2 % w/v carboxymethyl cellulose (CMC) in
distilled water by gavage (Table 1).  The dose volume was 40.4 mL/kg and
was divided into 2 parts administered approximately one hour apart.  The
control group was treated with 2 % w/v CMC in the same manner as the
test animals.  Body weights were recorded prior to dosing, on days 7 and
14 or at death.  The test animals were observed for clinical signs of
toxicity at least three times post-dosing and at least daily thereafter
for 14 days.  All decedent or euthanized animals were necropsied.   One
control male (No. 17-M) was found dead on day 2.  All other mice
survived the study. With the exception of one female (No. 10-F) that
failed to gain weight during the first week, all surviving animals
gained weight during the study.  In the vehicle control group (i.e., CMC
treated), there was no affect on weight gain. The oral LD50 for males,
females, and combined was greater than 5050 mg/kg (or > 1616 mg VIP3A
protein/kg body weight).

Classification: SUPPLEMENTAL. The VIP3A protein used in this study
differs from the VIP3A protein present in COT102 cotton by a two amino
acids, one at position 2 (aspartate replaces asparagine), another at
position 284 (lysine replaces glutamine).

Note: this study provides additional support for the conclusion that
Vip3Aa proteins are non-toxic to mammals.

45766503

Acute Oral Toxicity/ Single Dose Oral Toxicity Study with VIP3A-0199 in
Mice2	Twenty-seven male and 27 female CD-1® (ICR)BR mice were dosed
with VIP3A protein (Batch VIP3A-0199 containing ~ 54% by weight VIP3A
protein), produced in an E. coli over-expression system. The VIP3A
protein used as the test material differs from that present in cotton
Event COT102 by a single amino acid; glutamine substituted for lysine
(Q284K).  The mice were quarantined for 16 days and fasted approximately
4 hours prior to dosing.  The test material (5000 mg/kg body weight) was
dosed as a suspension of 200 mg/mL in 0.5% w/v carboxymethyl cellulose
(CMC) in deionized water by gavage (Table 1).  The dose volume was 25
mL/kg.  The control group was treated with 0.5 % w/v CMC in the same
manner and volume as the test animals.  Body weights were recorded prior
to dosing, and on day 8 for animals designated to be sacrificed on day
15, and on each animals’s respective day of necropsy (days 1, 2, or
15).  The animals were observed for clinical signs of toxicity
approximately 1, 2.5, 4, and 6 hours post dosing and at least daily
until sacrifice. Animals were observed for any abnormal behavior,
changes in posture or clonic / tonic movements. Mortality was observed
twice daily.  All animals were necropsied after sacrifice.  The organ
weight of the brain, kidneys, liver with drained gallbladder, and
stomach were recorded and organ to body weight and organ to brain weight
were calculated.  Histopathology was performed on brain, gallbladder,
heart, intestines (cecum, colon, duodenum, ileum, jejunum, and rectum),
kidneys, lesions, liver, lung, and stomach. All animals survived prior
to the scheduled sacrifice. All animals sacrificed on day 15 had normal
body weight gains.  All control and a few test animals sacrificed on day
1 and one male test and some control animals sacrificed on day 2 lost
weight prior to sacrifice. No significant differences considered to be
test material related in organ/body weight or organ/brain weight between
control and test animals were found.  The oral LD50 for males, females,
and combined was greater than 5000 mg/kg (or > 2700 mg VIP3A protein/kg
body weight).

Classification: SUPPLEMENTAL - The test material for this study,
VIP3A-0199, differs in sequence by one amino acid (Q284K) from that form
of the protein which is present in COT102.

Note: this study provides additional support for the conclusion that
Vip3Aa proteins are non-toxic to mammals.  	45766504

Acute Oral Toxicity/ Acute Oral Toxicity Study with Test Substance
VIP3A-0100 Protein in Mice2

The test animals (Sixteen male and 16 female Crl-1® (ICR)BR mice) were
quarantined for 9 days and fasted approximately 4 hours prior to dosing.
 The test material (5000 mg/kg body weight) was dosed as a suspension of
196 mg/mL in 0.5% w/v carboxymethyl cellulose (CMC) in deionized water
by gavage.  The dose volume was 25.5 mL/kg.  The control group was
treated with 0.5% w/v CMC in the same manner as the test animals.  Body
weights were recorded prior to dosing and on days 8 and 15 for animals
designated to be sacrificed on day 15.  The animals were observed for
clinical signs of toxicity approximately 1, 2.5, 4, and 6 hours post
dosing and at least daily until sacrifice.  Mortality was observed twice
daily.  All animals were necropsied after sacrifice.  The organ weight
of the brain, kidneys, liver with drained gallbladder, and stomach were
recorded and organ to body weight and organ to brain weight were
calculated.  Histopathology was performed on brain, gallbladder, heart,
intestines (cecum, colon, duodenum, ileum, jejunum, and rectum),
kidneys, lesions, liver, lung, and stomach. All animals sacrificed on
day 15 had normal body weight gains.  No test material related
macroscopic alterations were noted.  In addition, no significant
differences related to the test material in organ/body weight or
organ/brain weight between control and test animals were found. The oral
LD50 for males, females, and combined was greater than 5000 mg/kg (or >
3675 mg VIP3A protein/kg body weight).

Classification: Acceptable

45766505

Acute Oral Toxicity/ Single Dose Oral Toxicity Study with VIP3A-Enriched
Maize (Corn) Leaf Protein (LPPACHA-0199) in Mice2	VIP3A-Enriched Maize
(Corn) Leaf Protein (Sample Lot. No. LPPACHA-0199 containing ~ 0.36% by
weight VIP3A protein) was prepared from transgenic VIP3A maize (corn)
leaves. The mice were quarantined for at least 7 days and fasted
approximately 4 hours prior to dosing.  The test material (5000 mg/kg
body weight) was dosed as a suspension of 250 mg/mL in 0.5% w/v
carboxymethyl cellulose (CMC) in deionized water by gavage (Table 1).
The dose volume was 20 mL/kg.  The control group was treated with
Control Maize (Corn) Leaf Protein, Batch LPPACHA-0199C in 0.5% w/v CMC
in deionized water at a concentration of 250 mg/mL in the same manner as
the test animals.  Body weights were recorded prior to dosing, and on
days 7, 14, or at death.  The test animals were observed for clinical
signs of toxicity at least three times post-dosing and at least daily
thereafter for 14 days.  All decedent or euthanized animals were
necropsied. All mice survived the study, gained weight and appeared
normal during the study. The oral LD50 for males, females, and combined
was greater than 18 mg/kg VIP3A protein/kg body weight.  The net
concentration of VIP3A (18 mg / kg body weight) is significantly lower
than the prescribed 2000 to 5000 mg / kg body weight suggested in the
guideline requirements. At this concentration and with the mix of other
proteins present in the leaf preparation, no toxicity was evident in the
test animals.

Classification: SUPPLEMENTAL. Information is supportive, but not part of
guideline requirements; no further information required.

Note: this study provides additional support for the conclusion that
Vip3Aa proteins are non-toxic to mammals.

	45766506

In Vitro Digestibility of VIP3A Protein Under Simulated Mammalian
Gastric Conditions 2

VIP3A from recombinant maize (field corn) plants was prepared as sample
LPPACHA-0199 by extracting the leaves of recombinant corn plants and
concentrating the VIP3A by ammonium sulfate precipitation, dialysis of
the resulting salt, and lyophilization of the collected protein.  ELISA
showed VIP3A constituted ~0.36 % by weight of the sample and retained
insecticidal activity against sensitive lepidopteran species. VIP3A from
E. coli  was prepared as sample VIP3A-0100 in an E. coli strain
BL21DE3pLysS over-expression system.  The synthetic vip3A(a) gene was
cloned into the inducible over-expression pET-3a® vector.  Following
collection, purification, dialysis, and lyophilization, the sample was
estimated by ELISA to contain ~73.5% VIP3A by weight and it retained its
insecticidal activity against sensitive lepidopteran species. The
reactions were initiated by the addition of 80 µL of LPPACHA-0199 or
VIP3A-0100 to 320 µL of simulated gastric fluid containing pepsin
incubated at 37(C.  Immediately after sample addition, an aliquot was
removed and quenched with an equal volume of Laemmli buffer (pH not
reported) and inactivated at >75(C for 10 minutes.  Additional aliquots
were removed and treated as above following 2, 5, 10, 20, 30, and 60
minutes of incubation. Digestion of the protein samples was evaluated
using SDS-PAGE and Western blotting. The digestion of VIP3A protein in a
simulated gastric environment proceeds at a rapid rate and demonstrates
the lability of this protein to conditions typical of a monogastric
mammalian stomach. The presence of a small amount of immunoreactive
protein (approximately 6 to 9 kD) indicates that a portion or domain of
the protein is less readily digested in this environment, although these
bands do degrade beyond the point of immunorecognition with time.
Results of this study indicate VIP3A protein, whether isolated from
recombinant corn plants or from genetically modified E. coli, will be
rapidly digested in a simulated gastric environment.

Classification: ACCEPTABLE

45835805

Amino acid sequence comparison/ Vip3Aa19: Assessment of amino acid
sequence homology with known toxins.

Report No. SSB-122-064

The purpose of this study was to determine if Vip3Aa19 had any
significant amino acid sequence homology to known protein toxins.  No
relevant similarities between the Vip3Aa19 query sequence and known
protein toxins were found other than with other insect-specific
vegetative insecticidal proteins of B. thuringiensis.

Classification:  Acceptable; Supersedes MRID 457665-02

46885903

Amino acid sequence comparison/ Vip3Aa19: Assessment of amino acid
sequence homology with known allergens.

Report No. SSB-130-064	The purpose of this study was to determine if
Vip3Aa19 had any significant amino acid sequence homology to known
protein allergens.  Vip3Aa19 had no significant amino acid sequence
homology to known or putative allergenic proteins.

Classification: Acceptable; Supersedes MRID 457665-02

	46885906

Amino acid sequence comparison/ Vip3A as expressed in Event MIR162
maize: Assessment of amino acid sequence homology with known toxins3	The
purpose of the study was to determine if Event MIR162 Vip3A protein had
any significant amino acid sequence homology to known or putative
protein toxins.  The database identified 32 entries with E values below
6 x 10-6, of which 30 were vegetative insecticidal proteins of B.
thuringiensis and had E values of 0.0 to 1 x 10-10.  Two proteins were
identified as rhoptry proteins from Plasmodium yoelii, a pathogen that
causes malaria in rodents via erythrocyte binding and invasion (Ogun and
Holder, 1996).  Despite the pathogenic nature of P. yoelii, the low
overall sequence similarity between MIR162 Vip3A and the rhoptry
proteins (3.9 or 11.4% overall amino acid sequence identity) suggests
that the E values are of no biological significance (Doolittle, 1990).
Furthermore, a global protein alignment (Myers and Miller, 1988)
demonstrates that there are no more than three contiguous identical
amino acids between Vip3A and the rhoptry proteins.  Therefore, no
relevant similarities between the Event MIR162 Vip3A query sequence and
known protein toxins were found.

Classification: ACCEPTABLE

	46864808

Amino acid sequence comparison/ Vip3A as expressed in Event MIR162
maize: Assessment of amino acid sequence homology with known allergens3
The purpose of this study was to determine if Event MIR162 Vip3Aa20 had
any significant amino acid sequence homology to known or putative
protein allergens.  No significant sequence homology was found between
any sequential MIR162 Vip3A 80-amino acid peptides and any entry in the
SBI Allergen Database.  No alignments of eight or more contiguous
identical amino acids were identified between MIR162 Vip3A and proteins
in the SBI Allergen Database.  Therefore, no significant amino acid
sequence homology was found between the MIR162 Vip3A and any known or
putative protein allergens.

Classification: ACCEPTABLE

	46864809

Analysis of Vip3A or Vip3A-Like Proteins in Six Different Commercial
Microbial Bacillus thuringiensis Products	The purpose of this study was
to determine whether Vip3A or Vip3A-like proteins are detectable and
quantifiable in commercial formulations of Bacillus thuringiensis
(Bt)-based microbial insecticide products. ELISA (enzyme-linked
immunosorbent assay) and Western blot analyses were used to detect and
analyze Vip3A or Vip3A-like proteins in the formulations. Vip3A or
Vip3A-like proteins were detected in all six commercial products, with
concentrations ranging from a low of ca. 2.0 µg/g product to a high of
ca. 209 µg/g. Those products showing the highest protein concentrations
were all derived from the kurstaki subspecies of B. thuringiensis.

Classification: ACCEPTABLE 	47017613

Amino acid sequence comparison/ Vip3Aa19: Assessment of amino acid
sequence homology with known allergens	Two amino acid sequences
comparisons of Vip3Aa19 with known allergens were conducted using the
Syngenta Biotechnology, Inc (SBI) Allergen Database.  The results
indicate that Vip3Aa19 has no significant amino acid sequence homology
to known or putative allergenic proteins based on a search for greater
than 35% sequence identity over successive 80-amino acid peptides and a
search for eight or more contiguous identical amino acids.

Classification: ACCEPTABLE	47017617

II.B.2.  Human Health Assessment of Modified Cry1Ab Containing 26
Additional Amino Acids

A. Mammalian Toxicity and Allergenicity Assessment

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.

Syngenta has submitted acute oral toxicity data demonstrating the lack
of mammalian toxicity at high levels of exposure to the pure modified
Cry1Ab protein containing the additional 26 amino acid ‘Geiser
motif’.   The 26 amino acid sequence is found at the C-terminus of the
pro-toxin portion of the modified Cry1Ab protein.  The pro-toxin is
enzymatically cleaved in the insect gut to produce active Cry1Ab.  These
toxicity 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
158.2140). For microbial products, further toxicity testing (Tiers II &
III) and residue data are triggered by significant adverse acute effects
in studies such as the acute oral toxicity study, to verify the observed
adverse effects and clarify the source of these effects.

An acute oral toxicity study in mice indicated that modified Cry1Ab is
non-toxic to humans. Groups of five male and five female mice were given
0 or 1830 mg/kg bodyweight microbially-produced modified Cry1Ab by oral
gavage as a single dose.  There were no effects on clinical condition,
body weight, food consumption, clinical pathology, organ weight, or
macroscopic or microscopic pathology that were attributed to the test
substance.

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 modified Cry1Ab, even at
relatively high dose levels, the modified Cry1Ab protein is not
considered toxic.

Since modified Cry1Ab is a protein, allergenic potential was also
considered. Currently, no definitive tests for determining the
allergenic potential of novel proteins exist.  Therefore, EPA uses a
weight-of-evidence approach where the following factors are considered:
source of the trait; amino acid sequence comparison with known
allergens; and biochemical properties of the protein, including in vitro
digestibility in simulated gastric fluid (SGF) and glycosylation.  This
approach is consistent with the approach outlined in the Annex to the
Codex Alimentarius “Guideline for the Conduct of Food Safety
Assessment of Foods Derived from Recombinant-DNA Plants.”  The
allergenicity assessment for modified Cry1Ab follows:

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
modified Cry1Ab with known allergens showed no significant sequence
identity over 80 amino acids or identity at the level of eight
contiguous amino acid residues.

Digestibility.  Modified Cry1Ab was rapidly digested in simulated
gastric fluid containing pepsin.

Glycosylation.  Modified Cry1Ab expressed in cotton was shown not to be
glycosylated.

Conclusion.  Considering all of the available information, EPA has
concluded that the potential for modified Cry1Ab to be a food allergen
is minimal.

Although modified Cry1Ab was only shown not to be glycosylated in
cotton, it is unlikely to be glycosylated in any other crops because in
order for a protein to be glycoslyated, it needs to contain specific
recognition sites for the enzymes involved in glycosylation, and the
mechanisms of protein glycosylation are similar in different plants
(Lerouge et al., 1998).

B. Aggregate Exposures

Pursuant to FFDCA section 408(b)(2)(D)(vi), EPA considers available
information concerning aggregate exposures from the pesticide residue in
food and all other non-occupational exposures, including drinking water
from ground water or surface water and exposure through pesticide use in
gardens, lawns, or buildings (residential and other indoor uses).

The Agency 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. These considerations include dietary exposure under the
tolerance exemption and all other tolerances or exemptions in effect for
the plant-incorporated protectants 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. In addition, even if exposure can
occur through inhalation, the potential for modified Cry1Ab to be an
allergen is low, as discussed above.  Although the allergenicity
assessment focuses on potential to be a food allergen, the data also
indicate a low potential for modified Cry1Ab to be an inhalation
allergen.  Exposure via residential or lawn use to infants and children
is also not expected because the use sites for the modified Cry1Ab
protein is agricultural.  Dietary exposure may occur from ingestion of
processed cotton products but is expected to be very low because the
already low expression levels in the seed and would be reduced further
by the heat and pressure used for processing.  Also, dietary exposure
may theoretically occur through exposure in drinking water because plant
stubble may release modified Cry1Ab protein into ground water upon
decay.  This protein would not be expected to survive in the soil due to
microbial degradation, adherence to soil components and removal upon
drinking water treatment procedures.  In addition, oral toxicity testing
showed no adverse effects.

C. Cumulative Effects

Pursuant to FFDCA section 408(b)(2)(D)(v), EPA has considered available
information on the cumulative effects of such residues and other
substances that have a common mechanism of toxicity. These
considerations included the cumulative effects on infants and children
of such residues and other substances with a common mechanism of
toxicity.  Because there is no indication of mammalian toxicity from the
plant-incorporated protectant, EPA concludes that there are no
cumulative effects for the modified Cry1Ab protein.

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

1) Toxicity and Allergenicity Conclusions

The data submitted and cited regarding potential health effects for the
modified Cry1Ab protein includes the characterization of the expressed
modified Cry1Ab protein in cotton, as well as the acute oral toxicity
study, amino acid sequence comparisons to known allergens, and in vitro
digestibility of the protein. 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.

Adequate information was submitted to show that the modified Cry1Ab test
material derived from microbial culture was biochemically and
functionally equivalent to the protein in the plant.  Microbially
produced protein was used in the safety studies so that sufficient
material for testing was available.

The acute oral toxicity data submitted support the prediction that the
modified Cry1Ab protein would be non-toxic to humans.  As mentioned
above, when proteins are toxic, they are known to act via acute
mechanisms and at very low dose levels (Sjoblad et al., 1992). Since no
treatment-related adverse effects were shown to be caused by the Cry1Ab
protein, even at relatively high dose levels, the modified Cry1Ab
protein is not considered toxic.  Basing this conclusion on acute oral
toxicity data without requiring further toxicity testing and residue
data is similar to the Agency position regarding toxicity and the
requirement of residue data for the microbial Bacillus thuringiensis
products from which this plant-incorporated protectant was derived (See
40 CFR 158.2140). For microbial products, further toxicity testing and
residue data are triggered when significant adverse effects are seen in
studies such as the acute oral toxicity study.  Further studies verify
the observed adverse effects and clarify the source of these effects.

Residue chemistry data were not required for a human health effects
assessment of the subject plant-incorporated protectant ingredients
because of the lack of mammalian toxicity.  However, data submitted
demonstrated low levels of the modified Cry1Ab protein in cotton
tissues.

Since Cry1Ab is a protein, potential allergenicity is also considered as
part of the toxicity assessment.  Considering all of the available
information (1) modified Cry1Ab originates from a non-allergenic source;
(2) modified Cry1Ab has no sequence similarities with known allergens;
(3) modified Cry1Ab is not glycosylated; and (4) modified Cry1Ab is
rapidly digested in simulated gastric fluid; EPA has concluded that the
potential for modified Cry1Ab to be an allergen is minimal.

Neither available information concerning the dietary consumption
patterns of consumers (and major identifiable subgroups of consumers
including infants and children) nor safety factors that are generally
recognized as appropriate for the use of animal experimentation data
were evaluated. The lack of mammalian toxicity at high levels of
exposure to the modified Cry1Ab protein, as well as the minimal
potential to be an allergen, demonstrate the safety of the product at
levels well above possible maximum exposure levels anticipated.

The genetic material necessary for the production of the
plant-incorporated protectant active ingredient include the nucleic
acids (DNA, RNA) that encode these proteins and regulatory regions. The
genetic material (DNA, RNA) necessary for the production of the modified
Cry1Ab protein has been exempted from the requirement of a tolerance
under 40 CFR 174.507 (“Nucleic acids that are part of a
plant-incorporated protectant”).

2) Infants and Children Risk Conclusions

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) also 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 database unless EPA determines that a
different margin of safety will be safe for infants and children.

In this instance, based on all the available information, the Agency
concludes that there is a finding of no toxicity for the modified Cry1Ab
protein.  Thus, there are no threshold effects of concern and, as a
result, the provision requiring an additional margin of safety does not
apply. Further, the considerations of consumption patterns, special
susceptibility, and cumulative effects do not apply.

3) Overall Safety Conclusion

There is a reasonable certainty that no harm will result from aggregate
exposure to the U.S. population, including infants and children, to the
modified Cry1Ab protein and the genetic material necessary for its
production. This includes all anticipated dietary exposures and all
other exposures for which there is reliable information. The Agency has
arrived at this conclusion because, as discussed above, no toxicity to
mammals has been observed, nor any indication of allergenicity potential
for the plant-incorporated protectant.

E. Other Considerations

1) Endocrine Disruptors

The pesticidal active ingredient is a protein, derived from a source
that is not known to exert an influence on the endocrine system.
Therefore, the Agency is not requiring information on the endocrine
effects of this plant-incorporated protectant at this time.

2) Analytical Method(s)

A lateral flow enzyme-linked immunosorbent assay (ELISA) protocol has
been provided to the Agency for detecting modified Cry1Ab in cotton.
This analytical method will be independently validated as a condition of
registration for cotton product(s) containing modified Cry1Ab.

3) Codex Maximum Residue Level

No Codex maximum residue level exists for the plant-incorporated
protectant Bacillus thuringiensis modified Cry1Ab protein.

F.  Tolerance Exemptions

The data submitted and reviewed for modified Cry1Ab support the petition
for an exemption from the requirement of tolerance for Bacillus
thuringiensis modified Cry1Ab protein containing the additional 26 amino
acid sequence when used as a plant-incorporated protectant in or on the
food and feed commodities of cotton.

G.  Supporting Data

The human health studies submitted to support the safety of modified
Cry1Ab are summarized in Table 8 below.

Table 8. Summary of Modified Cry1Ab Human Health Data (reviewed in
Edelstein 2008 unless otherwise noted)

Study Type/Title

Summary

MRID #

Acute oral toxicity (OPPTS 870.1100)/ FLCRY1AB-0103: Single Dose Oral
Toxicity Study in the Mouse (AM7516/Regulatory/Report)

Groups of five male and five female mice were given 0 or 1830 mg/kg
bodyweight microbially-produced modified Cry1Ab (FLCRY1AB-0103) by oral
gavage as a single dose.  There were no effects on clinical condition,
body weight, food consumption, clinical pathology, organ weight, or
macroscopic or microscopic pathology that were attributed to the test
substance.

Classification: ACCEPTABLE

47017614

In vitro digestibility/ In vitro digestibility of full-length Cry1Ab
protein (test substances FLCRY1AB-0103 and IAPCOT67B-0106) under
simulated mammalian gastric conditions	The in vitro digestibility in
simulated gastric fluid of the modified Cry1Ab protein as expressed in
COT67B and from a bacterial source was investigated.  No intact
full-length modified Cry1Ab protein from bacterial- or plant-derived
sources was found one minute after incubation in simulated gastric
fluid.  An immunoreactive polypeptide fragment (~ 60,000 Da) in the
digestion mixture was visible in the 5 minute sample in the
plant-derived source and in the 10 minute sample in the
bacterial-derived source.  The study results indicate that the
full-length Cry1Ab protein is rapidly digested in simulated gastric
fluid; a 60 kDa fragment is formed, which also appears to be digestible,
but at a slower rate.

Classification: ACCEPTABLE	47017615

Heat stability/ Effect of temperature on the stability of full-length
Cry1Ab protein

The effect of temperature on the bioactivity of modified Cry1Ab was
investigated.  Heating of E. coli-derived modified Cry1Ab
(FLCRY1AB-0103) at 65˚C or 95˚C for 30 minutes substantially decreased
or eliminated the insecticidal activity of the protein.  No significant
effect on the protein’s insecticidal properties was found following
incubation for 30 minutes at temperatures ≤37˚C.

Classification: ACCEPTABLE

47017616

Amino acid sequence comparison/ Full-length Cry1Ab as expressed in Event
COT67B: Assessment of amino acid sequence homology with known allergens
Two amino acid sequences comparisons of modified Cry1Ab with known
allergens were conducted using the Syngenta Biotechnology, Inc (SBI)
Allergen Database.  The results indicate that modified Cry1Ab has no
significant amino acid sequence homology to known or putative allergenic
proteins based on a search for greater than 35% sequence identity over
successive 80-amino acid peptides and a search for eight or more
identical contiguous amino acids.

Classification: ACCEPTABLE 	47017619

II.B.3.  Human Health Assessment of Hygromycin B Phosphtransferase
(APH4)

The hygromycin B phosphotransferase (APH4) protein expressed in COT102 x
COT67B is covered by the exemption from the requirement of a tolerance
at 40 CFR 174.526 Hygromycin B phosphtransferase (APH4) marker protein
in all plants; exemption from the requirement of a tolerance.

Summary of new data submitted for APH4

MRID# 47017618—APH4 (Entrez Database accession No. CAA85741):
Assessment of Amino Acid Sequence Homology with Known Allergens:

Two amino acid sequences comparisons of APH4 with known allergens were
conducted using the Syngenta Biotechnology, Inc (SBI) Allergen Database.
 The results indicate that APH4 has no significant amino acid sequence
homology to known or putative allergenic proteins based on a search for
greater than 35% sequence identity over successive 80-amino acid
peptides and a search for eight or more contiguous amino acids.

Classification: ACCEPTABLE

II.B.4.  References

Crickmore, N., Zeigler, D.R., Schnepf, E., Van Rie, J., Lereclus, D.,
Baum, J, Bravo, A. and Dean, D.H., 2007.  Bacillus thuringiensis toxin
Nomenclature (2007).  Available at:
http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/

Edelstein, R., 2008.  Review of Human Health and Product
Characterization Data for Registration of B. thuringiensis Modified
Cry1Ab and Vip3Aa19 Proteins and the Genetic Material Necessary for
their Production in COT67B x COT102 Cotton.  Memorandum from R.
Edelstein to A. Reynolds, dated February 7, 2008.

Matten, S., 2007.  Review of Product Characterization and Human Health
Data for Plant- Incorporated Protectant Bacillus thuringiensis (Bt)
Insect Control Proteins Full-length Cry1Ab and Vip3Aa19 and the Genetic
Material Necessary for Their Production in Event COT67B, Event COT102,
and COT67B X COT102 Cotton in Support of the Experimental Use Permit
(EUP) (67979-EUP-T) and the Extension of the Temporary Tolerance
Exemption for Bacillus thuringiensis  Vip3A.  Memorandum from S. Matten
to A. Reynolds, dated April 4, 2007.

Food and Agriculture Organization of the United Nations and World Health

Organization, 2003. Foods Derived from Biotechnology.  Codex
Alimentarius.

Sec. 4, No.38, pg.16.

Lerouge, P. Cabanes-Macheteau, M., Rayon, C., Fichette-Lainé, A-C.,
Gomord, V., and Faye, L., 1998.  “N-Glycoprotein biosynthesis in
plants: recent developments and future trends,” Plant Molecular
Biology 38: 31-48, 1998.

Sjoblad, R.D., J.T. McClintock and R. Engler, 1992. Toxicological
considerations for

protein components of biological pesticide products.  Regulatory
Toxicol.

Pharmacol. 15:  3-9.

Waggoner, A., 2007.  Memorandum from A. Waggoner to M. Mendelsohn dated
February 8, 2007.

Wozniak, C.  2004.  EPA Review of the product characterization,
environmental fate and human health toxicity studies for the
insecticidal protein VIP3A, as expressed in upland cotton, for control
of lepidopteran insect pests, and the selective marker protein APH4.
Memorandum from C. Wozniak to L. Cole dated March 24, 2004.

II. C.	Environmental Hazard Assessment

Note:  EPA’s environmental assessment was conducted for Vip3Aa
proteins, which include the Vip3Aa19 protein as expressed in cotton.
“Full-length Cry1Ab” (FLCry1Ab) refers to the modified Cry1Ab (OECD
Unique Identifier SYN-IR67B-1) protein in VipCot.

Background

Vip3A is a novel class of recently discovered insecticidal proteins that
occur naturally in Bacillus thuringiensis (Bt), a gram-positive soil
bacterium (Estruch, et al. 1996).  The vegetative insecticidal proteins
are produced during vegetative bacterial growth and are secreted as
soluble proteins into the extracellular environment.  Syngenta Seeds,
Inc. has developed Event COT102, a cotton line that expresses an insect
control protein, known as Vip3Aa.  In addition, Syngenta Seeds, Inc. has
also developed Event COT67B, a cotton line that expresses a Bt insect
control Cry protein, known as full-length Cry1Ab. These proteins are
intended to control several lepidopteran pests of cotton including:
Helicoverpa zea (cotton bollworm), Heliothis virescens (tobacco
budworm), Spodoptera frugiperda (fall armyworm), Spodoptera exigua (beet
armyworm), and Trichoplusia ni (cabbage looper).

Syngenta Seeds, Inc. is requesting a Sec. 3 registration for Bt insect
control protein Vip3Aa as expressed in Event COT102 cotton, full-length
Cry1Ab (hereafter, referred to as FLCry1Ab) as expressed in Event COT67B
cotton, and its associated breeding stack, COT102 x COT67B [also known
as VipCot, EPA Reg. No. 67979-O] cotton (which combines Vip3Aa and
FLCry1Ab proteins), crossed via traditional breeding.  An  experimental
use permit (EUP) was granted by the Agency to conduct field tests on
Event COT102, Event COT67B, and its associated breeding stack COT102 x
COT67B (Matten, 2007).

Event COT102 cotton specifically expresses Vip3Aa19, a variant of the
naturally occurring Vip3Aa1 protein isolated from Bacillus thuringiensis
strain AB88, differing from the Vip3Aa1 protein by one amino acid.  The
same protein variant present in Event COT102 cotton is also expressed as
Vip3Aa19 in Syngenta’s experimental Event Pacha corn. The Agency
previously determined that “all proteins designated as Vip3Aa are more
than 95% identical,” and “there is sufficient information to support
the safety of all Vip3Aa proteins, provided that they do not have any
significant sequence similarity with known allergens” (Edelstein,
2008). Therefore, in addition to the data reviewed in this report, all
the previously submitted data developed for Vip3Aa protein can be cited
in support of the registration of Event COT102.

Although Vip3Aa protein shares no homology with FLCry1Ab or other known
Cry proteins, extensive testing by Syngenta has established that Vip3Aa
has demonstrated a similar toxicity against larvae of certain
lepidopteran species, including key pests of cotton.  While the modes of
action differ between the two proteins,  the general symptoms displayed
by sensitive lepidopteran larvae following ingestion of Vip proteins
resembles that caused by Cry proteins (i.e., cessation of feeding, loss
of gut peristalsis, overall paralysis of the insect, and death) (Yu, et
al, 1997).  Since the effects of Vip and Cry proteins are considered
similar, the studies submitted on non-target organisms for Event COT102
were conducted and evaluated according to the same environmental risk
assessment criteria of previously reviewed PIP products containing Cry
protein.

FLCry1Ab protein expressed in COT67B cotton and native Cry1Ab protein
are both derived from Bacillus thuringiensis subsp. kurstaki strain HD-1
(B.t.k.).  FLCry1Ab differs from the naturally occurring Cry1Ab protein
in that FLCry1Ab contains 26 additional consecutive amino acids
(described as the ‘Geiser motif”) in the C-terminal portion (Geiser
et al., 1986). The ‘Geiser motif’ is also expressed in another
registered PIP cotton product containing Cry1Ac.  FLCry1Ab protein in
Event COT67B is also similar to the truncated protein variants of Cry1Ab
as expressed in transgenic maize.  The Agency previously determined that
Syngenta’s Event Bt11 corn produces a truncated Cry1Ab protein that
has the same insecticidal active region of amino acids as FLCry1Ab
produced in COT67B cotton (Matten, 2007).  In addition, there are
numerous laboratory studies, field studies, and scientific literature on
the mode of action of Cry1Ab protein, Cry1Ab-expressing maize and
Cry1Ac-expressing cotton (US EPA, 2001b; Naranjo et al., 2005; Romeis et
al., 2006; Cattaneo et al., 2006; and Torres and Ruberson, 2007).  These
data provide a large weight-of-evidence that these proteins demonstrate
very similar insecticidal activity against several lepidopteran cotton
pests at concentrations found in transgenic plants. Furthermore, the
Agency also determined that field efficacy data submitted with the
registration application (MRID No. 470176-33) and reports provided with
the Public Interest Document (MRID No. 470176-35) demonstrate a similar
insecticidal spectrum of the truncated and full-length Cry1Ab proteins
(Martinez, 2008).  Therefore, the effects of truncated Cry1Ab proteins
are considered predictive of the effects of FLCry1Ab protein as
expressed in COT67B cotton to non-target organisms for the purposes of
the environmental risk assessment.

The Agency has conducted an environmental risk assessment of COT102 and
COT67B cotton lines expressing Vip3Aa and FLCry1Ab proteins.  The
general topics covered include gene flow to related wild plants,
potential of weediness, effects on wildlife, and fate of Vip3Aa and
Cry1Ab proteins in the environment.  The assessment is based on data
submitted to the Agency during the development of the cotton lines,
additional data submitted for registration, Federal Insecticide
Fungicide and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP)
recommendations, consultations with scientific experts, and public
comments on Plant-Incorporated Protectant (PIP) regulation.

II. C. 1.  Environmental Risk Assessment for COT102 and COT67B
(lepidopteran active)

A.  Tiered Testing and Risk Assessment Process

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  (US EPA, 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 (US EPA, 2000, 2001a, 2002,
and 2004).

Chronic studies: Since delayed adverse effects and/or accumulation of
toxins through the food chain are not expected to result from exposure
to proteins, protein toxins are not routinely tested for chronic effects
on non-target organisms.  However, the 30 day test duration requirement
does amount to subchronic testing when performed at field exposure test
doses. Proteins 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