Number range CHAPTER

**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**
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10

A. Product Characterization
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B. Human Health
Assessment\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...
25

C. Environmental Assessment
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43

D. Insect Resistance Management
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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]{.underline}**

**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]{.underline}

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]{.underline}

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]{.underline}

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]{.underline}

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 LD~50~ 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:

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

2.  Amino acid sequence. A comparison of the amino acid sequence of
    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.

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

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

5.  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]{.underline}

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]{.underline}

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]{.underline}: 95:5 external structured, unsprayed refuge;
> 150 ft wide, within ½ mile of edge of field.
>
> [Option 2]{.underline}: 80:20 external sprayed refuge; within 1 linear
> mile, preferably ½ mile, of edge of field.
>
> [Option 3]{.underline}: 95:5 embedded refuge; contiguous or within 1
> mile^2^ 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[^1], 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              | APH4                  |
|                       |                       |                       |
|                       | (µg/g dry weight      | (µg/g dry weight      |
|                       | [+]{.underline}       | [+]{.underline}       |
|                       | standard deviation)   | standard deviation)   |
+-----------------------+-----------------------+-----------------------+
| Leaves\*              | 44 [+]{.underline}    | \< 0.42 -- 8.2        |
|                       | 10 - 277              | [+]{.underline} 1.4   |
|                       | [+]{.underline} 41    |                       |
+-----------------------+-----------------------+-----------------------+
| Squares               | 116 [+]{.underline}   | 2.2 [+]{.underline}   |
|                       | 22                    | 0.4                   |
+-----------------------+-----------------------+-----------------------+
| Flowers               | 162                   | 1.68                  |
+-----------------------+-----------------------+-----------------------+
| Pollen                | 3.47                  | 64.3                  |
+-----------------------+-----------------------+-----------------------+
| Bolls                 | 19 [+]{.underline} 4  | \< 0.39               |
+-----------------------+-----------------------+-----------------------+
| Whole Plants          | 25 [+]{.underline} 4  | \< 0.37               |
+-----------------------+-----------------------+-----------------------+
| Seed                  | 7 + 2                 | 1.4 [+]{.underline}   |
|                       |                       | 0.3                   |
+-----------------------+-----------------------+-----------------------+
| Roots                 | 16 [+]{.underline} 2  | 0.53 [+]{.underline}  |
|                       |                       | 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       | **Summary**                                  | **   |
| Type/Title**  |                                              | MRID |
|               |                                              | \#** |
+===============+==============================================+======+
| Expression    | Transgenic cotton plants (COT102) and a      | 4583 |
| Levels/       | non-transgenic isoline (Coker 312) were      | 5801 |
| Quantitation  | grown concurrently in 2001 in Camilla, GA;   |      |
| of VIP3A and  | Maricopa, AZ; and Idalou, TX. Ten whole      |      |
| APH4 Protein  | transgenic plants (including roots) and two  |      |
| in Cotton     | control plants were harvested approximately  |      |
| Tissues and   | 2, 4, 9, 13, 15, and 22 week post-emergence  |      |
| Whole Plants  | (stages: four-leaf, squaring, first white    |      |
| Derived from  | bloom, peak bloom, first open boll,          |      |
| T             | pre-harvest, respectively). Tissue extracts  |      |
| ransformation | were analyzed for VIP3A and APH4 by ELISA.   |      |
| Event         | VIP3A protein was detected in COT102 whole   |      |
| COT102[^2]    | 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**               |      |
+---------------+----------------------------------------------+------+
| Cha           | Southern blot analysis and DNA sequencing    | 4583 |
| racterization | suggest that event COT102 has one transgene  | 5802 |
| of Inserted   | insertion site with a single copy of intact  |      |
| DNA/Molecular | *vip3A(a)* and *aph4* expression cassettes   |      |
| Cha           | (containing one copy of the *vip3A(a)* gene, |      |
| racterization | *aph4* gene, actin-2 promoter, and ubq3      |      |
| and Genetic   | promoter). DNA sequence alignment revealed   |      |
| Stability of  | an exact sequence match between the pCOT-1   |      |
| Event         | vector and event COT102, and showed the lack |      |
| COT102^b^     | 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**              |      |
+---------------+----------------------------------------------+------+
| Ch            | The *Bacillus thuringiensis* (*Bt*) VIP3A    | 4576 |
| aracteristics | insect control protein as expressed in       | 6501 |
| of *Bacillus  | transgenic cotton seed confers protection    |      |
| t             | against the bollworm complex and other       |      |
| huringiensis* | lepidopteran cotton pests. The seeds are     |      |
| VIP3A Protein | derived from transgenic cotton event COT102, |      |
| and VIP3A     | which contains the insecticidal gene via     |      |
| Cotton Plants | plasmid vector pCOT1. The product active     |      |
| Derived from  | ingredient is ≤0.0015 % dry weight *Bacillus |      |
| Event         | thuringiensis* VIP3A Protein and the genetic |      |
| COT102^b^     | 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.       |      |
+---------------+----------------------------------------------+------+
| Cha           | VIP3A protein produced in cotton plants      | 4583 |
| racterization | derived from transgenic cotton event         | 5812 |
| of the active | "COT102\" was characterized for its          |      |
| i             | biochemical and functional similarity with   |      |
| ngredient/Cha | VIP3A expressed in recombinant *Escherichia  |      |
| racterization | coli* and "Pacha" derived transgenic maize   |      |
| of VIP3A      | plants. Samples of purified VIP3A protein    |      |
| Protein       | from *E. coli* and maize were dissolved in   |      |
| Produced in   | buffer for analysis by SDS-PAGE and Western  |      |
| C             | blotting. VIP3A from cotton leaves was       |      |
| OT102-Derived | extracted following published procedures and |      |
| Cotton and    | prepared for SDS-PAGE and Western blotting.  |      |
| Comparison    | VIP3A proteins from all three sources were   |      |
| with VIP3A    | determined to have the predicted molecular   |      |
| Protein       | weight of ca. 89,000 and cross-reacted       |      |
| Expressed in  | immunologically with the same anti-VIP3A     |      |
| Both Maize    | antibody. No evidence of any                 |      |
| (Corn)        | post-translational modification of VIP3A was |      |
| Derived from  | observed in any of the three Vip3A protein   |      |
| Event PACHA   | sources. Peptides representing *ca*. 85%     |      |
| and           | (673/789) of the complete VIP3A amino acid   |      |
| Recombinant   | sequence were identified by mass spectral    |      |
| *Escherichia  | analysis of cotton produced VIP3A protein.   |      |
| coli*^b^      | 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**               |      |
+---------------+----------------------------------------------+------+
| []{.mark}     | []{.mark}                                    | [    |
|               |                                              | ]{.m |
| Expression    | Processing transgenic COT102 and control     | ark} |
| Level/        | Coker 312 cotton seeds resulted in similar   |      |
| Analysis of   | yields for the hulls, lint, kernels, refined | 4583 |
| Processed     | oil, and de-fatted meal. Analysis of the     | 5803 |
| COT102        | refined oil and de-fatted meal (non-toasted  |      |
| Cottonseed    | and toasted) by ELISA detected VIP3A protein |      |
| Products for  | in COT102 meal but not in oil, and not in    |      |
| Yield and     | meal or oil from control seeds. Analysis of  |      |
| Presence of   | both COT102 and Coker 312 de-fatted meal for |      |
| Gossypol and  | the plant toxin gossypol detected free       |      |
| Vip3A         | gossypol (HPLC method) and total gossypol    |      |
| Protein^b^    | (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.**                     |      |
+---------------+----------------------------------------------+------+
| The mode of   | This publication (Lee *et al.,* 2003), which | 4688 |
| action of the | examined the differences in the mechanism of | 0801 |
| *Bacillus     | insecticidal activity of Cry1Ab and Vip3A,   |      |
| t             | was submitted by the registrant to provide   |      |
| huringiensis* | additional product characterization data,    |      |
| vegetative    | specifically Vip3A's mode of action. The     |      |
| insecticidal  | submitted publication examined differences   |      |
| protein Vip3A | in the mechanism of insecticidal activity of |      |
| differs from  | Cry1Ab and Vip3A proteins. Ligand blotting   |      |
| that of       | showed that activated Cry1Ab and Vip3A-G     |      |
| Cry1Ab        | (Vip3A proteolytically cleaved with          |      |
| delta-        | lepidopteran gut juice) bound different      |      |
| endotoxin[^3] | 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**               |      |
|               |                                              |      |
|               | []{.mark}                                    |      |
+---------------+----------------------------------------------+------+
| []{.mark}     | []{.mark}                                    | [    |
|               |                                              | ]{.m |
| Cha           | The purpose of this study was to             | ark} |
| racterization | re-characterize the microbially produced     |      |
| of Test       | test substance, VIP3A-0204. The purity,      | 4701 |
| Sub           | integrity, and bioactivity of the test       | 7602 |
| stance/Re-Cha | substance were determined and compared with  |      |
| racterization | previous analyses after being stored *ca.*   |      |
| of Vip3A      | 15 months under desiccation at -20 °C. Total |      |
| Protein Test  | protein in VIP3A-0204 was quantified         |      |
| Substance     | spectrophotometrically, and the purity was   |      |
| (Vip3A-0204)  | 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 (LC~50~ of 34 ng       |      |
|               | Vip3A/cm^2^ 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**               |      |
+---------------+----------------------------------------------+------+
| Cha           | Molecular analysis of event COT 102 was      | 4701 |
| racterization | performed using restriction enzyme digestion | 7603 |
| of the        | and Southern blot analysis to determine the  |      |
| inserted DNA/ | number of inserts, copy number of functional |      |
| Additional    | elements, and the presence or absence of     |      |
| Molecular     | plasmid backbone sequences. This study also  |      |
| Cha           | assessed the inheritance and stability of    |      |
| racterization | the insert. Data from the Southern analyses  |      |
| of Event      | demonstrated that the BC4F1 generation of    |      |
| COT102 Cotton | COT102 cotton: (1) contains a single intact  |      |
| by Southern   | insert; (2) contains a single copy of the    |      |
| Analysis      | *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**               |      |
+---------------+----------------------------------------------+------+
| Inheritance   | The purpose of this study was to use ELISA   | 4701 |
| and           | to analyze the levels of expression of the   | 7609 |
| Stability/    | Vip3Aa19 and hygromycin B phosphotransferase |      |
| Stability of  | (APH4) proteins in leaves (collected at the  |      |
| Vip3Aa19 and  | 1^st^ white bloom stage) of three            |      |
| APH4 Protein  | generations (F1, BC1F1, and BC4F1) of Event  |      |
| Expression    | COT102 cotton. The levels of Vip3Aa19        |      |
| Across        | protein measured were comparable (ca. 60     |      |
| Multiple      | µg/g dry weight) in all three generations    |      |
| Generations   | analyzed. APH4 protein was detectable in all |      |
| of Event      | three generations analyzed, but the          |      |
| COT102 Cotton | 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 1^st^    |      |
|               | white bloom stage.                           |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+

**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 [+]{.underline}  |
|                                   | standard deviation)\*             |
+-----------------------------------+-----------------------------------+
| Leaves                            | 65 [+]{.underline} 9 -- 158       |
|                                   | [+]{.underline} 40                |
+-----------------------------------+-----------------------------------+
| Squares                           | 93 [+]{.underline} 13             |
+-----------------------------------+-----------------------------------+
| Flowers                           | 101                               |
+-----------------------------------+-----------------------------------+
| Pollen                            | 12.1                              |
+-----------------------------------+-----------------------------------+
| Bolls                             | 47 [+]{.underline} 7              |
+-----------------------------------+-----------------------------------+
| Whole Plants                      | 26 [+]{.underline} 2              |
+-----------------------------------+-----------------------------------+
| Seed                              | 29 [+]{.underline} 5              |
+-----------------------------------+-----------------------------------+
| Roots                             | 17 [+]{.underline} 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       | **Summary**                                  | **   |
| Type/Title**  |                                              | MRID |
|               |                                              | \#** |
+===============+==============================================+======+
| Cha           | The purpose of this study was to determine   | 4688 |
| racterization | the DNA sequence and contiguousness of the   | 5901 |
| of Inserted   | full length *cry1Ab* (*flcry1Ab*) gene       |      |
| DNA/ Harper,  | present in Syngenta's COT67B cotton and its  |      |
| B. (2006).    | inheritance ratio across generations. COT67B |      |
| Molecular     | cotton plants express a modified full length |      |
| cha           | Cry1Ab *Bacillus thuringiensis* protein      |      |
| racterization | (FLCry1Ab) that contain an additional 26     |      |
| of Event      | amino acids in the C-terminal portion of the |      |
| COT67B        | protein described as the "Geiser motif."     |      |
| cotton.       | FLCry1Ab confers resistance to certain       |      |
|               | lepidopteran insects in cotton. The T-DNA    |      |
| Report No.    | insert (via the pNOV4641 plasmid) in COT67B  |      |
| SS            | cotton was analyzed by Southern blots and    |      |
| B-125-06.[^4] | 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**               |      |
+---------------+----------------------------------------------+------+
| Expression    | The purpose of this study was to quantify    | 4688 |
| Levels/ Hill, | expression of Cry1Ab protein in Event        | 5902 |
| K. (2006).    | COT67B-derived cotton plants. Quantifiable   |      |
| Q             | levels of Cry1Ab protein in Event            |      |
| uantification | COT67B-derived cotton plants were determined |      |
| of Cry1Ab     | by enzyme-linked immunosorbent assay (ELISA) |      |
| protein in    | for various plant tissues and whole plants   |      |
| Event COT67B  | at five developmental stages in four         |      |
| cotton        | locations. Corresponding, near-isogenic,     |      |
| tissues and   | non-transgenic control cotton plants were    |      |
| whole plants. | analyzed in parallel. As expected, Cry1Ab    |      |
|               | protein was detected in all COT67B plant     |      |
| Report        | tissues (i.e., young leaves, old leaves,     |      |
| No.           | roots, flowers, pollen, bolls) except fiber  |      |
| SSB-022-06^d^ | and nectar. The concentrations of Cry1Ab in  |      |
|               | COT67B were similar between the four         |      |
|               | locations for each tissue type at each time  |      |
|               | point, although no specific conclusions      |      |
|               | about differences between locations can be   |      |
|               | made from the data. Where the concentrations |      |
|               | of Cry1Ab appeared variable, there were no   |      |
|               | consistent trends to indicate that the       |      |
|               | plants grown in a given location had higher  |      |
|               | or lower Cry1Ab concentrations. No           |      |
|               | statistical analysis was performed. Cry1Ab   |      |
|               | concentrations in most of the near-isogenic, |      |
|               | nontransgenic control 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 1^st^ 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.**             |      |
+---------------+----------------------------------------------+------+
| Cha           | The purity, integrity, and bioactivity of    | 4701 |
| racterization | *E. coli*-produced test substance            | 7604 |
| of test       | FLCRY1AB-0103, containing modified           |      |
| substance/Cha | full-length Cry1Ab, were determined          |      |
| racterization | initially and after *ca.* 5 months of being  |      |
| of Cry1Ab     | stored under desiccation at -20 °C. The      |      |
| Test          | purity of test substance FLCRY1AB-0103 was   |      |
| Substance     | determined to be *ca.* 86%, both before and  |      |
| FLCRY1AB-0103 | after storage, and Western blot analysis of  |      |
| and           | the test substance showed a dominant         |      |
| Certificate   | immunoreactive band corresponding to the     |      |
| of Analysis   | 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 LC~50~ of 3.7 ng           |      |
|               | Cry1Ab/cm^2^ 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**               |      |
+---------------+----------------------------------------------+------+
| Cha           | The purpose of this study was to             | 4701 |
| racterization | re-characterize the purity, integrity, and   | 7605 |
| of test       | bioactivity of microbially produced test     |      |
| sub           | substance FLCRY1AB-0103 (containing modified |      |
| stance/Re-Cha | full-length Cry1Ab) after storage at -20 °C  |      |
| racterization | for *ca.* 14 months. Total protein in test   |      |
| of Cry1Ab     | substance FLCRY1AB-0103 was quantified       |      |
| Test          | spectrophotometrically by measuring its      |      |
| Substance     | absorption at 280 nm (A~280~ method). The    |      |
| FLCRY1AB-0103 | purity of test substance FLCRY1AB-0103 was   |      |
|               | calculated from the total sample weight and  |      |
|               | the total protein as determined by the       |      |
|               | A~280~ 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**               |      |
+---------------+----------------------------------------------+------+
| []{.mark}     | []{.mark}                                    | [    |
|               |                                              | ]{.m |
| Cha           | The purpose of this study was to use various | ark} |
| racterization | biochemical and functional parameters to     |      |
| of Expressed  | demonstrate the biochemical equivalence      | 4701 |
| Substance/    | between the Cry1Ab protein expressed in      | 7608 |
| Cha           | transgenic Event COT67B cotton and the       |      |
| racterization | Cry1Ab protein contained in test substance   |      |
| of the Cry1Ab | FLCRY1AB-0103 prepared from an *E. coli*     |      |
| Protein       | over-expression system. Cry1Ab protein was   |      |
| Produced in   | extracted from COT67B cotton plant tissue    |      |
| Event         | and its apparent molecular weight,           |      |
| C             | immunoreactivity, glycosylation status, and  |      |
| OT67B-Derived | bioactivity were compared to the Cry1Ab      |      |
| Cotton Plants | protein from test substance FLCRY1AB-0103.   |      |
| and           | In addition, the microbial- and              |      |
| Comparison    | plant-derived Cry1Ab proteins were analyzed  |      |
| with Cry1Ab   | by peptide mass mapping and the N-terminal   |      |
| Protein       | amino acid sequence of Cry1Ab from test      |      |
| Produced in   | substance FLCRY1AB-0103 was determined.      |      |
| Recombinant   |                                              |      |
| *Escherichia  | The Cry1Ab proteins from COT67B and from     |      |
| coli*         | 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**               |      |
+---------------+----------------------------------------------+------+
| []{.mark}     | []{.mark}                                    | [    |
|               |                                              | ]{.m |
| Expression    | The purpose of this study was to use ELISA   | ark} |
| lev           | to analyze the levels of expression of the   |      |
| els/Stability | modified Cry1Ab protein in leaves (collected | 4701 |
| of Cry1Ab     | at open boll stage) of the F1, BC1F1, and    | 7610 |
| Protein       | BC4F1 generations of Event COT67B cotton.    |      |
| Expression    | Identical plant tissues from two             |      |
| Across        | near-isogenic, nontransgenic cotton plants   |      |
| Multiple      | (cotton line 2429) from the BC1F1 and BC4F1  |      |
| Generations   | generations were concurrently sampled and    |      |
| of Event      | analyzed to identify any potential           |      |
| COT67B Cotton | 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 μg/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**               |      |
+---------------+----------------------------------------------+------+
| Expression    | The purpose of this study was to quantify    | 4701 |
| Levels/       | Cry1Ab protein in linters, defatted toasted  | 7611 |
| Analysis for  | cottonseed meal, and once-refined cottonseed |      |
| the Presence  | oil derived from COT67B, and to determine    |      |
| of Cry1Ab     | Cry1Ab protein concentrations in the fuzzy   |      |
| Protein in    | seed used to generate these processed        |      |
| Linters,      | fractions. Quantification was carried out    |      |
| Toasted       | using an enzyme-linked immunosorbent assay   |      |
| Cottonseed    | (ELISA). The Cry1Ab extraction efficiencies  |      |
| Oil from      | were \>69% for fuzzy seed, linters, and      |      |
| Processed     | defatted toasted cottonseed meal from        |      |
| Seed of Event | COT67B. The mean concentrations of Cry1Ab    |      |
| COT67B Cotton | protein (corrected for extraction            |      |
| Expressing    | efficiency) in fuzzy seed, linters, and      |      |
| Full-Length   | defatted toasted cottonseed meal from COT67B |      |
| Cry1Ab        | were 25.1, 9.6, and 47.5 µg Cry1Ab/g,        |      |
| Protein       | 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**               |      |
+---------------+----------------------------------------------+------+

[]{.mark}

[]{.mark}

**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        | APH4            | Cry1Ab        |
|                 |                 |                 |               |
|                 | (µg/g dry       | (µg/g dry       | (µg/g dry     |
|                 | weight          | weight          | weight        |
|                 | [+]{.underline} | [+]{.underline} | [+            |
|                 | standard        | standard        | ]{.underline} |
|                 | deviation)\*    | deviation)\*    | standard      |
|                 |                 |                 | deviation)\*  |
+=================+=================+=================+===============+
| Leaves          | 55              | \<0.41 -- 6.3   | 70            |
|                 | [+]{.underline} | [+]{.underline} | [+            |
|                 | 7 -- 239        | 1.3             | ]{.underline} |
|                 | [+]{.underline} |                 | 14 -- 185     |
|                 | 46              |                 | [+            |
|                 |                 |                 | ]{.underline} |
|                 |                 |                 | 63            |
+-----------------+-----------------+-----------------+---------------+
| Squares         | 132             | 2.1             | 94            |
|                 | [+]{.underline} | [+]{.underline} | [+            |
|                 | 18              | 0.5             | ]{.underline} |
|                 |                 |                 | 10            |
+-----------------+-----------------+-----------------+---------------+
| Flowers         | 148             | 1.80            | 121           |
+-----------------+-----------------+-----------------+---------------+
| Pollen          | 3.06            | 74.7            | 10.7          |
+-----------------+-----------------+-----------------+---------------+
| Bolls           | 21              | \< 0.43         | 42            |
|                 | [+]{.underline} |                 | [+            |
|                 | 4               |                 | ]{.underline} |
|                 |                 |                 | 7             |
+-----------------+-----------------+-----------------+---------------+
| Whole Plants    | 25              | \< 0.40         | 29            |
|                 | [+]{.underline} |                 | [+            |
|                 | 7               |                 | ]{.underline} |
|                 |                 |                 | 7             |
+-----------------+-----------------+-----------------+---------------+
| Seed            | 7               | 1.6             | 27            |
|                 | [+]{.underline} | [+]{.underline} | [+            |
|                 | 1               | 0.4             | ]{.underline} |
|                 |                 |                 | 4             |
+-----------------+-----------------+-----------------+---------------+
| Roots           | 11              | 0.46            | 20            |
|                 | [+]{.underline} | [+]{.underline} | [+            |
|                 | 3               | 0.05            | ]{.underline} |
|                 |                 |                 | 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       | **Summary**                                  | **   |
| Type/Title**  |                                              | MRID |
|               |                                              | \#** |
+===============+==============================================+======+
| Cha           | Molecular analyses (restriction enzyme       | 4701 |
| racterization | digests and Southern blots) were performed   | 7606 |
| of Inserted   | to compare the integrity of the transgenic   |      |
| DNA/          | inserts in the cotton lines Event COT102     |      |
| Comparative   | cotton and Event COT67B cotton with the      |      |
| Southern      | transgenic inserts in stacked COT102 x       |      |
| Analysis of   | COT67B cotton, which was produced by         |      |
| Stacked       | conventional plant breeding of COT102 and    |      |
| COT102 x      | COT67B. The Southern blot data demonstrated  |      |
| COT67B        | 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**               |      |
+---------------+----------------------------------------------+------+
| Expression    | The purpose of this study was to use an      | 4701 |
| Levels/       | enzyme-linked immunosorbent assay (ELISA) to | 7607 |
| Comparison of | analyze tissues from cotton plants derived   |      |
| Transgenic    | from transformation Event COT102, Event      |      |
| Protein       | COT67B and from COT102 x COT67B in order to  |      |
| Expression in | compare the concentrations of Vip3Aa19,      |      |
| Event COT102, | hygromycin B phosphotransferase (APH4), and  |      |
| Event COT67B, | Cry1Ab proteins produced in the transgenic   |      |
| and Stacked   | plants. For the Vip3Aa19 and APH4 proteins,  |      |
| COT102 x      | the concentrations and patterns of           |      |
| COT67B Cotton | expression were generally similar between    |      |
| Lines         | 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**               |      |
+---------------+----------------------------------------------+------+
| Analytical    | The SeedChek Vip3A/FLCry1Ab lateral flow     | 4707 |
| Detection     | test kit was tested for the qualitative      | 4101 |
| Meth          | detection of modified Cry1Ab and Vip3A       |      |
| od/Analytical | proteins in cotton seed and cotton leaf. The |      |
| Detection     | study showed that the SeedChek kit is able   |      |
| Method for    | to detect Vip3A and Cry1Ab in both cotton    |      |
| the Detection | seed and cotton leaf, and no unexpected      |      |
| of Vip3A and  | cross reactivity with other transgenic       |      |
| FLCry1Ab      | varieties was observed.                      |      |
| Protein in    |                                              |      |
| Cotton        | **Classification: ACCEPTABLE**               |      |
| Tissues       |                                              |      |
| Derived from  |                                              |      |
| COT102 x      |                                              |      |
| COT67B Cotton |                                              |      |
| (VipCot       |                                              |      |
| Cotton)       |                                              |      |
+---------------+----------------------------------------------+------+

**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 LD~50~
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:

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

2.  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.

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

4.  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       | **Summary**                                  | **   |
| Type/Title**  |                                              | MRID |
|               |                                              | \#** |
+===============+==============================================+======+
| Summary of    | No significant adverse effects were observed | 4576 |
| Mammalian     | in male and female mice dose by gavage at    | 6502 |
| Toxicology    | approximately 3675 mg VIP3A/kg body weight   |      |
| Data for the  | (the highest dose tested) and the LD~50~ for |      |
| VIP3A and     | pure VIP3A protein was \>3675 mg/kg body     |      |
| APH4 Proteins | weight. The LD~50~ for pure APH4 protein in  |      |
| Produced by   | male and female mice was \>774 mg/kg body    |      |
| Transgenic    | weight. The allergen database compiled by    |      |
| VIP3A Cotton  | Syngenta needs to be better defined or       |      |
| Event         | described in order to ascertain the number   |      |
| COT102^2^     | 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.**                         |      |
+---------------+----------------------------------------------+------+
| Acute Oral    | Eleven male and 11 female HSD:ICR albino     | 4576 |
| Toxicity/     | mice were dosed with VIP3A protein (Lot no.  | 6503 |
| Acute Oral    | VIP3A-0196 containing \~ 32% by weight VIP3A |      |
| Toxicity of   | protein). The mice were quarantined for 5    |      |
| Vip3A Protein | days and fasted approximately 16 hours prior |      |
| in Mice^2^    | 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       |      |
|               | LD~50~ 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.**         |      |
+---------------+----------------------------------------------+------+
| Acute Oral    | Twenty-seven male and 27 female CD-1^®^      | 4576 |
| Toxicity/     | (ICR)BR mice were dosed with VIP3A protein   | 6504 |
| Single Dose   | (Batch VIP3A-0199 containing \~ 54% by       |      |
| Oral Toxicity | weight VIP3A protein), produced in an *E.    |      |
| Study with    | coli* over-expression system. The VIP3A      |      |
| VIP3A-0199 in | protein used as the test material differs    |      |
| Mice^2^       | 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 LD~50~ 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.**         |      |
+---------------+----------------------------------------------+------+
| Acute Oral    | The test animals (Sixteen male and 16 female | 4576 |
| Toxicity/     | Crl-1^®^ (ICR)BR mice) were quarantined for  | 6505 |
| Acute Oral    | 9 days and fasted approximately 4 hours      |      |
| Toxicity      | prior to dosing. The test material (5000     |      |
| Study with    | mg/kg body weight) was dosed as a suspension |      |
| Test          | of 196 mg/mL in 0.5% w/v carboxymethyl       |      |
| Substance     | cellulose (CMC) in deionized water by        |      |
| VIP3A-0100    | gavage. The dose volume was 25.5 mL/kg. The  |      |
| Protein in    | control group was treated with 0.5% w/v CMC  |      |
| Mice^2^       | 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 LD~50~ for males, females, and      |      |
|               | combined was greater than 5000 mg/kg (or \>  |      |
|               | 3675 mg VIP3A protein/kg body weight).       |      |
|               |                                              |      |
|               | **Classification: Acceptable**               |      |
+---------------+----------------------------------------------+------+
| Acute Oral    | VIP3A-Enriched Maize (Corn) Leaf Protein     | 4576 |
| Toxicity/     | (Sample Lot. No. LPPACHA-0199 containing \~  | 6506 |
| Single Dose   | 0.36% by weight VIP3A protein) was prepared  |      |
| Oral Toxicity | from transgenic VIP3A maize (corn) leaves.   |      |
| Study with    | The mice were quarantined for at least 7     |      |
| V             | days and fasted approximately 4 hours prior  |      |
| IP3A-Enriched | to dosing. The test material (5000 mg/kg     |      |
| Maize (Corn)  | body weight) was dosed as a suspension of    |      |
| Leaf Protein  | 250 mg/mL in 0.5% w/v carboxymethyl          |      |
| (             | cellulose (CMC) in deionized water by gavage |      |
| LPPACHA-0199) | (Table 1). The dose volume was 20 mL/kg. The |      |
| in Mice^2^    | 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   |      |
|               | LD~50~ 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.**         |      |
+---------------+----------------------------------------------+------+
| *In Vitro*    | VIP3A from recombinant maize (field corn)    | 4583 |
| Digestibility | plants was prepared as sample LPPACHA-0199   | 5805 |
| of VIP3A      | by extracting the leaves of recombinant corn |      |
| Protein Under | plants and concentra­ting the VIP3A by        |      |
| Simulated     | ammonium sulfate precipitation, dialysis of  |      |
| Mammalian     | the resulting salt, and lyophilization of    |      |
| Gastric       | the collected protein. ELISA showed VIP3A    |      |
| Conditions    | constituted \~0.36 % by weight of the sample |      |
| ^2^           | 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**               |      |
+---------------+----------------------------------------------+------+
| Amino acid    | A.  The purpose of this study was to         | 4688 |
| sequence      |     determine if Vip3Aa19 had any            | 5903 |
| comparison/   |     significant amino acid sequence homology |      |
| Vip3Aa19:     |     to known protein toxins. No relevant     |      |
| Assessment of |     similarities between the Vip3Aa19 query  |      |
| amino acid    |     sequence and known protein toxins were   |      |
| sequence      |     found other than with other              |      |
| homology with |     insect-specific vegetative insecticidal  |      |
| known toxins. |     proteins of *B. thuringiensis*.          |      |
|               |                                              |      |
| Report No.    | B.  **Classification: Acceptable; Supersedes |      |
| SSB-122-06^4^ |     MRID 457665-02**                         |      |
+---------------+----------------------------------------------+------+
| Amino acid    | C.  The purpose of this study was to         | 4688 |
| sequence      |     determine if Vip3Aa19 had any            | 5906 |
| comparison/   |     significant amino acid sequence homology |      |
| Vip3Aa19:     |     to known protein allergens. Vip3Aa19 had |      |
| Assessment of |     no significant amino acid sequence       |      |
| amino acid    |     homology to known or putative allergenic |      |
| sequence      |     proteins.                                |      |
| homology with |                                              |      |
| known         | D.  **Classification: Acceptable; Supersedes |      |
| allergens.    |     MRID 457665-02**                         |      |
|               |                                              |      |
| Report No.    |                                              |      |
| SSB-130-06^4^ |                                              |      |
+---------------+----------------------------------------------+------+
| Amino acid    | The purpose of the study was to determine if | 4686 |
| sequence      | Event MIR162 Vip3A protein had any           | 4808 |
| comparison/   | significant amino acid sequence homology to  |      |
| Vip3A as      | known or putative protein toxins. The        |      |
| expressed in  | database identified 32 entries with E values |      |
| Event MIR162  | below 6 x 10^-6^, of which 30 were           |      |
| maize:        | vegetative insecticidal proteins of *B.      |      |
| Assessment of | thuringiensis* and had E values of 0.0 to 1  |      |
| amino acid    | x 10^-10^. Two proteins were identified as   |      |
| sequence      | rhoptry proteins from *Plasmodium yoelii*, a |      |
| homology with | pathogen that causes malaria in rodents      |      |
| known         | *via* erythrocyte binding and invasion (Ogun |      |
| toxins^3^     | 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**               |      |
|               |                                              |      |
|               | E.                                           |      |
+---------------+----------------------------------------------+------+
| Amino acid    | The purpose of this study was to determine   | 4686 |
| sequence      | if Event MIR162 Vip3Aa20 had any significant | 4809 |
| comparison/   | amino acid sequence homology to known or     |      |
| Vip3A as      | putative protein allergens. No significant   |      |
| expressed in  | sequence homology was found between any      |      |
| Event MIR162  | sequential MIR162 Vip3A 80-amino acid        |      |
| maize:        | peptides and any entry in the SBI Allergen   |      |
| Assessment of | Database. No alignments of eight or more     |      |
| amino acid    | contiguous identical amino acids were        |      |
| sequence      | identified between MIR162 Vip3A and proteins |      |
| homology with | in the SBI Allergen Database. Therefore, no  |      |
| known         | significant amino acid sequence homology was |      |
| allergens^3^  | found between the MIR162 Vip3A and any known |      |
|               | or putative protein allergens.               |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Analysis of   | The purpose of this study was to determine   | 4701 |
| Vip3A or      | whether Vip3A or Vip3A-like proteins are     | 7613 |
| Vip3A-Like    | detectable and quantifiable in commercial    |      |
| Proteins in   | formulations of *Bacillus thuringiensis*     |      |
| Six Different | (Bt)-based microbial insecticide products.   |      |
| Commercial    | ELISA (enzyme-linked immunosorbent assay)    |      |
| Microbial     | and Western blot analyses were used to       |      |
| *Bacillus     | detect and analyze Vip3A or Vip3A-like       |      |
| t             | proteins in the formulations. Vip3A or       |      |
| huringiensis* | Vip3A-like proteins were detected in all six |      |
| Products      | 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**               |      |
+---------------+----------------------------------------------+------+
| Amino acid    | Two amino acid sequences comparisons of      | 4701 |
| sequence      | Vip3Aa19 with known allergens were conducted | 7617 |
| comparison/   | using the Syngenta Biotechnology, Inc (SBI)  |      |
| Vip3Aa19:     | Allergen Database. The results indicate that |      |
| Assessment of | Vip3Aa19 has no significant amino acid       |      |
| amino acid    | sequence homology to known or putative       |      |
| sequence      | allergenic proteins based on a search for    |      |
| homology with | greater than 35% sequence identity over      |      |
| known         | successive 80-amino acid peptides and a      |      |
| allergens     | search for eight or more contiguous          |      |
|               | identical amino acids.                       |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+

**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:

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

2.  Amino acid sequence. A comparison of the amino acid sequence of
    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.

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

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

5.  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       | **Summary**                                  | **   |
| Type/Title**  |                                              | MRID |
|               |                                              | \#** |
+---------------+----------------------------------------------+------+
| Acute oral    | Groups of five male and five female mice     | 4701 |
| toxicity      | were given 0 or 1830 mg/kg bodyweight        | 7614 |
| (OPPTS        | microbially-produced modified Cry1Ab         |      |
| 870.1100)/    | (FLCRY1AB-0103) by oral gavage as a single   |      |
| F             | dose. There were no effects on clinical      |      |
| LCRY1AB-0103: | condition, body weight, food consumption,    |      |
| Single Dose   | clinical pathology, organ weight, or         |      |
| Oral Toxicity | macroscopic or microscopic pathology that    |      |
| Study in the  | were attributed to the test substance.       |      |
| Mouse         |                                              |      |
| (AM7516/Regul | **Classification: ACCEPTABLE**               |      |
| atory/Report) |                                              |      |
+---------------+----------------------------------------------+------+
| In vitro      | The *in vitro* digestibility in simulated    | 4701 |
| d             | gastric fluid of the modified Cry1Ab protein | 7615 |
| igestibility/ | as expressed in COT67B and from a bacterial  |      |
| *In vitro*    | source was investigated. No intact           |      |
| digestibility | full-length modified Cry1Ab protein from     |      |
| of            | bacterial- or plant-derived sources was      |      |
| full-length   | found one minute after incubation in         |      |
| Cry1Ab        | simulated gastric fluid. An immunoreactive   |      |
| protein (test | polypeptide fragment (\~ 60,000 Da) in the   |      |
| substances    | digestion mixture was visible in the 5       |      |
| FLCRY1AB-0103 | minute sample in the plant-derived source    |      |
| and           | and in the 10 minute sample in the           |      |
| IA            | bacterial-derived source. The study results  |      |
| PCOT67B-0106) | indicate that the full-length Cry1Ab protein |      |
| under         | is rapidly digested in simulated gastric     |      |
| simulated     | fluid; a 60 kDa fragment is formed, which    |      |
| mammalian     | also appears to be digestible, but at a      |      |
| gastric       | slower rate.                                 |      |
| conditions    |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Heat          | The effect of temperature on the bioactivity | 4701 |
| stability/    | of modified Cry1Ab was investigated. Heating | 7616 |
| Effect of     | of *E. coli*-derived modified Cry1Ab         |      |
| temperature   | (FLCRY1AB-0103) at 65˚C or 95˚C for 30       |      |
| on the        | minutes substantially decreased or           |      |
| stability of  | eliminated the insecticidal activity of the  |      |
| full-length   | protein. No significant effect on the        |      |
| Cry1Ab        | protein's insecticidal properties was found  |      |
| protein       | following incubation for 30 minutes at       |      |
|               | temperatures ≤37˚C.                          |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Amino acid    | Two amino acid sequences comparisons of      | 4701 |
| sequence      | modified Cry1Ab with known allergens were    | 7619 |
| comparison/   | conducted using the Syngenta Biotechnology,  |      |
| Full-length   | Inc (SBI) Allergen Database. The results     |      |
| Cry1Ab as     | indicate that modified Cry1Ab has no         |      |
| expressed in  | significant amino acid sequence homology to  |      |
| Event COT67B: | known or putative allergenic proteins based  |      |
| Assessment of | on a search for greater than 35% sequence    |      |
| amino acid    | identity over successive 80-amino acid       |      |
| sequence      | peptides and a search for eight or more      |      |
| homology with | identical contiguous amino acids.            |      |
| known         |                                              |      |
| allergens     | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+

**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:]{.underline}

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[^5], 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.

[]{.mark}

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[^6].

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)[^7]. 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)[^8]. 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.[^9] 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[^10].

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 LD~50~ and to quantify the hazard. In
the definitive testing, the number of doses and test organisms evaluated
must be sufficient to determine an LD~50~ 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. LC~50~, ED~50~, or LD~50~ \>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 [^11] (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[^12] of the data
collected from 42 field studies indicated that non-target invertebrates
are generally more abundant in *Bt* cotton and *Bt* maize fields than in
non-transgenic fields managed with insecticides (Marvier, *et al*.,
2007). In addition, a comprehensive review of short and long term field
studies on the effects of invertebrate populations in *Bt* corn and
cotton fields indicated that no unreasonable adverse effects are taking
place as a result of wide scale *Bt* crop cultivation (Sanvido, *et al.*
2007). Another review of field tests published to date concluded that
the large-scale studies in commercial *Bt* cotton have not revealed any
unexpected non-target effects other than subtle shifts in the arthropod
community caused by the effective control of the target pests (Romeis
*et al*., 2006). Slight reductions in some invertebrate predator
populations are an inevitable result of all pest management practices,
which result in reductions in the abundance of the pests as prey.

Overall, the Agency is in agreement with the conclusions of these
studies and collectively, these results provide extensive data to
support that *Bt* crops have not caused long term environmental effects
on a population level to organisms not targeted by *Bt* proteins. Based
on these considerations, regulatory testing of the specialist predators
and parasitoids of target pests may eventually be considered
unnecessary.

**B. Environmental Exposure Assessment**

The EPA risk assessment is centered only on adverse effects at the field
exposure rates (1X EEC), and not on adverse effects at greater
concentrations. Although it is recommended that non-target testing be
conducted at a test dose 10 X the EEC whenever possible, the test dose
margin can be less than 10X where uncertainty in the system is low or
where high concentrations of test material are not possible to achieve
due to test organism feeding habits. High dose testing also may not be
necessary where many species are tested or tests are very sensitive,
although the concentration used must exceed 1X EEC. It is important to
note that Tier I screen testing is not "safety factor testing". In a
traditional "10X safety factor" test any adverse effect noted is a
"level of concern", whereas in the EPA environmental risk assessment
scenario any adverse effect is viewed as a concern only at 1X the field
exposure.

For the purposes of the non-target organism (NTO) studies submitted in
support of Event COT102 and Event COT67B, the test material dose levels
were based on the estimated concentration of Vip3Aa and full-length
Cry1Ab protein expressed in the tissue(s) that NTO would most likely be
exposed to in the environment (see Matten, 2007; Edelstein, 2008 for
protein expression levels). The Agency has determined that the NTOs most
likely to be exposed to the Vip3Aa and FLCry1Ab protein in transgenic
cotton fields were beneficial insects feeding on cotton pollen.
Consequently, test material dose levels were based on the maximum level
of measured protein expression in pollen (3.47 ug/g dwt for Vip3Aa and
12.1 ug/g dwt for Cry1Ab). The principal route of Vip3Aa and full-length
Cry1Ab protein exposure for soil-dwelling organisms (such as collembola,
earthworms, and/or rove beetles) is assumed to be from decomposing plant
tissue and plant exudates in soil. Consequently, the dose levels of the
test material were based on the maximum level of estimated protein
expression in the soil environment.

**C. Non-Target Wildlife Hazard Assessments for Event COT102 and Event
COT67B**

Two separate SAP reports (October 2000 and August 2002) recommended that
non-target testing of *Bt* Cry proteins should focus on invertebrate
species exposed to the crop being registered. Following SAP
recommendations, the EPA determined that non-target organisms with the
greatest exposure potential to Cry protein in transgenic corn fields are
beneficial insects, which feed on corn pollen and nectar, and soil
invertebrates, particularly Lepidoptera species. The Agency recommended
using this same approach for testing the effects of Vip protein in Event
COT102 and Cry protein in Event COT67B on beneficial insects in
transgenic cotton fields. Therefore, toxicity testing using the maximum
hazard dose on representative beneficial organisms from several taxa was
performed in support of both Section 3 FIFRA cotton registrations. The
toxicity of the Vip3Aa and Cry1Ab have been evaluated on several species
of invertebrates including the lady beetle, minute pirate bug,
collembola, daphnia, honey bee, rove beetle, and/or earthworm.
Reproductive and developmental observations were also examined in the
lady beetle, rove beetle, minute pirate bug, and honeybee studies.

As previously noted, Vip3Aa protein in Event COT102 and Cry1Ab protein
in Event COT67B are very host specific, conferring toxic effects on
cotton bollworm, tobacco budworm, fall armyworm, beet armyworm, and
cabbage looper. Number range CHAPTERDespite the October 2000 and August
2002 SAP's recommendations against testing of non-target species not
related to susceptible target pests, EPA has completed a risk assessment
on a range of non-target wildlife to comply with the Agency's published
non-target data requirements. In the absence of PIP-specific risk
assessment guidance, EPA requires applicants for PIP registrations to
meet the 40 CFR Part 158 data requirements for microbial toxins. These
requirements include birds, mammals, plants, and aquatic species. In
addition, earthworm, springtail, and/or rove beetle studies were
voluntarily submitted to the Agency to ascertain the potential effects
of Vip3Aa and FLCry1Ab proteins on beneficial decomposer species.

The October 2000 SAP recommended that while actual plant material is the
preferred test material, bacterial-derived protein is also a valid test
substance, particularly in scenarios where test animals do not normally
consume cotton plant tissue and where large amounts of Cry protein (Cry
protein concentrations that exceed levels present in plant tissue) are
needed for maximum hazard dose testing. For Event COT102, an insect
feeding study, which compared the relative potency of plant-derived
Vip3Aa protein in both Event COT102 cotton and Event Pacha corn to the
microbial-derived proteins, indicated that plant-derived protein was
similar in toxicity to the microbial-derived protein (MRID No. 458358-12
and Edelstein, 2008). Similarly, for Event COT67B, an insect feeding
study, which compared the relative potency of plant-derived FLCry1Ab
protein in COT67B cotton to the microbial-derived protein, indicated
that plant-derived protein was similar in toxicity to the
microbial-derived protein (MRID No. 470176-08 and Edelstein, 2008).
Therefore, these data indicate that the microbial-derived proteins for
each event are substantially equivalent to the plant-derived proteins
expressed in cotton plants based on the similar insecticidal activity
for studying any potential toxicity on NTOs for the purposes of the
environmental risk assessment.

The Agency has also determined that toxicity studies using corn-derived
plant material rather than cotton-derived plant material is acceptable
because cotton contains gossypol and other possible plant toxicants that
may adversely affect non-target organisms. Furthermore, the non-target
species in the cotton agroecosystem are comparable to those in corn;
Specifically for Vip3Aa protein toxicity tests, Event COT102 cotton
expresses the same *vip3A(a)* gene as is expressed in Event Pacha corn,
and the expression level of pollen of Event Pacha corn is much higher
than that of Event COT102 cotton.

In support of the COT102 registration, test substances used in the
submitted studies included bacterial-produced purified Vip3Aa19 and
Vip3Aa1 protein, in addition to Vip3Aa19 as expressed in COT102 cotton
pollen and Event Pacha maize grain, pollen, and leaves. Likewise, in
support of the COT67B registration, test substances used in the
submitted studies included bacterial-produced purified full-length
Cry1Ab and truncated Cry1Ab protein, in addition to Cry1Ab protein as
expressed in Event Bt11 maize grain, pollen, and leaves. The individual
results for each study on ecological effects for Vip3Aa and Cry1Ab are
summarized in Tables 9 and 10, respectively. The results are also
presented in a more descriptive format in subsequent sections of the
risk assessment document. Full reviews of each study for each event can
be found in the individual Data Evaluation Reports (DERs/MRID#s).

**Table 9.** Summary of environmental effects studies and waiver
justifications for COT102 submitted to comply with data requirements
published in 40 CFR § 158.2150(d).

+----------+------+-------------+----------------------------+--------+
| **Data   | *    | **Test      | **Results Summary and      | **MRID |
| Requi    | *OPP | Substance** | Classification**           | No.**  |
| rement** | TS** |             |                            |        |
|          |      |             |                            |        |
|          | *    |             |                            |        |
|          | *Gui |             |                            |        |
|          | deli |             |                            |        |
|          | ne** |             |                            |        |
+==========+======+=============+============================+========+
| Avian    | 885. | Vip3Aa19    | A 49-day dietary study     | 470    |
| dietary  | 4050 | maize grain | showed no adverse affects  | 176-23 |
| testing, |      |             | to broiler chickens when   |        |
|          |      | (Event      | fed a 50% diet composed of |        |
| broiler  |      | Pacha)      | Event Pacha maize grain    |        |
| chicken, |      |             | (containing VIP3A).        |        |
| *Gallus  |      |             | Therefore, the NOEC was    |        |
| dom      |      |             | 0.588 µg VIP3A/g corn feed |        |
| esticus* |      |             | and the LC~50~ was \>      |        |
|          |      |             | 0.588 µg VIP3A/g feed corn |        |
|          |      |             | grain.                     |        |
|          |      |             |                            |        |
|          |      |             | **Classification:          |        |
|          |      |             | Acceptable**               |        |
+----------+------+-------------+----------------------------+--------+
| Avian    | 885. | N/A         | **Acceptable waiver        | N/A    |
| i        | 4100 |             | rationale**                |        |
| njection |      |             |                            |        |
| testing  |      |             |                            |        |
+----------+------+-------------+----------------------------+--------+
| Avian    | 850. | Microbial   | A 14-day study showed no   | 457    |
| oral     | 2100 | Vip3Aa1     | adverse effects to         | 665-08 |
| testing, |      | (           | bobwhite quail from        |        |
| bobwhite |      | VIP3A-0198) | VIP3A-0198, after a single |        |
| quail,   |      |             | oral dose via gavage. The  |        |
|          |      |             | NOEL was 400 mg VIP3A/kg   |        |
| *Colinus |      |             | and the LD~50~ was \> 400  |        |
| virg     |      |             | mg VIP3A/kg bird body      |        |
| inianus* |      |             | weight.                    |        |
|          |      |             |                            |        |
|          |      |             | **Classification:          |        |
|          |      |             | Acceptable**               |        |
+----------+------+-------------+----------------------------+--------+
| Wild     | 885. | N/A         | **Acceptable bridging      | N/A    |
| mammal   | 4150 |             | rationale to acute oral    |        |
| testing  |      |             | toxicity test on mice      |        |
|          |      |             | (MRID No. 457665-05).**    |        |
+----------+------+-------------+----------------------------+--------+
| Fr       | 885. | Vip3Aa19    | A 30-day study showed no   | 470    |
| eshwater | 4200 | maize grain | adverse effects on         | 176-24 |
| fish     |      | (FF         | juvenile catfish after     |        |
| testing, | [    | PACHA-0100) | exposure to Vip3Aa protein |        |
|          | ]{.m |             | from Event Pacha corn      |        |
| channel  | ark} |             | grain. Therefore, the NOEC |        |
| catfish, |      |             | was 7.10 µg Vip3Aa19/g     |        |
| *I       |      |             | fish feed and the LC~50~   |        |
| ctalurus |      |             | was \> 7.10 µg Vip3Aa19/g  |        |
| pu       |      |             |                            |        |
| nctatus* |      |             | **Classification:          |        |
|          |      |             | Acceptable**               |        |
+----------+------+-------------+----------------------------+--------+
| Fr       | 885. | Vip3Aa19    | In a 48-hour static        | 457    |
| eshwater | 4240 | maize       | renewal limit bioassay,    | 921-01 |
| aquatic  |      | pollen      | VIP3A maize pollen         |        |
| inve     |      |             | (containing 83.8 µg VIP3A  |        |
| rtebrate |      | (PHO        | protein/g) had no adverse  |        |
| testing, |      | PACHA-0199) | effects on the survival of |        |
|          |      |             | *Daphnia magna,* when      |        |
| water    |      |             | suspended in 120 mg        |        |
| flea,    |      |             | pollen/L. The LC~50~ was   |        |
| *Daphnia |      |             | \> 83.8 µg VIP3A           |        |
| magna*   |      |             | protein/g.                 |        |
|          |      |             |                            |        |
|          |      |             | **Classification:          |        |
|          |      |             | Unacceptable.** The 885    |        |
|          |      |             | Series Guidelines call for |        |
|          |      |             | a 21 day study. The        |        |
|          |      |             | submitted 48 hour acute    |        |
|          |      |             | study is inadequate.       |        |
+----------+------+-------------+----------------------------+--------+
| E        | 885. | N/A         | **Acceptable waiver        | N/A    |
| stuarine | 4280 |             | rationale**                |        |
| and      |      |             |                            |        |
| marine   |      |             |                            |        |
| animal   |      |             |                            |        |
| testing  |      |             |                            |        |
+----------+------+-------------+----------------------------+--------+
| No       | 885. | N/A         | **Acceptable waiver        | N/A    |
| n-target | 4300 |             | rationale**                |        |
| plant    |      |             |                            |        |
| testing  |      |             |                            |        |
+----------+------+-------------+----------------------------+--------+
| No       | 885. | Microbial   | *Orius* *insidiosus*       | 468    |
| n-target | 4340 | Vip3Aa19    | nymphs fed a meat-based    | 648-14 |
| insect   |      |             | diet containing            |        |
| testing, |      | (           | microbial-derived 7.25 mg  |        |
| minute   |      | VIP3A-0104) | Vip3Aa19 protein/ g diet   |        |
| pirate/i |      |             | showed no adverse effects  |        |
| nsidious |      |             | after 21 days. The NOEC    |        |
| flower   |      |             | was 7.25 mg Vip3Aa19       |        |
| bug ,    |      |             | protein/ g and the LC~50~  |        |
| *Orius   |      |             | was \> 7.25 mg Vip3Aa19    |        |
| ins      |      |             | protein/ g.                |        |
| idiosus* |      |             |                            |        |
|          |      |             | **Classification:          |        |
|          |      |             | Acceptable**               |        |
+----------+------+-------------+----------------------------+--------+
| No       | 885. | Vip3Aa19    | *Coleomegilla maculata*    | 457    |
| n-target | 4340 | maize       | adults were fed a diet     | 665-09 |
| insect   |      | pollen      | containing 5.0% VIP3A      |        |
| testing, |      | (PHO        | maize pollen (containing   |        |
| pink     |      | PACHA-0100) | 144.8 µg VIP3A protein/g   |        |
| -spotted |      |             | pollen) for 21 days with   |        |
| lady     |      |             | no adverse effects         |        |
| beetle,  |      |             | observed. The NOEC was     |        |
| *Cole    |      |             | 7.24 µg VIP3A protein/g    |        |
| omegilla |      |             | pollen and the LC~50~ was  |        |
| m        |      |             | \> 7.24 µg/g VIP3A/g       |        |
| aculata* |      |             | pollen.                    |        |
|          |      |             |                            |        |
|          |      |             | **Classification:          |        |
|          |      |             | Acceptable**               |        |
+----------+------+-------------+----------------------------+--------+
| No       | 885. | Microbial   | *C. septempunctata* adults | 468    |
| n-target | 4340 | Vip3Aa19    | fed a 50% sucrose diet     | 848-02 |
| insect   |      | (           | containing 7250 µg         |        |
| testing, |      | VIP3A-0204) | Vip3Aa19 protein/g diet    |        |
| seven    |      |             | for showed no adverse      |        |
| -spotted |      |             | effects after 15 days. The |        |
| ladybird |      |             | NOEC was 7250 µg Vip3Aa19  |        |
| beetle,  |      |             | protein/g diet and the     |        |
| *Co      |      |             | LC~50~ was \> 7250 µg/g    |        |
| ccinella |      |             | Vip3Aa19 protein/g diet.   |        |
| septemp  |      |             |                            |        |
| unctata* |      |             | **Classification:          |        |
|          |      |             | Acceptable**               |        |
+----------+------+-------------+----------------------------+--------+
| No       | 885. | Microbial   | *Chyrsoperla carnea*       | 468    |
| n-target | 4340 | Vip3Aa19    | larvae fed a meat-based    | 848-15 |
| insect   |      | (           | diet containing 7250 µg    |        |
| testing, |      | VIP3A-0104) | Vip3Aa19 protein/g diet    |        |
| green    |      |             | showed no adverse effects. |        |
| l        |      |             | The NOEC of 7250 µg        |        |
| acewing, |      |             | Vip3Aa19 protein/g diet    |        |
| *Chy     |      |             | and the LC~50~ was \> 7250 |        |
| rsoperla |      |             | µg Vip3Aa19 protein/g diet |        |
| carnea*  |      |             | at day 14, when the        |        |
|          |      |             | control mortality reached  |        |
|          |      |             | 20%. There were no         |        |
|          |      |             | statistically significant  |        |
|          |      |             | differences between the    |        |
|          |      |             | VIP3A-0104 group and the   |        |
|          |      |             | negative control group.    |        |
|          |      |             |                            |        |
|          |      |             | **Classification:          |        |
|          |      |             | Acceptable**               |        |
+----------+------+-------------+----------------------------+--------+
| No       | 885. | Vip3Aa19    | Collembola were fed a diet | 458    |
| n-target | 4340 | maize       | containing 50% yeast and   | 358-10 |
| insect   |      | leaves      | 50% leaf tissue for 28     |        |
| testing, |      |             | days. No statistically     |        |
| coll     |      | (LL         | significant effects on     |        |
| embolan, |      | PACHA-0100) | survival or reproduction   |        |
| *        |      |             | were found among the test  |        |
| Folsomia |      |             | and negative control       |        |
| candida* |      |             | groups. The NOEC was 43.2  |        |
|          |      |             | µg Vip3Aa19 protein/g diet |        |
|          |      |             | and the LC~50~ was \> 43.2 |        |
|          |      |             | µg Vip3Aa19 protein/g      |        |
|          |      |             | diet.                      |        |
|          |      |             |                            |        |
|          |      |             | **Classification:          |        |
|          |      |             | Acceptable**               |        |
+----------+------+-------------+----------------------------+--------+
| Honeybee | 885. | Vip3Aa19    | Three-to-five day old      | 458    |
| testing, | 4380 | maize       | honeybee larvae were       | 358-09 |
| Honeybee |      | pollen      | administered a single dose |        |
| larvae,  |      |             | of *ca.*2 mg of pollen     |        |
|          |      | (PHO        | moistened with a drop of   |        |
| *Apis    |      | PACHA-0199) | 30% sucrose solution       |        |
| me       |      |             | (containing the equivalent |        |
| llifera* |      |             | of 168 µg of Vip3Aa) in    |        |
|          |      |             | their individual brood     |        |
|          |      |             | cells. After 19 days,      |        |
|          |      |             | there were no significant  |        |
|          |      |             | differences between the    |        |
|          |      |             | treatment and control      |        |
|          |      |             | groups in survival to      |        |
|          |      |             | capping, survival to       |        |
|          |      |             | emergence of adults, and   |        |
|          |      |             | the behavior and           |        |
|          |      |             | morphology of the emerged  |        |
|          |      |             | adults. The NOEL was 83.8  |        |
|          |      |             | µg Vip3Aa19 protein/g diet |        |
|          |      |             | and the LD~50~ was \> 83.8 |        |
|          |      |             | µg Vip3Aa19 protein/g      |        |
|          |      |             | diet.                      |        |
|          |      |             |                            |        |
|          |      |             | **Classification:          |        |
|          |      |             | Acceptable**               |        |
+----------+------+-------------+----------------------------+--------+
| E        | OECD | Vip3Aa19    | Adult earthworms were      | 457    |
| arthworm | G    | maize       | exposed to artificial soil | 921-02 |
| ~~t~~    | uide | leaves      | containing 3.60 µg VIP3A   |        |
| oxicity, | line |             | protein/g soil for 14      |        |
|          | 207, | (LP         | days. No mortality or      |        |
| *Eisenia | 850. | PACHA-0199) | differences in body        |        |
| foetida* | 6200 |             | weights were observed in   |        |
|          |      |             | the test group. The NOEC   |        |
|          |      |             | was 3.60 µg VIP3A          |        |
|          |      |             | protein/g soil and the     |        |
|          |      |             | LC~50~ \> 3.60 µg VIP3A    |        |
|          |      |             | protein/g soil.            |        |
|          |      |             |                            |        |
|          |      |             | **Classification:          |        |
|          |      |             | Acceptable**               |        |
+----------+------+-------------+----------------------------+--------+
| Soil     | 885. | Vip3Aa19    | Results of this            | 470    |
| fate and | 5200 | maize       | degradation study indicate | 176-30 |
| deg      |      | leaves      | that the DT~50~ of 16 mg/g |        |
| radation |      |             | concentration of the       |        |
|          |      | (LP         | Vip3Aa19 test material     |        |
|          |      | PACHA-0199) | protein do not persist in  |        |
|          |      |             | various types of soil from |        |
|          |      |             | 6 days to 12.6 days via    |        |
|          |      |             | measuring the loss of      |        |
|          |      |             | bioactivity in black       |        |
|          |      |             | cutworm.                   |        |
|          |      |             |                            |        |
|          |      |             | **Classification:          |        |
|          |      |             | Acceptable**               |        |
+----------+------+-------------+----------------------------+--------+

**Table 10.** **Summary of environmental effects studies and waiver
justifications for COT67B submitted to comply with data requirements
published in 40 CFR § 158.2150 (d).**

+---------+------+-----------+------------------------------+--------+
| **Data  | *    | **Test    | **Results Summary and        | **MRID |
| Requir  | *OPP | Su        | Classification**             | No.**  |
| ement** | TS** | bstance** |                              |        |
|         |      |           |                              |        |
|         | *    |           |                              |        |
|         | *Gui |           |                              |        |
|         | deli |           |                              |        |
|         | ne** |           |                              |        |
+=========+======+===========+==============================+========+
| Avian   | 885. | Bt11      | A 42-day dietary study       | 4565   |
| dietary | 4050 | maize     | showed no deleterious        | 251-01 |
| t       |      | grain     | effects on broiler chicken   |        |
| esting, |      |           | survival or carcass yield    |        |
|         |      |           | when fed a 50% diet composed |        |
| broiler |      |           | of Bt11 maize grain          |        |
| c       |      |           | (containing Cry1Ab).         |        |
| hicken, |      |           |                              |        |
| *Gallus |      |           | **Classification:            |        |
| dome    |      |           | Acceptable**                 |        |
| sticus* |      |           |                              |        |
+---------+------+-----------+------------------------------+--------+
| Avian   | 885. | N/A       | **Acceptable waiver          | N/A    |
| in      | 4100 |           | rationale**                  |        |
| jection |      |           |                              |        |
| testing |      |           |                              |        |
+---------+------+-----------+------------------------------+--------+
| Avian   | 850. | Bt176     | A 14-day study on bobwhite   | 433    |
| oral    | 2100 | Maize     | quail showed no adverse      | 236-09 |
| t       |      | leaf      | effects after a single oral  |        |
| esting, |      | protein   | dose of Bt176 grain,         |        |
| b       |      |           | containing Cry1Ab. The NOEL  |        |
| obwhite |      | (LP       | was 140 mg Cry1Ab/kg         |        |
| quail,  |      | 176-0194) | bodyweight and the LD~50~    |        |
|         |      |           | was \> 140 mg Cry1Ab/kg      |        |
| *       |      |           | bodyweight.                  |        |
| Colinus |      |           |                              |        |
| virgi   |      |           | **Classification:            |        |
| nianus* |      |           | Acceptable**                 |        |
+---------+------+-----------+------------------------------+--------+
| Wild    | 885. | N/A       | **Acceptable bridging        | N/A    |
| mammal  | 4150 |           | rationale to acute oral      |        |
| testing |      |           | toxicity test on mice (MRID  |        |
|         |      |           | No. 47017614)**              |        |
+---------+------+-----------+------------------------------+--------+
| Fre     | 885. | Microbial | A 30-day study showed no     | 470    |
| shwater | 4200 | FLCry1Ab  | adverse effects to juvenile  | 176-25 |
| fish    |      |           | channel catfish. The NOAEC   |        |
| t       | [    | (FLCRY    | was 7.10 µg FLCry1Ab/g fish  |        |
| esting, | ]{.m | 1AB-0103) | feed and the LC~50~ was \>   |        |
|         | ark} |           | 7.10 µg FLCry1Ab/g fish      |        |
| channel |      |           | feed.                        |        |
| c       |      |           |                              |        |
| atfish, |      |           | **Classification:            |        |
| *Ic     |      |           | Acceptable**                 |        |
| talurus |      |           |                              |        |
| pun     |      |           |                              |        |
| ctatus* |      |           |                              |        |
+---------+------+-----------+------------------------------+--------+
| Fre     | 885. | Bt176     | In a 48-hour static renewal  | 433    |
| shwater | 4240 | maize     | limit bioassay, Event 176    | 236-10 |
| aquatic |      | pollen    | maize pollen containing      |        |
| invert  |      | (PHO      | 12.36 µg Cry1Ab protein/g    |        |
| ebrate, |      | 176-0194) | had no adverse effects on    |        |
| water   |      |           | the survival of *Daphnia     |        |
| flea    |      |           | magna,* when suspended in    |        |
| n       |      |           | 150 mg pollen/L. The LC~50~  |        |
| eonate, |      |           | was \> 12.36 µg Cry1Ab       |        |
| *       |      |           | protein/L.                   |        |
| Daphnia |      |           |                              |        |
| magna*  |      |           | **Classification:            |        |
|         |      |           | Unacceptable.** The 885      |        |
|         |      |           | Series Guidelines call for a |        |
|         |      |           | 21 day study. The submitted  |        |
|         |      |           | 48 hour acute study is       |        |
|         |      |           | inadequate                   |        |
+---------+------+-----------+------------------------------+--------+
| Es      | 885. | N/A       | **Acceptable waiver          | N/A    |
| tuarine | 4280 |           | rationale**                  |        |
| and     |      |           |                              |        |
| marine  |      |           |                              |        |
| animal  |      |           |                              |        |
| testing |      |           |                              |        |
+---------+------+-----------+------------------------------+--------+
| Non     | 885. | N/A       | **Acceptable waiver          | N/A    |
| -target | 4300 |           | rationale**                  |        |
| plant   |      |           |                              |        |
| testing |      |           |                              |        |
+---------+------+-----------+------------------------------+--------+
| Non     | 885. | Microbial | *Orius laevigatus* had no    | 470    |
| -target | 4340 | FLCry1Ab  | adverse effects after fed    | 176-28 |
| insect  |      |           | meat-based artificial diets, |        |
| t       |      | (FLCRY    | containing either 1.0039 mg  |        |
| esting, |      | 1AB-0103) | FLCry1Ab/g diet or 1.0039 mg |        |
| pr      |      | and       | FLCry1Ab + 0.1950 mg         |        |
| edatory |      |           | Vip3Aa19/g diet for 14 days. |        |
| bug,    |      | Microbial | Only the results from the    |        |
| *Orius  |      | Vip3Aa19  | first study were valid. The  |        |
| laev    |      |           | NOEC for *O. laevigatus* was |        |
| igatus* |      | (VI       | 1003.9 µg FLCry1Ab/g diet    |        |
|         |      | P3A-0204) | and 1003.9 µg FLCry1Ab +     |        |
|         |      |           | 195.0 µg Vip3Aa19/g diet for |        |
|         |      |           | Event COT67 and Event COT102 |        |
|         |      |           | x COT67B cotton leaves,      |        |
|         |      |           | respectively. Furthermore,   |        |
|         |      |           | the LC~50~ was greater than  |        |
|         |      |           | 1003.9 µg FLCry1Ab/g diet    |        |
|         |      |           | and 1003.9 µg FLCry1Ab +     |        |
|         |      |           | 195.0 µg Vip3Aa19/g diet for |        |
|         |      |           | COT67B and COT102 x COT67B   |        |
|         |      |           | cotton, respectively.        |        |
|         |      |           |                              |        |
|         |      |           | **Classification:            |        |
|         |      |           | Supplemental- see discussion |        |
|         |      |           | below (Section III.2.e.ii)** |        |
+---------+------+-----------+------------------------------+--------+
| Non     | 885. | Microbial | *Coleomegilla maculata*      | 470    |
| -target | 4340 | FLCry1Ab  | larvae were fed prepared     | 176-26 |
| insect  |      |           | diets containing bee pollen, |        |
| t       |      | (FLCRY    | *Ephestia* eggs, and either  |        |
| esting, |      | 1AB-0103) | FLCRY1AB-0103 (containing    |        |
| pink-   |      | and       | 1000 µg FLCry1Ab protein/g   |        |
| spotted |      |           | diet) or FLCRY1AB-0103 and   |        |
| lady    |      | Microbial | VIP3A-0204 (containing 1000  |        |
| beetle, |      | Vip3Aa19  | µg FLCry1Ab and 250 µg       |        |
| *Coleo  |      |           | Vip3Aa protein/g diet) for   |        |
| megilla |      | (VI       | 21 days. No adverse effects  |        |
| ma      |      | P3A-0204) | were observed on larval,     |        |
| culata* |      |           | pupal, or adult survival     |        |
|         |      |           | from either test material    |        |
|         |      |           | diet. The NOAEC for FLCry1Ab |        |
|         |      |           | was 1000 µg FLCry1Ab/g diet  |        |
|         |      |           | and the LC~50~ was greater   |        |
|         |      |           | than 1000 µg FLCry1Ab/g      |        |
|         |      |           | diet. The NOAC for           |        |
|         |      |           | FLCry1Ab + Vip3Aa19 proteins |        |
|         |      |           | tested in combination was    |        |
|         |      |           | 1000 µg FLCry1Ab/g diet +    |        |
|         |      |           | 250 µg Vip3Aa19/g diet and   |        |
|         |      |           | the LC~50~ was greater than  |        |
|         |      |           | 1000 µg FLCry1Ab/g diet +    |        |
|         |      |           | 250 µg Vip3Aa19/g diet.      |        |
|         |      |           |                              |        |
|         |      |           | **Classification:            |        |
|         |      |           | Acceptable**                 |        |
+---------+------+-----------+------------------------------+--------+
| Non     | 885. | Microbial | *A. bilineata* adults were   | 470    |
| -target | 4340 | FLCry1Ab  | fed a meat diet containing   | 176-27 |
| insect  |      |           | 1298.7 g FLCry1Ab protein/g  |        |
| t       |      | (FLCRY    | diet for 35 days with a      |        |
| esting, |      | 1AB-0103) | LC~50~ \> 1298.7 g FLCry1Ab  |        |
| rove    |      |           | /g. Reproductive effects     |        |
| beetle, |      |           | were also assessed by        |        |
| *Al     |      |           | counting the number of       |        |
| eochara |      |           | second-generation adult      |        |
| bil     |      |           | beetles emerging from        |        |
| ineata* |      |           | parasitized pupae of the     |        |
|         |      |           | onion fly (*Delia antique).* |        |
|         |      |           | There were no differences    |        |
|         |      |           | noted between the treatment  |        |
|         |      |           | and negative control groups. |        |
|         |      |           |                              |        |
|         |      |           | **Classification:            |        |
|         |      |           | Acceptable**                 |        |
+---------+------+-----------+------------------------------+--------+
| Non     | 885. | Ly        | Collembola were fed a diet   | 458    |
| -target | 4340 | ophilized | containing 50% yeast and 50% | 358-10 |
| insect  |      | Bt11      | Bt11 leaf tissue for 28      |        |
| t       |      | maize     | days. No statistically       |        |
| esting, |      | leaf      | significant effects on       |        |
| colle   |      |           | survival or reproduction     |        |
| mbolan, |      | (LLB      | were found among the test    |        |
| *F      |      | t11-0100) | and negative control groups. |        |
| olsomia |      |           | The NOEC for the survival    |        |
| c       |      |           | and reproduction of *F.      |        |
| andida* |      |           | candida* was 17.1 µg Cry1Ab  |        |
|         |      |           | protein/g diet and the       |        |
|         |      |           | LC~50~ was \> 17.1 µg Cry1Ab |        |
|         |      |           | protein/g diet.              |        |
|         |      |           |                              |        |
|         |      |           | **Classification:            |        |
|         |      |           | Acceptable**                 |        |
+---------+------+-----------+------------------------------+--------+
| H       | 885. | Microbial | Honeybees were exposed via   | 470    |
| oneybee | 4380 | FLCry1Ab  | oral ingestion to            | 176-29 |
| t       |      |           | microbial-derived FLCry1Ab   |        |
| esting, |      | (FLCRY    | test material in a sucrose   |        |
|         |      | 1AB-0103) | solution, using in-hive      |        |
| *Apis   |      |           | commercial bee feeders. The  |        |
| mel     |      |           | treatments consisted of: a   |        |
| lifera* |      |           | sucrose solution containing  |        |
| larvae, |      |           | 107.82 mg/L FLCRY1AB-0103    |        |
| adults, |      |           | test material/g sucrose      |        |
| and     |      |           | solution (representing 92.4  |        |
| whole   |      |           | µg FLCry1Ab/mL and 10X EEC   |        |
| hive    |      |           | in FlCry1Ab in Event COT67B  |        |
| con     |      |           | pollen), a negative control  |        |
| ditions |      |           | of 50% w/v sucrose solution, |        |
|         |      |           | or a positive control of     |        |
|         |      |           | 6.35 g/L diflubenzuron       |        |
|         |      |           | insect growth regulator in   |        |
|         |      |           | sucrose solution. The test   |        |
|         |      |           | consisted of a single        |        |
|         |      |           | application of one liter of  |        |
|         |      |           | the appropriate solution per |        |
|         |      |           | hive and the hives were      |        |
|         |      |           | observed for 24 days for     |        |
|         |      |           | percent successful brood     |        |
|         |      |           | development to adults and    |        |
|         |      |           | colony conditions. There was |        |
|         |      |           | no significant difference in |        |
|         |      |           | mortality between the test   |        |
|         |      |           | and negative control groups  |        |
|         |      |           | for cells with eggs and      |        |
|         |      |           | young or old larvae. There   |        |
|         |      |           | was also no significant      |        |
|         |      |           | difference in pre- and       |        |
|         |      |           | post-test hive conditions    |        |
|         |      |           | between the test and         |        |
|         |      |           | negative control treatments. |        |
|         |      |           | Results for the positive     |        |
|         |      |           | control treatment were       |        |
|         |      |           | significantly different from |        |
|         |      |           | the other treatments. Adult  |        |
|         |      |           | bees were not affected by    |        |
|         |      |           | any of the treatments.       |        |
|         |      |           | Despite some experimental    |        |
|         |      |           | shortcomings, there is       |        |
|         |      |           | enough certainty to indicate |        |
|         |      |           | exposure of the FLCry1Ab to  |        |
|         |      |           | adult worker honeybees and   |        |
|         |      |           | larvae, via direct and       |        |
|         |      |           | incidental oral ingestion.   |        |
|         |      |           | Furthermore, the results of  |        |
|         |      |           | the study may be considered  |        |
|         |      |           | as weight-of-evidence for    |        |
|         |      |           | determining effects on       |        |
|         |      |           | honeybees for the purposes   |        |
|         |      |           | of the environmental risk    |        |
|         |      |           | assessment. Therefore, the   |        |
|         |      |           | NOEL was 92.4 µg FLCry1Ab/mL |        |
|         |      |           | and the LD~50~ was greater   |        |
|         |      |           | than 92.4 µg FLCry1Ab/mL.    |        |
|         |      |           |                              |        |
|         |      |           | **Classification:            |        |
|         |      |           | Acceptable-for the purposes  |        |
|         |      |           | of the environmental risk    |        |
|         |      |           | assessment**                 |        |
+---------+------+-----------+------------------------------+--------+
| Soil    | 885. | Microbial | The degradation of FLCry1Ab  | 470    |
| fate    | 5200 | FLCry1Ab  | protein (incorporated at a   | 176-31 |
| and     |      |           | rate equivalent to 80 µg     |        |
| degr    |      | (FLCRY    | FLCry1Ab/g dry wt soil) in a |        |
| adation |      | 1AB-0103) | sandy loam soil was assessed |        |
|         |      |           | by measuring the loss of     |        |
|         |      |           | bioactivity to European corn |        |
|         |      |           | borer. The estimated DT50    |        |
|         |      |           | and DT90 values were 17 and  |        |
|         |      |           | 52 days, respectively,       |        |
|         |      |           | indicating that FLCry1Ab     |        |
|         |      |           | protein in plant residues    |        |
|         |      |           | incorporated into sandy loam |        |
|         |      |           | soil is not likely to        |        |
|         |      |           | persist or accumulate in     |        |
|         |      |           | soil.                        |        |
|         |      |           |                              |        |
|         |      |           | **Classification:            |        |
|         |      |           | Acceptable**                 |        |
+---------+------+-----------+------------------------------+--------+
| 3-year  | 885. | Soil from | Soil samples were collected  | 460    |
| Soil    | 5200 | Bt11 corn | from five fields,            | 224-01 |
| Degr    |      | c         | representing four different  |        |
| adation |      | ultivated | soil types, in five          |        |
|         |      | fields    | different states, in which   |        |
|         |      |           | *Bt* corn expressing Cry1Ab  |        |
|         |      |           | had been grown for three     |        |
|         |      |           | consecutive years. Results   |        |
|         |      |           | showed that European corn    |        |
|         |      |           | borer (ECB) larvae exhibited |        |
|         |      |           | no toxic response to a diet  |        |
|         |      |           | mixture, containing 15% *Bt* |        |
|         |      |           | corn soil. Overall, results  |        |
|         |      |           | support use of corn          |        |
|         |      |           | expressing the Cry1Ab        |        |
|         |      |           | protein does not result in   |        |
|         |      |           | the accumulation and         |        |
|         |      |           | persistence of this protein  |        |
|         |      |           | in soil.                     |        |
|         |      |           |                              |        |
|         |      |           | **Classification:            |        |
|         |      |           | Acceptable**                 |        |
+---------+------+-----------+------------------------------+--------+

**1. Non-target Wildlife Study Summaries for COT102 expressing Vip3Aa**

**a. Avian species**

Published data and studies on file at EPA show that consumption of *Bt*
plants have no measurable deleterious effects on avian species. However,
to comply with published data requirements, the following studies were
submitted to EPA in support of Vip3Aa protein as expressed in Event
COT102 product registration. The broiler chicken study was published in
a peer-reviewed journal and not subject to GLP standards, while the
Northern Bobwhite quail study was GLP compliant. When considered
together, these studies meet EPA data requirements for avian species
risk assessment.

i.  **Broiler Chicken (MRID No. 470176-23)**

For the first 49 days of life, commercial broiler chickens (*Gallus
domesticus*) were fed a prepared diet based on 50% corn grain from
transgenic Event Pacha containing VIP3A protein, grain from an isoline
non-transgenic corn, or grain from one of two locally grown reference
corns. There were no treatment-related differences for mortality, body
weight, feed conversion ratio, carcass yield, or clinical chemistry
parameters. The diet containing VIP3A had no deleterious effects on
broiler performance or carcass yield. A separate study determined the
concentration of the transgenic Event Pacha grain as 0.588 µg Vip3Aa19/g
feed for this study (MRID No. 470176-20). Therefore, the NOEC was 0.588
µg VIP3A/g feed and the 49-day LC~50~ for broilers is greater than 0.588
µg VIP3A/g feed.

**Conclusions/Recommendations:** No adverse effects were observed on
*Gallus domesticus* after a 49-day chronic dietary study after exposure
to a 50% diet containing Event Pacha corn grain, expressing VIP3A. The
NOEC was 0.588 µg VIP3A /g feed and the LC~50~ for broilers is greater
than 0.588 µg VIP3A /g feed. Based on the information presented, this
study is acceptable.

ii. **Northern Bobwhite Quail (MRID No. 457665-08)**

Five male and five female (*Colinus virginianus*) quails were
administered a single oral dose of 2000 mg VIP3A-0198 /kg, via gelatin
capsules. The VIP3A-0198 test substance (microbial-derived protein)
represented 400 mg VIP3A /kg body weight. No mortalities occurred during
the study period. There were no clinical signs of toxicity in any birds
during the study. There were no statistically significant changes in
body weights after dosing. Additionally, gross pathological examinations
of all birds at study termination revealed no abnormalities. The results
indicate that the NOEL was 400 VIP3A mg/kg and the 14-day LD~50~ was \>
400 VIP3A mg/kg body weight for northern bobwhite for 14 days.

**Conclusions/Recommendations:** No adverse effects or mortalities were
found after a 14-day acute oral study after exposure to the test
substance (VIP3A-0198, microbial-derived containing Vip3Aa1). The NOEL
was 400 VIP3A mg/kg and the 14-day LD~50~ was \> 400 VIP3A mg/kg body
weight for northern bobwhite for 14 days. Based on the information
presented, this study is acceptable.

**b. Wild mammalian species**

Mammalian wildlife exposure to Vip3Aa protein is considered likely;
however, mammalian toxicology information gathered to date on *Bt* Cry
and Vip proteins does not show a hazard to wild mammals. In addition,
acute oral toxicity studies submitted to EPA in support of the COT102
registration indicated that no significant toxicity was seen when
rodents were exposed to microbial-derived Vip3Aa19 (VIP3A-0100) protein
at the maximum hazard dose level (MRID No. 457665-05). Therefore, no
hazard from COT102 cotton expressing Vip3Aa protein to mammalian
wildlife is anticipated and data on wild mammal testing is not required
for this registration.

**c. Aquatic species**

There is no reported toxicity to aquatic organisms from exposure to
anti-coleopeteran Cry proteins in *Bt* plants. However, a published
laboratory study with lepidopteran-active Cry proteins has revealed that
the leaf shredding (caddis fly) trichopteran, *Lepidostoma liba*, had
50% lower growth rate when fed *Bt* corn litter (Rosi-Marshall, et al.
2007). Two previous field study reports by the same authors did not find
adverse effects on head stream invertebrates. The Agency's position on
this matter is that until Tier III and Tier IV field studies are
performed, there is not enough information to assert that sufficient
corn plant litter enters streams to cause unreasonable adverse effects
on stream invertebrate populations or communities (See Section B.I.
above - Tiered Testing Hazard and Risk Assessment Process). Two years
ago the Iowa State University and the University of Maryland received
Research grants to study the effects of *Bt* corn cultivation on streams
and to develop methods for aquatic hazard assessment. The results of
these studies are pending. When the study reports are reviewed the
Agency will respond with action commensurate with the outcome of the
studies. Therefore, the Agency's current position is that there is no
evidence to conclude that there is sufficient aquatic exposure to Cry
proteins in corn plant litter to result in adverse effects on stream
invertebrate populations or communities. In regards to *Bt* cotton plant
litter expressing lepidopteran-active Vip proteins, the Agency maintains
the same position at this time.

Farmed fish may be exposed to *Bt* protein in fish feed. However, *Bt*
protein activity is generally destroyed during typical fish food
manufacturing processes due to protein degradation from with the high
temperatures. Consequently, exposure of farmed fish to active *Bt*
proteins is not expected. Overall, aquatic animal exposure to *Bt* crops
is extremely small.

i.  **Freshwater fish - Channel Catfish (MRID No. 470176-24)**

The objective of this study was to determine the potential for adverse
effects of Vip3Aa protein to freshwater fish, using the channel catfish,
*Ictalurus punctatus*, as a representative test species, in a 30-day
feeding study. The study compared survival and growth of juvenile
channel catfish fed commercial fish feed formulated with transgenic
maize grain with test substance FFPACHA-0100 (containing 7.1 µg Vip3Aa19
protein/g diet) or with non-transgenic maize grain for 30 days. Both
feeds contained approximately 50% maize grain by weight. The diet was
formulated using a "cold pelleting" process to minimize exposure to
temperatures that might degrade VIP3A protein. The formulation, nutrient
composition, characterization, homogeneity, and stability of the fish
feed test substance was also analyzed. After 30 days, there was no test
material-related mortality. Fish fed either the VIP3A maize grain or the
control maize grain gained equal amounts of weight, and no abnormal
behavior was observed in either group. The activity and stability of
VIP3A in grain and fish feed was confirmed via fall armyworm insect
bioassay and analyzed by ELISA to confirm the presence and amount of the
test material. There were no adverse effects on growth or behavior of
juvenile catfish exposed for 30 days. Therefore, the NOEC was 7.1 µg
Vip3Aa19/g diet and the 30-day LC~50~ was greater than 7.1 µg Vip3Aa19/g
diet fish feed made from Event Pacha maize grain.

**Conclusions/Recommendations:** No observed adverse effects were noted
in *Ictalurus punctatu* after exposure to Vip3Aa via commercial feed
formulated from Event Pacha grain*.* The NOEC was 7.1 µg Vip3Aa19/g diet
and the LC~50~ was greater than 7.1 µg Vip3Aa19/g diet. Based on the
information presented, this study is acceptable.

ii. **Freshwater aquatic invertebrates (MRID No. 457921-01)**

The objective of this study was to determine the potential for acute
effects to the aquatic organism, *Daphnia magna*, during a static
renewal exposure to VIP3A via the Pacha maize pollen. The test was
conducted as a limit test using test substance PHOPACHA-0199, containing
83.8 µg VIP3A protein/g pollen. Daphnids were exposed to a single
nominal test concentration of 120 mg pollen/L for 48 hours with renewal
of the test solution at approximately 24 hours. Two control groups were
included: a group in water exposed to pollen (120 mg/L) from
non-transgenic, near-isogenic maize, and an assay control group exposed
to water only. Each treatment was replicated three times and each
replicate contained 10 neonate daphnids. Observations of mortality,
immobility and other sub-lethal effects were made during the test. At
test termination, there was 100% survival in each group with no sign of
immobilization or any other adverse effects. Therefore, the NOEC was 120
mg VIP3A pollen/L and the LC~50~ was greater than 120 mg VIP3A pollen/L.

**Conclusions/Recommendations:** Results of the 48-hour limit test
showed the LC~50~ was greater than 120 mg PHOPACHA-0199/L, representing
10.1 µg VIP3A /L. Based on the information presented, this study is
unacceptable. The 48 hour test duration is not sufficient to show
mortality for *Bt* toxins. The mode of action of the toxin would take
more than 48 hours for target insect pests to succumb to Cry proteins,
therefore, mortality or reproductive effects to aquatic invertebrates
(e.g., daphnids) are not expected to show within 48 hours. Because Vip
proteins are also derived from *Bt* and susceptible species display
similar symptoms upon ingestion, a 7-14 day *Daphnia* study (OPPTS
Guideline 885.4240 Series) must be performed. This study can be
submitted as a condition of registration. Alternatively, a dietary study
of the effects on an aquatic invertebrate, representing the functional
group of a leaf shredder in headwater streams, can be performed and
submitted in lieu of the 7-14 day *Daphnia* study.

**iii. Estuarine and marine animals -** *Waiver granted*

Estuarine and marine animal studies were not required for this product,
because of the low probability that estuarine or marine systems will be
exposed to Vip3Aa protein produced in event COT102 cotton plant tissues
and pollen.

**d.** **Terrestrial and aquatic plant species -** *Waiver granted*

Plant toxicity studies were not required for this product because the
active ingredient is an insect toxin (*Bt* δ-endotoxin) that has never
shown any toxicity to plants.

**e. Invertebrate species**

The Vip3Aa protein is meant to target species within the order
Lepidoptera (moths and butterflies). *Bt* toxins are known to have a
limited host range, however, to address any unforeseen change in
activity spectrum as a result of laboratory protein synthesis and to
fulfill the published registration data requirements EPA requires that
test species used for non-target insect evaluations should include
several species that are not related to the target pests. Earthworm
studies are also recommended.

i.  **Ladybird beetle**

**MRID No. 457665-09**

The purpose of this study was to determine the potential dietary effects
of the Vip3Aa protein on the mortality and development of the ladybird
beetle, *Coleomegilla maculata*. The protocol for the non-target lady
beetle study was based on OPPTS Guideline 885.4340. Eight- to nine-day
old ladybird beetles were exposed to Vip3Aa via Pacha maize pollen test
substance (PHOPACHA-0100), incorporated into an artificial diet at 5%
weight by weight (w/w). The negative control diet comprised 5% w/w
pollen from non-transgenic, near-isogenic maize, and a positive control
diet contained 50 µg thiobendacarb/g diet. The treatment and control
groups each comprised three replicates of 25 beetles, which received
fresh diet daily. After 21 days, there were no statistically significant
differences in survival, development, and growth between the treatment
and negative control groups (P≤0.05), while there was 100% mortality in
the positive control group. Therefore, the NOEC was 7.24 µg Vip3Aa19/g
diet and the LC~50~ was greater than 7.24 µg Vip3Aa19/g diet.

**Conclusions/Recommendations:** The results indicate that Vip3Aa
protein had no adverse effect on the survival, development, and growth
of the ladybird beetles. The NOEC was 7.24 µg Vip3Aa19/g of diet and the
LC~50~ was greater than 7.24 µg Vip3Aa19/g of diet. This study was
previously reviewed and found acceptable (Rose and Vaituzis, 2003).

**MRID No. 468848-02**

The objective of this study was to determine the potential dietary
effects of Vip3Aa protein on the mortality and development of the
five-spotted ladybird beetle, *Coccinella septempunctata.* The test
substance, VIP3A-0204, was produced by recombinant *E. coli*
fermentation system and contained 7.25 mg Vip3Aa19/g before addition to
a 50% sucrose diet. The negative control diet comprised of sucrose only,
and a positive control diet contained 0.3333 mg dimethoate/g diet.
Treatment and control groups, each comprising of 40 beetles, were fed
fresh diet daily and the endpoints evaluated were survival and
development through 15 days. At study end, mortality in the
Vip3Aa-treated group was not statistically significantly different from
that of the untreated controls (0% vs. 5%, respectively). Positive
control mortality was 100%. The NOEC was 7.25 mg Vip3Aa19 protein/g diet
and the LC~50~ was \> 7.25 mg Vip3Aa19 protein/g diet.

**Conclusions/Recommendations:** No adverse effects were seen in *C.
septempunctata* after exposure to Vip3Aa protein in a sucrose diet*.*
The NOEC was 7.25 mg Vip3Aa19 protein/g diet and LC~50~ was \> 7.25 mg
Vip3Aa19 protein/g diet. This study was previously reviewed and found
acceptable (Milofsky and Vaituzis, 2007).

ii. **Minute pirate bug (MRID No. 468848-14)**

The purpose of this study was to determine the potential dietary effects
of Vip3Aa protein on mortality and development of *Orius insidiosus*,
the minute pirate bug or insidious flower bug.

The test substance was VIP3A-0104, a 63.1 % pure preparation of
microbial-derived Vip3Aa19. The test substance was dissolved in buffer
and incorporated at a rate of 11.49 mg/g diet (7.25 mg Vip3Aa19/g of
artificial diet \-- approximately 310X the highest mean concentration of
Vip3Aa in COT102) and was continuously supplied to predatory bug (*Orius
insidiosus*) nymphs for 21 days. Control nymphs were fed untreated diet,
and positive control nymphs were fed diet treated with 10 µg
teflubenzuron/g of diet. At study end, mortality in the Vip3Aa treated
nymphs was not significantly different from that of the untreated
controls (15% vs. 13%, respectively). Positive control mortality was
100%. The NOEC was 7.25 mg Vip3Aa19 protein/g diet and the LC~50~ value
was determined to be greater than 7.25 mg Vip3Aa19 protein/g diet.

**Conclusions/Recommendations:** No adverse effects were seen in *Orius
insidiosus* after exposure to Vip3Aa protein in an artificial diet*.*
The NOEC was 7.25 mg Vip3Aa19 protein/g diet and the LC~50~ value was
determined to be greater than 7.25 mg Vip3Aa19 protein/g diet. This
study was previously reviewed and found acceptable (Milofsky and
Vaituzis, 2007).

**iii. Green Lacewing (MRID No. 468848-15)**

The purpose of this study was to determine the potential dietary effects
of Vip3Aa protein on mortality and development of *Chrysoperla carnea*
larvae*,* the green lacewing. The test substance, VIP3A-0104, consisted
of 7.25 mg aVip3Aa19/g of artificial diet was continuously supplied to
green lacewing (*Chrysoperla carnea*) larvae for 21 days. Control larvae
were fed untreated diet, and positive control larvae were fed diet
treated with 10 µg teflubenzuron/g diet. At study end, mortality in the
Vip3Aa-treated larvae was not statistically significantly different from
that of the untreated controls (37.5% vs. 35.0%, respectively). Positive
control mortality was 100%. Although the control mortality exceeded the
25% criterion for the test to be considered valid, mortality did not
differ significantly between the test and control groups. Furthermore,
the control mortality was \<25% through day 21, which was judged to be a
sufficient exposure period to observe acute and developmental effects on
lacewing larvae. Therefore, the NOEC was 7.25 mg Vip3Aa19 protein/g diet
and the LC~50~ value was greater than 7.25 mg Vip3Aa19 protein/g diet.

**Conclusions/Recommendations:** No adverse effects were seen in
*Chrysoperla carnea* after exposure to Vip3Aa protein mixed in an
artificial diet*.* The NOEC was 7.25 mg Vip3Aa19 protein/g diet and the
LC~50~ value was determined to be greater than 7.25 mg Vip3Aa19
protein/g diet. This study was previously reviewed and found acceptable
(Milofsky and Vaituzis, 2007).

> **iv. Collembola (MRID No. 458358-10)**

The purpose of this study was to determine the potential dietary effects
of Vip3Aa protein on mortality and reproduction on *Folsomia candida*
(springtail; Collembola). The test substances included: LLPACHA-0100,
containing 43.4 µg Vip3Aa19 protein/g leaf tissue diet from Event Pacha,
distilled water as a negative control and thiodicarb as a positive
control. There were 4 replicates of 10 juvenile collembola per replicate
per treatment and fresh diet was provided daily. Vip3Aa protein had no
detectable impact on the survival or reproduction of the collembola
after 28 days of continuous exposure. The NOEC of lyophilized Vip3Aa
protein from Event Pacha corn leaves was 50% of the diet which was the
highest concentration tested. Therefore, the NOEC was 43.4 µg Vip3Aa19
protein/g diet and the LC~50~ was greater than 43.4 µg Vip3Aa19
protein/g diet.

**Conclusions/Recommendations:** No adverse effects were seen on
*Folsomia candida* after exposure to Vip3Aa protein in Event Pacha maize
leaf tissue. The NOEC was 43.4 µg Vip3Aa19 protein/g diet and the LC~50~
was greater than 43.4 µg Vip3Aa19 protein/g diet. This study was
previously reviewed and found acceptable (Rose and Vaituzis, 2003).

> **v. Honeybee** **(MRID No. 458358-09)**

The objective of this study was to evaluate potential dietary effects of
transgenic Vip3Aa pollen from Event Pacha corn on honeybee larvae (*Apis
mellifera*) survival and adult emergence in a single dose study. The
test substance (PHOPACHA-0199) contained 2 mg of pollen moistened with
30% sucrose solution and was estimated to contain 83.8 µg Vip3Aa19/g
pollen. The study included three controls: one group of larvae were fed
2 mg inbred maize pollen (PIPACHA-0299C) moistened with 30% sucrose
solution, one group received 2 mg inbred maize pollen (PIPACHA-0299C)
moistened with 30% sucrose solution and mixed with potassium arsenate at
1000 ppm (positive control), and the third group received no treatment
at all. Eighty, three- to five-day old larvae (four replicates of 20)
were allowed to consume the pollen and then returned to their source
hives for capping of the brood cells by nurse bees. The hives were
maintained under natural environmental conditions. After 19 days, mean
survival to capping and mean survival to adult emergence were 76.3% in
the Vip3Aa corn pollen group and 77.5% in the control corn pollen group.
Mean survival to capping and mean survival to adult emergence were 87.5%
for the negative control group. There were no statistically significant
differences among these three study groups. Mean survival to capping and
mean survival to adult emergence were 20% in the positive control group,
which was statistically significantly lower than in the other two study
groups. No behavioral or morphological abnormalities were noted among
the emerged adult bees, and no differences in mean emergence times were
observed. Therefore, no adverse effects from Vip3Aa pollen in Event
Pacha corn were seen on honeybee larvae and adult emergency. The NOEC
was 83.8 µg Vip3Aa19/g pollen and the LC~50~ was greater than 83.8 µg
Vip3Aa19/g pollen.

**Conclusions/Recommendations:** No adverse effects from Vip3Aa pollen
in Event Pacha corn were seen on the survival of *Apis mellifera*
honeybee larvae and adult emergence. The NOEC was 83.8 µg Vip3Aa19/g
pollen and the LC~50~ was greater than 83.8 µg Vip3Aa19/g pollen. This
study was previously reviewed and found acceptable (Rose and Vaituzis,
2003).

> **vi. Earthworm (MRID No. 457921-02)**

The objective of this study was to evaluate the potential effects of
Vip3Aa from Event Pacha administered to earthworms (*Eisenia fetida*)
via an artificial soil substrate during a 14-day exposure period. The
testing was conducted based on OPPTS Series 850.6200 Earthworm
Sub-chronic Toxicity Test and OECD Guideline 207. In the test,
earthworms were exposed to a single concentration of VIP3A protein
derived from Event Pacha maize leaf (test substance LPPACHA-0199) and
incorporated into an artificial soil substrate at 3.60 µg VIP3A/g soil.
There were no mortalities in the assay control group, buffer control
group, or VIP3A protein group. Analysis of the test soil showed that
VIP3A was present in the soil and was biologically active against
*Agrotis ipsilon* (black cutworm). Therefore, no adverse effects on
earthworms were observed after exposure to VIP3A protein via Event Pacha
maize leaf tissue. The NOEC was 3.60 µg VIP3A protein/kg dry soil and
the14-day LC~50~ for earthworms was determined to be greater than 3.60
µg VIP3A protein/kg dry soil.

**Conclusions/Recommendations:** No adverse effects from Vip3Aa maize
leaf tissue in Event Pacha were seen on the survival of *Eisenia
fetida,* via an artificial soil substrate after 14 days. The NOEC was
3.60 µg VIP3A protein/kg dry soil and the14-day LC~50~ for earthworms
was determined to be greater than 3.60 µg VIP3A protein/kg dry soil.
Based on the information presented, this study is acceptable.

**2. Non-target Wildlife Study Summaries for COT67B expressing
FLCry1Ab**

**a. Avian species**

Published data and studies on file at EPA show that consumption of *Bt*
plants have no measurable deleterious effects on avian species. However,
to comply with published data requirements, the following studies were
submitted to EPA in support of Event COT67B registration. The broiler
chicken study was published in a peer-reviewed journal and not subject
to GLP standards, while the Northern Bobwhite quail study was GLP
compliant. When considered together, these studies meet EPA data
requirements for avian species.

i.  **Broiler Chicken (MRID No. 456521-01)**

For the first 42 days of life, commercial broiler chickens (*Gallus
domesticus*) were fed a prepared diet based on 50% grain from either
transgenic Bt11 corn containing Cry1Ab protein, transgenic Bt11 corn
sprayed with Liberty herbicide, grain from an isoline, non-transgenic
corn, or grain from a locally grown reference corn. There were no
treatment-related differences for mortality, body weight, feed
conversion ratio, carcass yield, or clinical chemistry parameters. The
corn diet containing the test substance had no deleterious effects on
broiler performance or carcass yield in this study. It should also be
noted that the concentration of Cry1Ab in Bt11 grain used to formulate
the diet was 0.8 µg/g seed, however, the concentration of Cry1Ab in the
feed was not determined. In a similar broiler chicken study, the
concentration of Cry1Ab in Bt176 corn was less than 0.005 µg/g grain.
Therefore, while an official NOEC was not determined, exposure to Cry1Ab
was very likely during the experiment since it is expected that Cry1Ab
in Bt11 grain would behave similarly to Cry1Ab in Bt176 grain during
preparation of broiler diets.

**Conclusions/Recommendations:** No adverse effects were found in the
42-day dietary study with *Gallus domesticus* when fed transgenic Bt11
grain, containing Cry1Ab. This study was previously reviewed and found
acceptable (Hunter and Vaituzis, 2007).

ii. **Northern Bobwhite Quail (MRID No. 433236-09)**

Five male and five female juvenile bobwhite quails (*Colinus
virginianus*) were administered a single oral dose of 140 mg of Cry1Ab
protein/kg body weight, via oral gavage. The test substance was
LP176-0194 (Bt176 maize leaf protein). No mortalities occurred during
the study period. There were no clinical signs of toxicity in any birds
during the study. There were no statistically significant changes in
body weights at any weighing interval (3, 7 or 14 days) after dosing.
Additionally, gross pathological examinations of all birds at study
termination revealed no abnormalities. The results indicate that the
NOEL was 140 mg of Cry1Ab protein/kg body weight and the 14-day LD~50~
was greater than 140 Cry1Ab mg/kg body weight for bobwhite quail.

**Conclusions/Recommendations:** No adverse effects were found in the
14-day dietary study with *Colinus virginianus* when administered a
single oral dose of Vip3A*.* The NOEL was 140 mg of Cry1Ab protein/kg
body weight and the 14-day LD~50~ was greater than 140 Cry1Ab mg/kg body
weight for bobwhite quail. This study was reassessed in the 2001 *Bt*
PIPs Reassessment BRAD (US EPA, 2001b).

**b. Wild mammalian species**

Mammalian wildlife exposure to Cry1Ab protein is considered likely;
however, mammalian toxicology information gathered to date on *Bt* Cry
proteins does not show a hazard to wild mammals. In addition, acute oral
toxicity studies submitted to EPA in support of the COT67B registration
indicated that no significant toxicity was seen when rodents were
exposed to microbial-derived full-length Cry1Ab (FLCRY1AB-0103) protein
at the maximum hazard dose level (MRID No. 470176-14). Therefore, no
hazard to mammalian wildlife is anticipated and data on wild mammal
testing is not required for this registration.

**c. Aquatic species**

There is no reported toxicity to aquatic organisms from exposure to
anti-coleopeteran Cry proteins in *Bt* plants. However, a published
laboratory study with lepidopteran-active Cry proteins has revealed that
the leaf shredding (caddis fly) trichopteran, *Lepidostoma liba*, had
50% lower growth rate when fed *Bt* corn litter (Rosi-Marshall, et al.
2007). Two previous field study reports by the same authors did not find
adverse effects on headwater stream invertebrates. The Agency's position
on this matter is that until Tier III and Tier IV field studies are
performed, there is not enough information to assert that sufficient
corn plant litter enters streams to cause unreasonable adverse effects
on stream invertebrate populations or communities (See Section B.I.
above - Tiered Testing Hazard and Risk Assessment Process). Two years
ago the Iowa State University and the University of Maryland received
Research grants to study the effects of *Bt* corn cultivation on streams
and to develop methods for aquatic hazard assessment. The results of
these studies are pending. When the study reports are reviewed, the
Agency will respond with action commensurate with the outcome of the
studies. Therefore, the Agency's current position is that there is no
evidence to conclude that there is sufficient aquatic exposure to Cry
proteins in corn plant litter to result in adverse effects on stream
invertebrate populations or communities. In regards to
lepidopteran-active *Bt* cotton plant litter, the Agency maintains the
same position at this time.

Farmed fish may be exposed to *Bt* protein in fish feed. However, *Bt*
protein activity is generally destroyed during typical fish food
manufacturing processes due to protein degradation in high temperatures
associated and consequently, exposure of farmed fish to active *Bt*
proteins is not expected. Overall, aquatic animal exposure to *Bt* crops
is negligible.

> **i. Freshwater fish- Channel Catfish (MRID No. 470176-25)**

The objective of this study was to determine the potential for adverse
effects of full-length Cry1Ab to freshwater fish, using the channel
catfish, *Ictalurus punctatus*, as a representative test species in a 28
day feeding study. The study compared survival and growth of juvenile
channel catfish fed commercial catfish diet containing a purified
preparation of FLCRY1AB-0103 (a microbial-derived test substance,
representing 15.4 µg FLCry1Ab protein/g diet) or standard untreated diet
for 28 days. The diet was formulated using a "cold pelleting" process to
minimize exposure to temperatures that might degrade FLCry1Ab protein.
After 28 days, no mortalities or abnormalities were seen in fish either
fed the test material or control diet. The activity and stability of
FLCry1Ab fish feed was confirmed via European corn borer insect bioassay
and analyzed by ELISA to confirm the presence and amount of the test
material in a separate study. Overall, there were no adverse effects and
no mortality observed for juvenile catfish fed with the commercial
catfish diet containing FLCry1Ab after 28 days. The NOAEC was 15.4 µg
FLCry1Ab/g fish food diet and the LD~50~ was greater than 15.4 µg
FLCry1Ab/g diet for juvenile channel catfish.

**Conclusions/Recommendations:** No observed adverse effects were noted
in *Ictalurus punctatus.* Therefore, the NOAEC was 15.4 µg FLCry1Ab/g
fish food diet and the LD~50~ was greater than 15.4 µg FLCry1Ab/g diet
for juvenile channel catfish. Based on the information presented, this
study is acceptable.

> **ii. Freshwater aquatic invertebrates (MRID No. 433236-10)**

The objective of this study was to determine the potential for acute
effects to the aquatic organism, *Daphnia magna*, during a static
renewal exposure to Cry1Ab via the Bt176 maize pollen test substance
(PHO176-0194- containing 12.36 µg Cry1Ab/g). The test was conducted as a
limit test using one test concentration, representing 1.85 µg Cry1Ab/L.
Daphnids were exposed to a single nominal test concentration of 150 mg
pollen/L for 48 hours with renewal of the test solution at approximately
24 hours. Two control groups were included: a group in water exposed to
pollen (150 mg/L) from non-transgenic, near-isogenic maize, and an assay
control group exposed to water only. Each treatment was replicated three
times and each replicate contained 10 neonate daphnids. Observations of
any mortality, immobility and other sub-lethal effects were recorded. At
test termination there was 100% survival in each group with no sign of
immobilization or other effects. The NOEC was 150 mg PHO176-0194/L and
the LC~50~ was greater than 150 mg PHO176-0194/L, representing 1.85 µg
Cry1Ab/L.

**Conclusions/Recommendations:** After 48 hours, the results of the
limit test showed no adverse effects to Daphinia. The NOEC was 150 mg
PHO176-0194/L and the LC~50~ was greater than 150 mg PHO176-0194/L,
representing 1.85 µg Cry1Ab/L. However, based on the information
presented, this study is unacceptable. The 48 hour test duration is not
sufficient to show mortality for *Bt* toxins. The mode of action of the
toxin would take more than 48 hrs. for target insect pests to succumb to
Cry proteins, therefore, mortality or reproductive effects to aquatic
invertebrates e.g., daphnids, are not expected to show within 48 hours.
Therefore, a 7-14 day Daphnia study (OPPTS Guideline 885.4240 Series)
needs to be performed. This study can be submitted as a condition of
registration. Alternatively, a dietary study of the effects Cry1Ab on an
aquatic invertebrate, representing the functional group of a leaf
shredder in headwater streams, may be performed and submitted in lieu of
the 7-14 day Daphnia study.

**iii. Estuarine and marine animals-***Waiver granted*

Estuarine and marine animal studies were not required for this product,
because of the low probability that estuarine or marine systems will be
exposed to Cry1Ab protein produced in event COT67B cotton plant tissues
and pollen.

**d.** **Terrestrial and aquatic plant species-***Waiver granted*

Plant toxicity studies were not required for this product because the
active ingredient is an insect toxin (*Bt* endotoxin) that has never
shown any toxicity to plants.

**e. Invertebrate species**

The Cry1Ab protein is meant to target species within the order
Lepidoptera (moths and butterflies). *Bt* toxins are known to have a
limited host range; however, to address any unforeseen change in
activity spectrum as a result of laboratory protein synthesis and to
fulfill the published registration data requirements, EPA requires that
test species used for non-target insect evaluations should include
several species that are not related to the target pests. Earthworm
studies are also recommended.

i.  **Ladybird beetle (MRID No. 470176-26)**

The purpose of this study was to determine the potential dietary effects
of FLCry1Ab protein test alone and FLCry1Ab and Vip3Aa19 tested in
combination on the survival and development of the pink-spotted ladybird
beetle (*Coleomegilla maculata*). The protocol for the non-target lady
beetle study was based on OPPTS Guideline 885.4340. Five-day old, second
instar ladybird beetles were exposed to a diet of bee pollen,
*Esphestia* (moth) eggs, and either FLCRY1AB-0103 test material (at 1000
µg FLCry1Ab/g diet) or FLCRY1AB-0103 + VIP3A-0204 test materials (at
1000 µg FLCry1Ab/g diet + 250 µg Vip3Aa19/g diet). The negative control
diet was the pollen and moth egg diet only, and a positive control diet
contained 250 µg potassium arsentate/g diet. The treatment and control
groups each comprised of 40 beetles, which received fresh diet every
other day. After 21 days, there were no statistically significant
differences in larval, pupal, and adult survival between the treatment
and negative control groups (P≤0.05), while there was 100% mortality in
the positive control group. Therefore, the NOAEC for FLCry1Ab was 1000
µg FLCry1Ab/g diet and the LC~50~ was greater than 1000 µg FLCry1Ab/g
diet. The NOAC for FLCry1Ab + Vip3Aa19 proteins tested in combination
was 1000 µg FLCry1Ab/g diet + 250 µg Vip3Aa19/g diet and the LC~50~ was
greater than 1000 µg FLCry1Ab/g diet + 250 µg Vip3Aa19/g diet.

**Conclusions/Recommendations:** The results indicate that the FLCry1Ab
protein tested alone or in combination with Vip3Aa19 had no adverse
effect on the survival, development, and growth of the ladybird beetles.
In conclusion, the NOAEC for FLCry1Ab was 1000 µg FLCry1Ab/g diet and
the LC~50~ was greater than 1000 µg FLCry1Ab/g diet. The NOAC for
FLCry1Ab + Vip3Aa19 proteins tested in combination was 1000 µg
FLCry1Ab/g diet + 250 µg Vip3Aa19/g diet and the LC~50~ was greater than
1000 µg FLCry1Ab/g diet + 250 µg Vip3Aa19/g diet. Based on the
information presented, this study is acceptable.

ii. **Minute pirate bug (MRID No. 470176-28)**

The purpose of this study was to determine the potential dietary effects
of FLCry1Ab protein as expressed in Event COT67B and FLCry1Ab and
Vip3Aa19 proteins tested in combination, as expressed in Event COT102 X
COT67B, on mortality and development of *Orius laevigatus*, a predatory
bug which is closely-related and ecologically very similar to *O.
insidiosus*.

The report contained two dietary studies studying the effects on *O.
laevigatus*, after exposure via meat-based artificial diets containing
either FLCry1Ab insecticidal protein alone or in combination with
Vip3Aa19 insecticidal protein. Only the results of the second run of the
first sudy were considered valid and are presented in this summary.
After 14 days, *O. laevigatus* fed 1.0039 mg FLCry1Ab/g diet (7X the
maximum concentration in COT67B cotton leaves) had pre-imaginal
mortality of 17.95%. *O. laevigatus* fed the combined proteins of 1.0039
mg FLCry1Ab + 0.1950 mg Vip3Aa19/g diet (corresponding to 10X the
highest mean concentrations of FLCry1Ab and Vip3Aa19 found in COT67B and
COT02 cotton leaves) had pre-imaginal mortality of 39.47%, which was a
statistically significant increase in mortality. The control
pre-imaginal mortality was 12.82%, while the toxic reference standard
had 100% mortality, as expected. The NOEC for *O. laevigatus* was 1003.9
µg FLCry1Ab/g diet and 1003.9 µg FLCry1Ab + 195.0 µg Vip3Aa19/g diet for
Event COT67 at 7X EEC and Event COT102 x COT67B at 10X EEC cotton
leaves, respectively. Furthermore, the LC~50~ was greater than 1003.9 µg
FLCry1Ab/g diet and 1003.9 µg FLCry1Ab + 195.0 µg Vip3Aa19/g diet for
COT67B and COT102 x COT67B cotton, respectively.

**Conclusions/Recommendations:** The overall results of the two studies
were inconsistent due to the high control mortality, implicating the use
of *Orius laevigatus* is equivocal, as a representative indicator
species for studying the effects of *Bt* PIP proteins. In the only valid
study, there was a statistically significant increase in mortality of
*O. laevigatus* exposed to FLCYR1AB-0103 + VIP3A-0204 at 10X EEC for
COT102 x COT67B cotton leaves, which may represent an interaction
between FLCry1Ab and Vip3Aa19. However, the EPA established Level of
Concern (LOC) is 50% mortality when tested at 5X EEC and a less than 50%
mortality effect at the MHD is indicative of a minimal risk for the
purposes of the environmental risk assessment (US EPA, 1998). Therefore,
no adverse effects on *O. laevigatus* are expected at concentrations
encountered in field crops. The NOEC for *O. laevigatus* was 1003.9 µg
FLCry1Ab/g diet and 1003.9 µg FLCry1Ab + 195.0 µg Vip3Aa19/g diet for
Event COT67 at 7X EEC and Event COT102 x COT67B at 10X EEC cotton
leaves, respectively. Furthermore, the LC~50~ was greater than 1003.9 µg
FLCry1Ab/g diet and 1003.9 µg FLCry1Ab + 195.0 µg Vip3Aa19/g diet for
COT67B and COT102 x COT67B cotton, respectively. Based on the
information submitted, this study is supplemental for the purposes of
the environmental risk assessment.

In addition, a three-year field study conducted on Event Bt11 x Event
Pacha maize (expressing Cry1Ab and VIP3A proteins) showed no differences
on densities of non-target arthropod communities, including *Orius
insidiosus,* when compared with an isogenic conventional corn control
(Dively *et al.* 2005). The results also showed that biodiversity and
community-level responses were not significantly affected by expression
of the stacked VIP3A and Cry1Ab proteins.

When the results of the second run of the first study on *O. laevigatus*
are considered in combination with the three-year field Dively, *et al.*
(2005) study, the weight-of-evidence indicates there are no adverse
effects on *Orius* species from FLCry1Ab protein as expressed in COT67B
or its associated stacked product, Event COT102 x COT67B cotton. The
Agency also notes that there are several published field studies on the
effects of *B*t crops on insect predators showing no significant
differences in the density of beneficial insects, including *Orius
insidiosus* (Pilcher *et a*l., 1997a, 1997b, and 2005; and Al-Deeb *et
al*., 2001).

**iii. Rove Beetle (MRID No. 470176-27)**

The purpose of this study was to determine any reproductive effects of
FLCry1Ab protein on *Aleochara bilineata* (rove beetle). In a laboratory
bioassay, adult rove beetles (*Aleochara bilineata*) were exposed to a
prepared meat diet containing 1298.7 g FLCRY1AB-0103/g of diet for 35
days. The FLCry1Ab concentration fed to the beetles was approximately 10
times that which occurs in fresh leaf tissue of Event COT67B cotton
plants. A negative control diet and a reference control diet were also
included in the test. To assess reproduction of the beetles, onion fly
(*Delia antique*) pupae were provided to be parasitized by the beetles
during the test. Second-generation beetles emerging from the parasitized
pupae were counted until emergence stopped on test day 86. The results
of the reproductive success of the beetles showed no statistically
significant differences between the number of beetles that emerged from
the FLCry1Ab test treatment, when compared to the control. The IOBC
validity criteria were met (Grimm, *et al.,* 2000) and the stability and
bioactivity of the test material in the prepared diet were also
confirmed. Therefore, no adverse effects were noted on the reproductive
effects of FLCry1Ab protein on *A. bilineata*. Furthermore, the NOEC was
1000 µg FLCry1Ab/g diet for the reproduction of *Aleochara bilineata*
and the LC50 was greater than 1000 µg FLCry1Ab/g diet, when exposed
orally via a treated meat-based diet

**Conclusions/Recommendations:** No adverse effects were noted on the
reproductive effects of FLCry1Ab protein on rove beetles. Therefore, the
NOEC was 1000 µg FLCry1Ab/g diet for the reproduction of *Aleochara
bilineata* and the LC~50~ was greater than 1000 µg FLCry1Ab/g diet, when
exposed orally via a treated meat-based diet. Based on the information
presented, this study is acceptable.

> **iv. Collembola (MRID No. 458358-10)**

The purpose of this study was to determine the potential dietary effects
of Cry1Ab protein on mortality and reproduction on *Folsomia candida*
(springtail; Collembola). The treatments included: 17.1 µg Cry1Ab/g diet
of equal parts LLBt11-0100 test substance (lyophilized leaf from Bt11
maize) and yeast, a control diet containing equal parts yeast and
lyophilized leaves of non-transgenic, near-isogenic mazie, a diet to
control the effects of maize leaves consisting of yeast only, and a
positive control of yeast with 500 µg thiodicarb/g diet. There were 4
replicates of 10 juvenile collembola per replicate per treatment and
fresh diet was provided daily.

After 28 days, mean survival was 83%, 78%, and 80% in the
LLBt11-0100-treated group, the non-transgenic maize leaf-treated group,
and the group fed yeast only, respectively. The mean survival for the
positive control group was 3%, which was statistically significant from
the other treatment groups. The mean number of juveniles was 446.5,
343.5, and 218.5 in the LLBt11-0100-treated group, the non-transgenic
maize leaf-treated group, and the group fed yeast only, respectively.
The positive control was significantly different from the other groups.
Therefore, Cry1Ab protein had no detectable impact on the survival or
reproduction of the collembola after 28 days of continuous exposure. The
NOEC for the survival and reproduction of *F. candida* of lyophilized
Bt11 corn leaves was 17.1 µg Cry1Ab protein/g diet and the LC~50~ was
greater than 17.1 µg Cry1Ab protein/g diet.

**Conclusions/Recommendations:** No adverse effects of Cry1Ab were
observed on *Folsomia candida* from Bt11 corn leaf tissue. The NOEC for
the survival and reproduction of *F. candida* of lyophilized Bt11 corn
leaves was 17.1 µg Cry1Ab protein/g diet and the LC~50~ was greater than
17.1 µg Cry1Ab protein/g diet. This study was previously reviewed and
found acceptable (Vaituzis.and Rose, 2000).

> **v. Honeybee** **(MRID No. 470176-29)**

A semi-field whole-hive feeding study was conducted based on the
recommendations in EPPO Bulletin 22 (Oomen, *et al.,* 1992), in
accordance with UK Good Laboratory Practice regulations of 1999 and OECD
principles \[Revised 1997\].

The objective of this study was to evaluate potential dietary effects of
transgenic microbial-derived full-length Cry1Ab on honeybee (*Apis
mellifera*) larvae survival, adult emergence, exposed adult worker bee
survival, and whole-hive conditions in a semi-field study. Honeybees
were exposed, via oral ingestion using in-hive commercial bee feeders.
The treatments consisted of: a sucrose solution containing 107.82 mg/L
FLCRY1AB-0103 test material/g sucrose solution (representing 92.4 µg
FLCry1Ab/mL and 10X EEC in FlCry1Ab in Event COT67B pollen), a negative
control of 50% w/v sucrose solution, or a positive control of 6.35 g/L
diflubenzuron insect growth regulator in sucrose solution. The test
consisted of a single application of one liter of the appropriate
solution per hive and the hives were observed for 24 days for percent
successful brood development to adults and colony conditions. There was
no significant difference in mortality between the test and negative
control groups for brood development. There was also no significant
difference in pre- and post-test hive conditions between the test and
negative control treatments. Results for the positive control treatment
were significantly different from the other treatments for brood
development and hive condition (as indicated by the significantly
reduced mean percentage of comb covered by life stages). Adult bees were
not affected by any of the treatments. These results indicate direct and
incidental ingestion of FLCry1Ab proteins did not adversely affect brood
development, exposed worker bees, and the hive condition. Therefore, the
NOEL was 92.4 µg FLCry1Ab/mL and the LD~50~ was greater than 92.4 µg
FLCry1Ab/mL.

**Conclusions/Recommendations:** No adverse effects were observed after
a single-dose application of FLCRY1AB-0103 test material mixed with a
sucrose solution were observed on *Apis mellifera* honeybee larvae,
adult emergence, exposed adult worker bee survival, and whole-hive
conditions after 24 days. Despite some experimental shortcomings, there
is enough certainty to indicate exposure of the FLCry1Ab to adult worker
honeybees and larvae, via direct and incidental oral ingestion.
Therefore, the NOEL was 92.4 µg FLCry1Ab/mL and the LD~50~ was greater
than 92.4 µg FLCry1Ab/mL.Therefore, the NOEL was 92.4 µg FLCry1Ab/mL and
the LD~50~ was greater than 92.4 µg FLCry1Ab/mL. Therefore, this study
is rated acceptable for the purposes of the environmental risk
assessment.

In addition to this study, a recent meta-analysis of 25 studies that
independently assessed potential effects of *Bt* Cry proteins on
honeybee survival showed that *Bt* Cry proteins used in genetically
modified crops commercialized for control of lepidopteran and
coleopteran pests do not negatively affect the survival of either
honeybee larvae or adults in laboratory settings (Duan, *et al.,* 2008).
A semi-field study also showed no adverse effects of *Bt* corn pollen
containing high levels of Cry1Ab protein on adult honeybee survival,
foraging frequency, behavior or brood development during the 7-day
period of pollen shed and no adverse effects on brood development after
an additional 30 days following pollen shed (Schur *et al.,* 2000).

Therefore, the weight-of-evidence demonstrates that there are no adverse
effects of FLCry1Ab protein on honeybee brood development and adults in
either the laboratory or field setting. This conclusion was determined
by the two semi-field studies (showing no adverse effects of FLCry1Ab
and *Bt* Cry1Ab on brood development, adult survival, and whole hive
conditions) in combination with the meta-analysis of various laboratory
studies (demonstrating no adverse effects of *Bt* Cry proteins on
honeybee larvae and adults).

**3. Soil Fate**

Soil organisms may be exposed to Vip3Aa and FLCry1Ab protein through
contact with cotton plant roots (by direct feeding), cotton plant root
exudates, incorporation of above-ground plant tissues into soil
following harvest, or by soil-deposited pollen. Some evidence suggests
that soils which are high in clays and humic acids are more likely to
bind Cry protein. However, neutral pH soils tend to have high microbial
activity and microbes contribute to Cry protein degradation. In
addition, a study on the release of Cry proteins in the root exudates of
*Bt* cotton has shown that no Cry proteins were detected immunologically
or by larvicidal assay in any soil or hydroponic solution in which *Bt*
cotton had been grown (Saxena and Stotzky, 2001). The weight of evidence
indicates that Cry proteins do not accumulate in soil to arthropod-toxic
levels. Because Vip and Cry proteins are both toxins derived from
soil-inhabiting bacteria, *Bacillus thuringiensis* and found in
commercial microbial insecticides (De Maagd *et al.*, 2003 and Graser
and Song, 2006), Vip protein degradation would also be similar to Cry
protein degradation. Nonetheless, the Agency required the following soil
fate evaluations to support the Event COT102 and COT67B *Bt* cotton
registrations.

**MRID No. 470176-30**

The purpose of this study was to investigate the degradation of Vip3Aa
protein in various types of soils (clay, sandy clay loam, sandy loam,
silt loam, and artificial soils) by assessing the loss of bioactivity,
via insect bioassay. The test substance LPPACHA-0199 (maize leaf
protein, containing ca. 0.36% Vip3Aa19) was incorporated at
concentrations of 16 or 4 Vip3Aa19 mg/g of soil and incubated under
controlled conditions for 29 days. During the incubation, soil samples
were collected weekly and used in black cutworm (BCW, *Agrotis ipsilon*)
bioassays to determine biological activity of the test substance against
the insect over time. The loss of bioactivity was measured by BCW
mortality, which was used to estimate the DT50 (time to dissipation of
50% of the initial bioactivity) of the 16 mg/g concentration of the test
material in each soil. The estimated DT50 values ranged from 6.0 days in
the silt loam to 12.6 days in one of the clays, indicating that Vip3Aa
protein in plant residues incorporated into soil is not likely to
persist or accumulate in soil.

**MRID No. 470176-31**

The purpose of this study was to investigate the degradation of FLCry1Ab
protein in a viable microbial agricultural soil typical of a
cotton-growing region by assessing the loss of bioactivity, via insect
bioassay. The test substance FLCRY1AB-0103 (microbial-derived protein,
containing 103 µg FLCRYCRY1AB-0103/g soil) was applied to sandy loam
soil at a rate equivalent to 80 µg FLCry1Ab/g dry wt of soil, which
would be 160 times the estimated soil concentration that would result
from incorporation of pre-harvest stage COT67B cotton crop residue in
the field. The soil was incubated under controlled conditions for 0, 1,
3, 7, 14, 30, 62, 94, or 120 days after dosing, with samples collected
at each time point for use in the bioassays. The dosed soil samples were
incorporated into insect diet at a concentration of 10% (w/v) and
provided to first instar European corn borer (ECB, *Ostrinia nubilalis*)
larvae for approximately five days. Degradation of FLCry1Ab was assessed
by the loss of bioactivity, measured by ECB mortality. Mortality was
plotted against incubation time to estimate the DT50 and DT90 (time to
dissipation of 50% and 90% of the initial bioactivity, respectively) of
the test material in the soil. The estimated DT50 and DT90 values were
17 and 52 days, respectively, indicating that FLCry1Ab protein in plant
residues incorporated into sandy loam soil is not likely to persist or
accumulate in soil.

**Conclusions/Recommendations:** These studies utilized field soil
spiked with purified insecticidal protein derived from either plant- or
microbial-derived protein. This approach is useful because dose
responses can be easily quantified. However, the degradation and
accumulation of *Bt* Cry proteins found within decaying plant tissue may
behave differently than proteins in artificially spiked soil. Because
Vip protein is derived from *Bt* and display similar insecticidal
activity, the behavior of Vip protein is expected to be similar to Cry
proteins as well. Thus, the presence of low levels of *Bt* Cry and Vip
proteins in the soil (at or below the level of detection) is anticipated
until all plant tissue is 'mineralized'. However, the reviewed data show
that Cry and Vip proteins will be quickly degraded upon release from
decaying plant tissue. In addition, a study that evaluated Cry1Ab
protein accumulation in a field with three years of continuous Cry1Ab
field corn production showed that the protein had not accumulated in
soil to a level that would elicit a toxic response from ECB larvae, a
species that is highly susceptible to Cry1Ab protein (MRID No.
460224-01; Milofsky and Vaituzis, 2006).

Based on FIFRA Scientific Advisory Panel recommendations and public
comments, the Agency has required three year soil fate studies for the
currently registered Cry protein producing crops grown in a variety of
soils and environmental conditions, as a condition of registration. The
results of these studies show that there is no detectable Cry protein
accumulation in agricultural soils during commercial planting of
currently registered Cry protein producing crops (Milofsky and Vaituzis,
2006).

More recently, a comprehensive review of all available scientific data
on ecological effects of commercially grown GM crops over the last ten
years was completed (Sanvido, *et al*. 2007). The review concluded "none
of the laboratory or field studies suggest accumulation of *Bt-*toxins
in soil over several years of cultivation" and "experience from
commercial cultivation indicates that *Bt-*toxin will not persist for
long periods under natural conditions." The Agency agrees with these
conclusions.

Collectively, the long-term field studies for *Bt* crops also confirm
the previous SAP conclusion that "bioaccumulation is not expected to
occur with transgenic proteins because *biodegredation mechanisms for
proteins are ubiquitous*" (US EPA, 2000). More importantly, the numerous
laboratory studies that demonstrated rapid protein degradation in soil
of *Bt* proteins produced in *Bt* crops (when performed under realistic
environmental conditions) are can be considered predictive that *Bt*
protein in soil is not likely to persist or accumulate in soil after
continuous cultivation.

In light of these published findings and the rapid degradation of Vip3Aa
and FLCry1Ab proteins in soil as demonstrated in the insect bioassays,
there is no indication that the proteins expressed in Event COT102 and
Event COT67B are likely to persist or accumulate in soil after
continuous cultivation. Therefore, no additional long-term field studies
are required for these PIP products.

**4.** **Effects on Soil Microorganisms**

Numerous published studies indicate that exposure to Cry protein
produced in *Bt* PIP crop plants does not adversely affect soil
microorganisms (Sanvido *et al*., 2007). Although a minimal transient
increase and shift in microbial populations may result from the presence
of transgenic plant tissue in soil, no adverse effects have been
attributed to the Cry protein. In addition, comparisons of microbial
biomass in FLCry1Ab dosed and undosed soil prior to and during the study
showed that microbial activity was maintained throughout the test
period. Vip protein had similar DT~50~ or degradation time to Cry
proteins and these proteins are both *Bt* toxins.

[]{.mark}

In addition, there are several ongoing U.S. Department of Agriculture
and EPA Office of Research and Development funded research projects
evaluating the effects of Cry protein crops on soil microbial flora. If
adverse effects are seen from this or any other research, the Agency
will take appropriate action to mitigate potential risks.

With regard to the impact of genetically engineered crops on soil, it is
important to note that agricultural practices themselves cause large
changes in soil and soil microbial composition. Furthermore, factors
such variations in seasons and weather, plant growth stage, and plant
varieties, independent of being genetically engineered, are also
responsible for significant shifts in soil microbial communities. Most
studies with genetically engineered crops to date have shown minor or no
effects on soil microbes beyond the variation caused by the factors
listed above.

**5. Horizontal Transfer of Transgenes from *Bt* Crops to Soil
Organisms**

The EPA has evaluated the potential for horizontal gene transfer (HGT)
from *Bt* crops to soil organisms and has considered possible risk
implications if such a transfer were to occur. Genes that have been
engineered into *Bt* crops are mostly found in, or have their origin in,
soil-inhabiting bacteria. Soil is also the habitat of anthrax, tetanus
and botulinum toxin-producing bacteria. Transfer of these genes and/or
toxins to other microorganisms or plants has not been detected.
Furthermore, several experiments (published in scientific journals),
that were conducted to assess the likelihood of HGT, have been unable to
detect gene transfer under typical environmental conditions. Horizontal
gene transfer to soil organisms has only been detected with very
promiscuous microbes under laboratory conditions designed to favor
transfer.

As a result of these findings, which suggest that HGT is at most an
artificial event, and the fact that the Bt toxins engineered into COT102
and COT67B were derived from soil-inhabiting bacteria, EPA has concluded
that there is no risk of HGT from Vip3Aa or Cry1Ab producing cotton.

**6. Gene Flow and Weediness Potential**

Movement of transgenes from crop plants into weeds is a significant
concern, due to uncertainty regarding the effect that a new pest
resistance gene may have on plant populations in the wild. Under FIFRA,
the Agency has reviewed the potential for gene capture and expression of
Cry proteins in commercial *Bt* cotton by wild or weedy relatives of
cotton in the United States, its possessions or territories. Because Vip
proteins are *Bt* toxins and have similarities to Cry proteins in its
insecticidal activity on similar target species, the Agency maintains
the same approach in evaluation of gene flow and weediness potential.

There is a possibility for gene transfer in locations where wild or
feral cotton relatives exist. Therefore, EPA requires stringent sales
and distribution restrictions on *Bt* cotton within these areas to
preclude outcrossing or hybridization from the crop to sexually
compatible relatives. There are only four areas in the United States and
its territories wherein cultivated cotton has the opportunity to
outcross to wild or feral species, which are genetically compatible: (1)
southern Arizona, (2) Hawaiian islands, (3) southern Florida and 4)
Puerto Rico. *G. thurberi* (Arizona Wild Cotton) is present in the
elevated regions of Arizona and does not grow in areas of commercial
cotton production. *G. thurberi* is a diploid and produces sterile,
triploid progeny when crossed with the tetraploids *G. hirsutum* or *G.
barbadense*. In the very south of Florida, feral *G. hirsutum* exists in
apparently self-sustaining populations. Since these would readily cross
with cultivated cotton, sale of *Bt*-Cotton is restricted south of
Interstate 60. There is currently no commercial cotton production in the
southern part of Florida. Evidence from germplasm collections indicates
that feral *G. barbadense* and possibly *G. hirsutum* exist in the U.S.
Virgin Islands. There is presently no production of commercial cotton in
either of these places; hence, outcrossing is not an issue. For a
detailed review of the Agency's assessment of the potential for gene
capture and expression of *Bt* endotoxins by wild or weedy relatives of
cotton in the U.S., its possessions or territories, see the EPA
Biopesticides Registration Action Document (BRAD) for the *Bacillus
thuringiensis* (*Bt*) Plant-Incorporated Protectants, dated October 15,
2001.

**7. Impacts on Endangered Species**

The primary route of exposure to Vip3Aa and FLCry1Ab proteins in cotton
is through ingestion of cotton tissue or pollen. There are no reports of
threatened or endangered species feeding on cotton plants; therefore,
such species would not be exposed to cotton tissue containing these
proteins. Since Vip3Aa and FLCry1Ab proteins have not been shown to have
toxic effects on mammals, birds, plants, aquatic species, insects and
other invertebrate species at the Estimated Environmental Concentration
(EEC), a \"may affect\" situation for endangered land and aquatic
species is not anticipated. As previously noted, there is a possibility
for gene transfer in locations where wild or feral cotton relatives
exist. As a result, EPA requires stringent sales and distribution
restrictions on *Bt* cotton within these areas to preclude outcrossing
or hybridization from the crop to sexually compatible relatives.
Therefore, EPA does not expect that any threatened or endangered species
will be affected by outcrossing to wild relatives or by competition with
such entities.

There are extensive data that demonstrate the lack of hazard of Cry1Ab
to non-Lepidoptera and the environmental safety of Bt11 corn (US EPA,
2001b). Because of the selectivity of Vip3Aa and FLCry1Ab proteins for
lepidopteran species, endangered species concerns are mainly restricted
to the order Lepidoptera. Examination of an overlay map showing the
county level distribution of endangered/threatened lepidopteran species
(currently listed by the U.S. Fish and Wildlife Service) relative to
cotton production counties in the United States clearly indicated that
any potential concern regarding range overlap with cotton production was
mainly restricted to the Kern primrose sphinx moth (*Euproserpinus
euterpe*). However, cotton is not a host plant for this species nor do
host-range considerations place habitat in or near cotton fields.

Likewise, other insect species in the orders Diptera, Hemiptera,
Coleoptera, Donata, and Orthoptera that are listed as
endangered/threatened species are found in dune, meadow/prairie or open
forest habitats and are not closely associated with row crop production,
often times due to the specificity of the habitat of their host plants.
Furthermore, the reviewed toxicological data shows the relative
insensitivity of a range of insects in non-lepidopteran orders to the
Vip3Aa and FLCry1Ab proteins, indicating that COT102 and COT67B cotton
plants are not likely to have detrimental effects on non-lepidopteran
insects included on the endangered/threatened species list.

In light of the above considerations (based on no spatial and temporal
overlap), the Agency has determined that registered uses of Event COT102
and Event COT67B cotton plants will have No Effect (NE), direct or
indirect, on endangered and threatened species or their habitat as
listed by the United States Fish and Wildlife Service (USFWS) and the
National Marine Fisheries Services (NMFS), including mammals, birds or
terrestrial and aquatic plants and invertebrate species. Therefore, no
consultation with the USFWS is required under the Endangered Species
Act.

**II. C. 2. Environmental Risk Assessment for Event COT102 and Event
COT67B**

The EPA uses a Maximum Hazard Dose Tiered system for biopesticide
non-target wildlife hazard assessment. When no adverse effects at the
maximum hazard screening dose are observed, the Agency concludes that
there are no unreasonable adverse effects from the use of the pesticide.

**A. Direct effects**

At present, the Agency is aware of no identified significant adverse
effects of Vip3Aa and/or FLCry1Ab proteins on the abundance of
non-target beneficial organisms in any population in the field
environment, whether they are pest parasites, pest predators, or
pollinators. Further, the EPA believes that cultivation of Event COT102
and/or Event COT67B cotton may have fewer adverse impacts on non-target
organisms than use of chemical pesticides for cotton production, because
under normal circumstances, COT102 and COT67B cotton requires
substantially fewer applications of chemical pesticides, compared to
production of non-*Bt* cotton. Fewer chemical insecticide applications
generally result in increased populations of beneficial organisms that
control secondary pests, such as aphids and leafhoppers. In addition, no
adverse effect on Federally-listed endangered and threatened species is
expected from the proposed lepidopteran-resistant cotton registration
(see part 7 in the preceding section above). Furthermore, the EPA has
determined that there is no significant risk of gene capture and
expression of Vip3Aa and FLCry1Ab protein by wild or weedy relatives of
cotton in the U.S., its possessions, or territories (see part 6 in the
preceding section above). Available data do not indicate that Cry or Vip
proteins have any measurable adverse effect on microbial populations in
the soil (see part 4 in the preceding section above), nor has horizontal
transfer of genes from transgenic plants to soil bacteria been
demonstrated (see part 5 in the preceding section above). In conclusion,
this risk assessment finds no hazard to the environment at the present
time from cultivation of Event COT102 and Event COT67B cotton in support
for the Sec. 3 registration.

**B. Indirect effects:**

The purpose of using PIP plants is the same as for any other pest
management tactic, i.e., to reduce pest populations below economic
injury levels. As a result, the abundance of pest insects should be
significantly reduced and this will have corresponding implications for
those organisms that exploit these pests as prey and hosts. Thus, the
potential for these indirect ecological effects on biological control
organisms should not be regarded as a unique ecological risk associated
with the PIP crop. Some reductions, however, should be expected if the
pest management strategy is effective. Since PIP crops are often grown
in vicinity with conventional crops to prevent resistance build-up by
the target pest(s), specialist antagonists can persist in these
'refuges', in other crops and in non-crop habitats and retain the
potential for recolonization of the PIP crop area. Based on these
considerations, regulatory testing of the specialist predators and
parasitoids of target pests may eventually be considered unnecessary.

**II. C. 3. Supplemental Data Needed to Confirm COT102 and COT67B
Non-Target Hazard Assessment**

The Agency has sufficient information to believe that there is no risk
from the proposed uses of Event COT102 and Event COT67B cotton to
non-target wildlife, aquatic, and soil organisms. In previous Section 3
registrations of PIPs, the Agency required registrants to conduct
post-registration long term invertebrate population/community studies
and Cry protein accumulation in soils studies. However, the issue of
long range effects of cultivation of these Cry proteins on the
invertebrate community structure in corn and cotton fields has since
been adequately addressed by the meta-analysis of field studies
performed during the last 10 years (Marvier, *et al*. 2007; Sanvido, *et
a*l. 2007). No unexpected adverse effects on invertebrate community
structure were reported. The Agency is in agreement with these
conclusions Likewise, no unexpected accumulation of Cry or Vip proteins
in agricultural soils was seen in published studies (Icoz and Stotzky,
2007; Sanvido, *et al.* 2007) and in numerous studies submitted directly
to the EPA for the currently registered Cry proteins (Milofsky, 2006;
Part 3 in the preceding section above).

However, in light of recently published laboratory studies showing
reduced growth in shredding caddis flies exposed to anti-lepidopteran
Cry1A protein corn litter (Rosi-Marshall, et al. 2007), additional
aquatic invertebrate data are required. The submitted *Daphnia magna*
study is unacceptable because it is an 850 Series OPPTS Guideline study.
The 48 hour duration of this study is not sufficient to detect
mortality. It takes more than 48 hours for the target pests to succumb
to *Bt* δ-endotoxins, such as Cry or Vip proteins, therefore 48 hours is
also not expected to show mortality or reproductive effects on
*Daphnia.* A 7-14 day *Daphnia* study as per the OPPTS Series 885.4240
guideline must be performed (see Tables 11 and 12) for Event COT102 and
Event COT67B. Alternatively, a dietary study of the effects on an
aquatic invertebrate, representing the functional group of a leaf
shredder in headwater streams, may be performed and submitted in lieu of
the 7-14 day Daphnia study. These studies can be submitted as a
condition of registration.

**Table 11.** Supplemental non-target data requirements for COT102
expressing Vip3Aa

  -------------------------- --------------------------------------------
  **Testing Category**       **Type of Data**

  Aquatic invertebrate       A 7-14 day *Daphnia* study as per the OPPTS
                             885.4240 guideline has to be submitted as a
                             condition of registration. Alternatively, a
                             dietary study of the effects on an aquatic
                             invertebrate, representing the functional
                             group of a leaf shredder in headwater
                             streams, can be performed and submitted in
                             lieu of the 7-14 day *Daphnia* study.
  -------------------------- --------------------------------------------

**Table 12.** Supplemental non-target data requirements for COT67B
expressing FLCry1Ab

  -----------------------------------------------------------------------
  **Testing Category**       **Type of Data**
  -------------------------- --------------------------------------------
  Aquatic invertebrate       A 7-14 day *Daphnia* study as per the OPPTS
                             885.4240 guideline has to be submitted as a
                             condition of registration. Alternatively, a
                             dietary study of the effects on an aquatic
                             invertebrate, representing the functional
                             group of a leaf shredder in headwater
                             streams, can be performed and submitted in
                             lieu of the 7-14 day *Daphnia* study.

  -----------------------------------------------------------------------

**II. C. 4.** **Event COT102 X COT67B (VipCot) Environmental Risk
Assessment**

**SUMMARY**

Syngenta Seeds, Inc. developed COT102 x COT67B cotton (VipCot) by
conventional breeding of transgenic event COT102 cotton and transgenic
event COT67B cotton, which express the Vip3Aa and FLCry1Ab insecticidal
proteins, respectively, for control of certain lepidopteran pests.
Vip3Aa is a protein variant of Vip3Aa1 (originally identified in
*Bacillus thuringiensis* strain AB88), from which it differs by one
amino acid substitution. FLCry1Ab is a δ-endotoxin identical to a
protein produced by *B. thuringiensis* subsp. *kurstaki* HD-1 except for
an additional 26 amino acids at the C-terminal region. The VipCot stack
expresses both the Vip3Aa and FLCry1Ab proteins.

It was previously established by the Agency that the relative potency of
plant-produced Vip3Aa and full-length Cry1Ab proteins is similar to
their corresponding microbial-produced proteins, indicated that
plant-produced protein was similar in toxicity to the microbial-produced
protein (Matten, 2007 and Edelstein, 2008). Each event also had
comparable protein expression levels to the COT102 x COT67B breeding
stack (MRID No. 470176-07 and Edelstein, 2008).

Although the general symptomatology of Vip3Aa displayed by sensitive
lepidopteran larvae following ingestion of *Bt* δ-endotoxins resembles
that of Cry proteins (Yu *et al.*, 1997), Vip3Aa contains significantly
different receptor binding properties than the Cry proteins (Lee *et*
*al.*, 2003). Therefore, since the proteins have different modes of
action, the predicted effect of the mixture was calculated using a model
called independent joint action[^13] (Raybould, 2007; Colby, 1967). The
observed and expected mortalities were compared over a range of
concentrations. Since there is no test to identify statistical
significance, the predicted dose response curves were compared with the
expected dose response curves. If there is greater mortality than
expected over the range of concentrations in a sensitive pest species,
the hypothesis of synergism is falsified and subsequently it is likely
that there will be no synergism of the mixture against non-target
organisms.

Syngenta submitted additional data on the potential synergistic
interaction between Vip3Aa and FLCry1Ab proteins and are summarized in
this report to support the hypothesis of no synergism between the two
proteins. If no synergism is indicated, then development of new
non-target species data are not required because the reviewed non-target
data and the environmental risk assessments for the single indicated PIP
lines are applicable to the COT102 x COT67B cotton line. The results of
ecological effects studies[^14] submitted in support of the Section 3
full-commercial registration of Event COT012 and Event COT67B were
previously summarized in Tables 1 and 2, respectively, and presented in
a more descriptive format in previous sections of this risk assessment
document.

**Synergism Studies**

The purpose of these studies was to characterize the potential for
interaction between the lepidopteran-active proteins Vip3Aa and
FLCry1Ab. The Vip3Aa and FLCry1Ab proteins were tested alone and in
combination against tobacco budworm (TBW, *Heliothis virescens*) and
cotton bollworm (CBW, *Helicoverpa zea*), respectively, in diet
incorporation studies.

**MRID No. 470176-21**

Four laboratory feeding bioassays were conducted to assess any
synergistic or antagonistic interactions between Vip3Aa and full-length
Cry1Ab proteins in a key lepidopteran pest, tobacco budworm (*Heliothis
virescens*). Five dilution series of the test materials were prepared in
buffer for each test: one series each of Vip3Aa and FLCry1Ab alone, and
three series of the two proteins mixed together in different ratios (up
to 1600 µg/mL Vip3Aa and 100 µg/mL FLCry1Ab together). The treatments
were applied to non-transgenic cotton leaves which were fed to *H.
virescens* larvae. Interaction between the two test materials was
assessed by comparing the larval mortality observed for the mixed
proteins with the predicted responses based on the bioassay of each
protein individually. The predicted responses were calculated based on
the assumption of "independent action" (Raybould, 2007) and there was no
evidence of either a synergistic or an antagonistic interaction between
Vip3Aa and FLCry1Ab in *H. virescens*, indicating that the effect of a
mixture of Vip3Aa and FLCry1Ab on non-target Lepidoptera can be
predicted from the effects of the individual proteins alone.

**MRID No. 470176-22**

Three laboratory feeding bioassays were conducted to assess any
synergistic or antagonistic interactions between Vip3Aa and full-length
Cry1Ab proteins in a key lepidopteran pest, cotton bollworm
(*Helicoverpa zea*). Five dilution series of the test materials were
prepared in buffer for each test: one series each of Vip3Aa and FLCry1Ab
alone, and three series of the two proteins mixed in different ratios
(up to 25,600 ng/cm2 Vip3Aa and 12,800 ng/cm2 FLCry1Ab together). The
test materials were added to standard lepidopteran diet and fed to *H.
zea* larvae. Interaction between the two test materials was assessed by
comparing the larval mortality observed for the mixed proteins with the
predicted responses based on the bioassay of each protein individually.
Since previous evidence indicates that Vip3Aa and FLCry1Ab act at
different binding sites, the predicted responses were calculated based
on the assumption of "independent action" (Raybould, 2007). The results
were compared and there was no evidence of either a synergistic or an
antagonistic interaction between Vip3Aa and FLCry1Ab in *H. zea*,
indicating that the effect of a mixture of Vip3Aa and FLCry1Ab on
non-target Lepidoptera can be predicted from the effects of the
individual proteins alone.

**Conclusions/Recommendations:** The results of the interaction studies
of the combined proteins (Vip3Aa and FLCry1Ab) indicate that there is no
change in the level of activity among susceptible insects. Collectively
these data provide evidence that Vip3Aa and FLCry1Ab proteins do not
interact in an antagonistic or synergistic manner. These studies, along
with the single-species, NTO toxicity testing and the Vip3Aa and Cry1Ab
protein long-term field studies, reviewed for the parental Event COT102
and Event COT67B, indicate its associated breeding stack, COT102 x
COT67B cotton, will not result in any unexpected interaction related to
an antagonistic or synergistic action to target and non-target insects.
Therefore, it is extremely unlikely that the Vip3Aa and FLCry1Ab
proteins contained in a single plant will impart any hazard to
non-target organisms exposed to these hybrids in the environment. In
conclusion, the Agency has determined that the environmental risk
assessment of Event COT102 expressing Vip3Aa protein and Event COT67B
expressing FLCry1Ab protein indicate there will be no unreasonable
adverse effects to the environment, including federally-listed
threatened and endangered species, by VipCot (COT102 x COT67B) cotton
hybird, crossed via traditional breeding.

**CONCLUSION**

The environmental risk assessment indicates for the VipCot cotton
breeding stack (COT102 x COT67B), based on prior assessments conducted
on Vip3Aa and Cry1Ab proteins individually, that no unreasonable harm
will result to the environment or any federally-listed threatened or
endangered species from commercial cultivation of COT102 x COT67B
cotton. 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 Vip3Aa and
FLCry1Ab proteins, is that the number of chemical insecticide
applications for non-target pest control will be reduced for management
of multiple pest problems.

**II. C. 5.** **References**

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Dively, G.P. (2005). Impact of transgenic VIP3A x Cry1Ab
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Edelstein, R. (2008). Review of Human Health and Product
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Estruch, J.J., G.W. Warren, M.A. Mullins, G.J. Nye, J.A. Craig, and M.G.
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Icoz, I, and G. Stotzky (2007). Cry3Bb1 protein from *Bacillus
thuringiensis* in root exudates and biomass of transgenic corn does not
persist in soil. Transgenic Research, September 13, 2007.

Lee, M.K., F.S. Walters, H. Hart, N. Palekar and J-S Chen. (2003). The
mode of action of the

*Bacillus thuringiensis* vegetative insecticidal protein Vip3A differs
from that of Cry1Ab δ-endotoxin. *App. Environ. Micro.* 69(8):
4648-4657.

Martinez, J. (2008). Review of Insect Resistance Management (IRM) for
Sec 3 registration for VipCot™ (COT102 x COT67B). U.S. Environmental
Protection Agency. Washington, D.C. Memorandum dated February11, 2008.

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cotton and maize on nontarget invertebrates. *Science* 316: 1475--1477.

Matten, S. and J. Kough (2007). Review of Product Characterization and
Human Health Data for PIP *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 EUP. Biopesticides and Pollution
Prevention Division. U.S. Environmental Protection Agency. Washington,
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Milofsky, T. and Z. Vaituzis (2006). Review the soil fate study
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Milofsky, T. and Z. Vaituzis (2007b). Environmental Risk Assessment for
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Pilcher, C. D., M. E. Rice, J. J. Obrycki & L. C. Lewis. (1997b). Field
and laboratory evaluations of transgenic *Bacillus thuringiensis* corn
on secondary Lepidopteran pests (Lepidoptera: Noctuidae). *J. Econ.
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Pilcher *et al.* (2005) Impact of transgenic *Bacillus thuringiensis*
corn and crop phenology on five nontarget arthropods. *Environmental
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Raybould, A. (2007) Environmental Risk Assessment of Genetically
Modified Crops:

General Principles and Risks to Non-target Organisms *BioAssay* 2:8, pg.
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Rose R. and Z. Vaituzis. (2003). Review of non-target terrestrial
arthropod studies (lady

beetle, honey bee, Collembola & green lacewing) submitted by Syngenta
Seeds, Inc. to EPA for the registration of *Bacillus thuringiensis*
VIP3A protein expressed in cotton. U.S. Environmental Protection Agency.
Washington, D.C. Memorandum dated November 18, 2003.

Rose *et al.* (ed.) (2007). White Paper on Tier-Based Testing for the
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expressing *Bacillus*

*thuringiensis* toxins and biological control. *Nature Biotechnology*
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**II. D. Insect Resistance Management**

**1. Background**

Syngenta's VipCot cotton is a pyramided transgenic cotton trait that
expresses full-length Cry1Ab insecticidal protein (COT67B event) and
Vip3Aa19 (COT102 event). The Vip3A is different from Cry proteins as it
is produced during vegetative growth of the bacteria, does not form
parasporal crystal proteins, and is secreted (but not processed upon
secretion) from the cell as a soluble protein. Its physical
manifestations of intoxication, however, resemble those of Cry proteins
(gut paralysis and lysis of midgut epithelial cells) (Schnepf *et al.*
1998). Activated Vip3A does not bind to the same receptors as Cry1Ab
(APN and cadherin-like receptor); the two Bt proteins do not seem to
share binding sites. Lee et al. (2003) have investigated the mode of
action of the Vip3A protein and determined that it involves a number of
steps much like the mode of actions for the δ-endotoxins. Following
ingestion by the lepidopteran target pest, the Vip3A protein becomes
soluble in the gut and is then processed into four dominant bands
(retaining activity). The authors propose that this processing is
required for the bioactivity of the toxin (activation step). Interaction
with the midgut epithelium is the next likely step in the mode of action
of Vip3A. However, Vip3A does not bind to APN and cadherin-like
glycoprotein receptors as does Cry1Ab (as already stated by Schnepf et
al. (1998)) and supported by the researchers' competition study. Upon
binding to midgut epithelial receptors, data supports the existence of a
pore-forming step that creates ion channels which are structurally and
functionally distinct from those of Cry1Ab. Direct structural
information is missing for Vip3A; however, preliminary data do not
support the notion that the two proteins share similar domain
organization or an α-helical bundle region.

COT102 cotton expresses the vegetative insecticidal protein (VIP3A),
which was isolated from *Bacillus thuringiensis* strain AB88. The cotton
line Coker 312 (*Gossypium hirsutum* L. cv Coker 312) was transformed
via *Agrobacterium* transformation procedures with synthetic *vip3A(a)*
gene encoding VIP3A protein and the selectable marker gene *aph4*
encoding the enzyme APH4. The transformation event that produced the
transgenic cotton line, designated COT102, was transformed with plasmid
pCOT1. COT102 is intended to protect cotton from feeding by the primary
lepidopteran pests: tobacco budworm (*Heliothis virescens*, TBW), cotton
bollworm (*Helicoverpa zea*, CBW), and pink bollworm (*Pectinophora
gossypiella*, PBW). Based on cotton insect loss data from 1991-2000, the
primary target pests, TBW, CBW, and PBW, account for more than 77% of
the yield loss and 84% of the insecticide use due to lepidopteran
infestation in cotton.

In 2004, BPPD reviewed an earlier IRM plan for COT102 and determined
that the registrant did not provide sufficient data to formulate an IRM
strategy (BPPD, 2004). Specifically, The BPPD IRM team concluded that
data or published literature was needed to address the pest biology of
each target pest; additional data was required to make high-dose claim
for COT102 using two of the five techniques described by 1998 FIFRA
Science Advisory Panel; baseline susceptibility and diagnostic
concentrations needed to be established for all target pests; estimates
of initial resistant allele frequency as well as models of evolution of
resistance for Vip3A19 should be provided; additional cross-resistance
data was required for target pests using Vip3A19, Cry1Ac, and Cry2Ab2;
and specific monitoring plans, remedial action strategies, grower
education program, compliance assurance program and research activities
for COT102 needed to be provided.

A revised IRM plan (MRID 470176-34) was submitted by Syngenta to support
registration of VipCot in 2006 and has been reviewed by BPPD (see
Martinez 2008 a, b). An additional e-mail correspondence clarifying
aspects of the dose evaluation was submitted by Syngenta (Reed 2008).
The details of resistance management plan as reviewed by BPPD are
described below.

**2. Pest Biology**

A clear understanding of pest biology and ecology is essential to the
development of a sound IRM plan. The target pests of VipCot (TBW, CBW,
and PBW) have been well studied since Bt cotton was first introduced and
is available in the scientific literature. A summary of the biology and
ecology for these insects can be found in the Agency's 2001 Bt crop
reassessment document (EPA 2001).

**3. Dose**

The determination of dose, or the amount of toxin expressed by the
transgenic crop relative to the susceptibility of the target pests, is a
critical component of IRM. Models have shown that a high-dose of toxin,
coupled with a non-transgenic refuge to provide a supply of susceptible
insects, is the most effective strategy for delaying resistance in Bt
crops. The high-dose/refuge strategy assumes that resistance to Bt is
recessive and is conferred by a single locus with two alleles resulting
in three genotypes: susceptible homozygotes (SS), heterozygotes (RS),
and resistant homozygotes (RR). The high-dose/refuge strategy also
assumes that there will be a low initial resistance allele frequency and
extensive random mating between resistant and susceptible adults. In
practice, a high-dose PIP should express sufficient quantities of toxin
to kill all susceptible insects (SS) as well as heterozygous insects
with one resistance allele (RS). Lower dose PIPs might allow for
survival of insects with at least one susceptibility allele (SS or RS),
although effective IRM may still be possible with a suitable refuge
strategy. To be able to demonstrate high-dose, it is recommended that
registrants generate data by at least two of the five laboratory and
field approaches as outlined by the SAP (1998) and described by the
Agency in the 1998 Bt Plant-Pesticides and Resistance Management
document (EPA, 1998) and 2001 Biopesticide Registration Action document
(EPA, 2001).

The 1998 SAP defined high-dose as a level of toxin 25 times greater than
is needed to kill all susceptible insects. The SAP also outlined five
techniques to determine high dose: 1) Serial dilution bioassay with
artificial diet containing lyophilized tissues of Bt plants using
tissues from non-*Bt* plants as controls; 2) Bioassays using plant lines
with expression levels approximately 25-fold lower than the commercial
cultivar determined by quantitative ELISA or some more reliable
technique; 3) Survey large numbers of commercial plants in the field to
make sure that the cultivar is at the LD~99.9~ or higher to assure that
95% of heterozygotes would be killed (see Andow & Hutchison 1998); 4)
Similar to #3 above, but would use controlled infestation with a
laboratory strain of the pest that had an LD~50~ value similar to field
strains; and 5) Determine if a later larval instar of the targeted pest
could be found with an LD~50~ that was about 25-fold higher than that of
the neonate larvae. If so, the later stage could be tested on the Bt
crop plants to determine if 95% or more of the later stage larvae were
killed.

It must be noted that both the high-dose definition and verification
techniques were developed in 1998 when all of the registered Bt crops
were single toxin products targeted against lepidopteran pests. In
recent years, PIPs in Bt cotton have been approved that contain two
genes targeted at the same insect pest. These "pyramided" products can
be beneficial for IRM since target pests must overcome two toxins to
develop field resistance to the PIP. The benefits are greatest for two
toxins with unrelated modes of action (i.e. binding to different Bt
receptor sites in the midgut) that are expressed at high-doses in the
plant (Roush 1994).

For pyramided products, the dose of each toxin should be evaluated
separately. This can be easily accomplished if the pyramided product is
created through conventional breeding \-- in this case, the dose of the
single toxin products has already been established and the combined dose
in the pyramided PIP can be determined with comparative efficacy
studies. However, for pyramids created by non-conventional breeding
(e.g. recombinant DNA techniques), defining the dose can be more
complicated since single toxin lines may not be available (or
commercialized) for comparisons. The dual toxins can also be evaluated
collectively to determine an "effective" high-dose. In some examples,
each toxin by itself may not supply a high-dose, but in combination a
sufficient control (\>95% of heterozygotes) is provided and can be
considered high-dose.

To evaluate dose, Syngenta conducted a number of laboratory and field
studies with diet bioassays and COT67B, COT102, and VipCot cotton plant
material. Three sets of experiments were conducted: 1) bioassays with
the single proteins expressed in lyophilized plant material and both
proteins expressed in lyophilized plant material and combined as VipCot
to determine target pest susceptibility (TBW, CBW, PBW), 2) field tests
on VipCot and COT67B plants and control plants (artificial infestation)
during the 2005 and 2006 growing seasons, and 3) tolerance assays with
single proteins expressed in fresh plant material and both proteins
expressed in fresh plant material and combined as VipCot to determine
susceptibility of neonates as compared to fourth instar larvae (N.C.
State University lab colony).

*Verification Methods*:

1\) Bioassays (Syngenta's submission, MRID 470176-34, Appendix 1) using
US EPA method #1 (serial dilution assays) were conducted by two
laboratories (Jealott's Hill International Research Center and Syngenta
Biotechnology, Inc) using lyophilized tissue from terminal leaves and
bolls of COT102, COT67B, VipCot, and Coker 312 cotton. Mortality data
for *H. zea*, *H. virescens*, and *P. gossypiella* were collected.

At 25X dilution, percent mortality on COT102 for TBW ranges from 66.7-
95.0%, and for CBW mean corrected mortality reported (one lab only) is
72.4%. For PBW, mean corrected mortality reported (by one lab only) is
16.7%. These data seem to indicate that COT102 alone has minimal
efficacy against PBW and an efficacy against TBW ranging from
intermediate to high. COT102 efficacy against CBW does not appear to
quite as high judging from these test results. Based on the range of
susceptibility observed in CBW for other Bt PIPs, it can be expected
that the range of susceptibility for COT102 in this target pest is
rather wide as well and that 72% mortality likely represents an upper
value in this potential range.

At 25X dilution, percent mortality on COT67B for TBW and PBW is reported
to be 100% and for CBW ranges from 98.3- 100%. The dilution bioassay for
VipCot was conducted at SBI only; their results suggest that at 25X
dilution, mortality on VipCot for TBW, CBW, and PBW is 100%. Based on
this verification method alone, VipCot provides an 'effective' high-dose
against all three target pests, while COT102 provides no high-dose to
all three target pests and COT67B expresses a high-dose against TBW and
PBW and is nearly high dose for CBW.

2\) Unlike the artificial diet bioassay, the second method to verify
high-dose was conducted on VipCot and COT67B plants in the field (US EPA
method #4) using artificial infestations of laboratory strains of TBW
and CBW (MRID 470176-34, Appendix 2; additional clarification provided
in Reed 2008). The purpose of this approach was to determine whether the
dose of toxins expressed in VipCot and COT67B was at or greater than the
LD~99~ for the key target pests. Specifically, TBW survival was measured
on both COT67B and VipCot, while CBW survival was measured on VipCot
cotton only.

In 2005, two field trials were conducted for COT67B of which one was
held in Mississippi and the other in Florida. Unreplicated blocks of
\>1,000 plants were planted with one control block of Coker 312 plants
at each site. Infestation with TBW was simulated by spraying eggs onto
cotton plants (greater infestation was conducted on Bt variety due to
low survival expectancy). Survival on control plants was estimated by
collecting leaves containing TBW eggs from Coker 312 plants (50% and 75%
of field inspected in MS and FL, respectively) and counting successful
larval hatching. Survival of TBW larvae on COT67B plants was estimated
by visually assessing Bt plants for larval presence in each field in MS
and FL, respectively. When survivors were found, the plants were marked
and reexamined for further larval survival after four to seven days.
Syngenta states in their report that in MS and FL one and two surviving
TBW larvae, respectively, were found after seven days of artificial
infestation (7DAI) and zero survivors after 14 days (14DAI). The second
set of observations (14 DAI) may not be as reliable as the initial
assessment of survivors because it appears that bolls were not 'caged'
to prevent larval loss or escape. Thus, BPPD will base its review and
conclusion on the first set of observations (7DAI).

In 2006, six VipCot cotton field trials were held at three locations in
the US (1,600 -2,000 plants each trial) and, like for COT67B, did not
get replicated. Experimental design, artificial infestation for TBW and
CBW, egg hatching, and larval survival estimation methods resembled
those of 2005. The number of survivors of TBW and CBW (7DAI) were 32 and
21, respectively, at the Texas site and 14 and 5, respectively, at the
Louisiana site. For Mississippi, no CBW and TBW survivors on VipCot
plants were reported. In Tables 2 and 3 of Appendix 2 (MRID 470176-34),
BPPD notes that there is large difference in number of insects observed
in the control plots 7DAI (i.e. 363 TBW observed, TX) and 14DAI (i.e. 17
TBW observed, TX). Based on such a decrease of survivors in the control
plots, BPPD concludes that other outside effects add to the natural
mortality when laboratory reared insects are exposed to cotton plants
(i.e. fed on artificial diets for many generations and no longer fit to
survive on natural host). Thus, the low number of TBW survivors reported
from Bt-plants 7DAI is very likely confounded by this additional 'non-Bt
exposure' mortality. Similar results were reported for CBW, and
therefore, the same conclusion is extended to this pest. Based on the
discrepancy observed in survivorship on control plants, BPPD concludes
that the 2006 data alone are inconclusive. However, given the results
observed in 2005 for TBW, it can be concluded that COT67B likely
provides a high-dose for TBW under this method.

3\) The third verification method for high-dose was conducted as a
tolerance bioassay with purified toxins as well as leaf disks to
determine if a later larval instar of the targeted pest could be found
with an LD~50~ that was approximately 25-fold higher than that of the
neonate larvae (US EPA method #5) on VipCot, COT 102, and COT67B plants
for CBW and on COT102 for TBW (MRID 470176-34, Appendix 3). The SBI lab
conducted the purified toxin (with Vip3A and FLCry1Ab) and leaf disk
study (on COT102, COT67B, and VipCot) for CBW; JH lab conducted the
purified toxin (with Vip3A) and leaf disk study (on COT102) for TBW.

*Leaf Disk Bioassays for CBW*

While Syngenta reports 100% mortality for both neonates and older CBW
larvae for leave disk bioassays (method #5), Syngenta does not mention
that control mortality for neonates ranged from 0% to 81% for neonates.
While neonates may have only served as a reference, it is inappropriate
to list these results to support the conclusion that VipCot, as well as
its individual events, is highly effective against CBW. However, control
mortality for later instar larvae was much lower and ranged from 0% to
28% (only one control had very high mortality results). The results from
leaf disk bioassays show that later instar larvae of CBW are
experiencing 100% mortality when exposed to VipCot, COT102, and COT67B.

*Leaf Disk Bioassays for TBW*

Mortality for TBW later instar larvae was 100% when tested on COT102.
Control mortality results for later instar larvae were 14%, 15%, and
27%; treatment mortality for later instars was 100%. The results from
leave disk bioassays show that older larvae of TBW are experiencing 100%
mortality when exposed to COT102.

*Tolerance Assays for CBW*

Syngenta reports that the tolerance assay conducted with the purified
toxin Vip3A and tested on CBW did not allow estimating LC~50~ for later
instars because the estimates were well in excess of the highest
concentration tested. Syngenta concludes that later instars are at least
25X more tolerant to Vip3A than neonate larvae. BPPD recognizes that
there are several issues with the tolerance assay data reported: 1)
Mortality data for neonates are highly variable from bioassay to
bioassay, and LC~50~ estimates for CBW neonates range from 504.6ng/cm^2^
to 2669ng/cm^2^. 2) Mortality data for neonates *within* any test are
not steadily increasing with increasing toxin concentrations but show a
fluctuating trend and data gaps at some concentrations tested. Mortality
data for later instar larvae follow the same trend as described for
neonates under 1 and 2. 3) Where an LC~50~ could be estimated for both
CBW neonates and later instars, the difference between estimates is only
8 fold as opposed to the desirable 25-fold factor. The variability
reported in estimated LC~50~s for neonates and mortality data for
neonates and older instars are most likely due to CBW's wide range in
response to Bt toxins. Overall, the tolerance assay data support that it
is difficult to achieve mortality in neonates and older instars of CBW
when exposed to Vip3A.

Two tolerance bioassays were conducted with FLCry1Ab on CBW (Tables 6
and 7, Appendix 3). In these tests, mortality data for later instars and
neonates were more consistently increasing with increasing
concentrations. Later instar LC~50~ estimates were greater than neonate
LC~50~ estimates by more than 25-fold; BPPD concludes that older CBW
larvae are \>25-fold more tolerant than neonates when exposed to
FLCry1Ab.

*Leaf Disk Assays for TBW*

For the leaf disk assays, Syngenta reported 100% mortality for older TBW
larvae. Although control mortality for older larvae was somewhat higher
than desirable (ranging from 15%-27%), BPPD concludes that COT102
appears to have very good activity against TBW.

*Tolerance Assays for TBW*

Syngenta reports that the tolerance bioassays conducted with Vip3A on
TBW neonate and 2^nd^ instar larvae show a 36-fold difference in
susceptibility/ LC~50~ estimates. BPPD would like to add that there were
two estimated LC~50~s for older larvae of which one showed a 21-fold and
the other 36-fold difference from neonate susceptibility. Overall, BPPD
concludes that older TBW larvae are approximately ≥25-fold more tolerant
than neonates when exposed to Vip3A alone.

4\) Method #4 was conducted to verify high-dose in the field on COT67B
plants (and COT69D plants) under artificial infestation of laboratory
strains of PBW (MRID 470176-34, Appendix 4). The purpose of this
approach was to determine whether the dose of toxins expressed in COT67B
was at or greater than the LD~99~ for this key target pest.

The experiment was conducted at the University of California, Desert
Research and Extension Center. The experiment was set up as a randomized
complete block design with four replicates, COT67B plants as treatment,
and Coker 312 plants as controls. One-hundred bolls per plot were
artificially infested (twice) with PBW eggs supplied by USDA/ARS in
Phoenix, AZ. Eight days after infestation, 75 bolls from each plot got
harvested and evaluated for damage such as warts, mines, dead larvae,
and exit holes (indicating survival of larva). Surviving larvae and exit
hole data collected on Coker 312 plants served as a baseline to assess
infestation levels and PBW populations.

No live larva larger than 1^st^ instar was found on COT67B cotton bolls.
One exit hole in one boll was found out of 1,120 bolls of COT67B towards
the later time of the season and is possibly attributed to lower
expression levels of the toxin in aged plants. Mortality on COT67B
plants is estimated at 99.9%. Mortality in Coker 312 is significantly
lower and estimated at 40.3% with first instar larvae included. These
data support the conclusion that COT67B expresses a high-dose against
PBW.

Overall, BPPD's conclusions for the VipCot dose trials are as follows:

-   COT67B expresses a probable high-dose against TBW (methods 1 and 4)
    and CBW (methods 1 and 5).

-   VipCot expresses a probable high-dose against TBW (using method 1;
    method 4 was considered inconclusive) and CBW (methods 1 and 5).

-   COT102 does not appear to express a high-dose against any of three
    target pests when tested with method 1, but has a probable high dose
    against TBW and CBW with method 5.

-   COT67B expresses a high-dose against PBW based on data from
    verification methods 1 and 4.

-   VipCot expresses a high-dose against PBW based on data from
    verification method 1.

Table 13. BPPD's High-Dose Determinations for TBW, CBW, and PBW

  ------------- ------------ ------------ ------------ ------------ ------------ ------------ ------------ ------------
  **Species**   **Method 1**                           **Method 4**              **Method 5**              

                **COT102**   **COT67B**   **VipCot**   **COT67B**   **VipCot**   **COT102**   **COT67B**   **VipCot**

  **TBW**       No high-dose High-dose    High-dose    Probable     \*\*\*       Probable     \-\--        \-\--
                                                       high-dose                 high-dose                 

  **CBW**       No high-dose Near high    High-dose    \-\--        \*\*\*       High-dose    High-dose    High-dose
                             dose                                                                          

  **PBW**       No high-dose High dose    High-dose    High dose    \-\--        \-\--        \-\--        \-\--
  ------------- ------------ ------------ ------------ ------------ ------------ ------------ ------------ ------------

Shaded fields indicate high-dose determinations by BPPD for single
toxins or stacked *Bt* product

\*\*\* indicates that results were inconclusive for a definitive dose
determination

\-\-- indicates *Bt* variety not tested under a particular method

**4. Cross-Resistance Potential**

Analyses of resistance to Bt Cry proteins indicate that cross-resistance
occurs most often with proteins that are similar in structure
(Tabashnik, 1994; Gould et al., 1995). While direct structural
information of the Vip3A protein expressed in VipCot is missing (Lee et
al. 2003), this novel Bt protein does not share any sequence homology
with the known Bt Cry protein genes, and the predicted secondary
structure give no indication of a similar domain organization or
α-helical bundle region within the polypeptide sequence of Vip3A as
exists for the Cry proteins. Protein folding blasts reveal that Vip3A
may be a pore forming protein that has a structure of β-barrels
(Syngenta unpublished data). In order to further investigate the
potential for cross-resistance of Vip3A to Cry proteins, Syngenta
examined the mode of action of Vip3A at selected steps critical to the
mode of action of Bt Cry proteins: proteolytic activation, receptor
binding, and pore forming.

The first piece of analysis relates to the proteolytic activation in
both Bt toxins and shows that Vip3A and Cry proteins are proteolytically
activated upon solubilization in the midgut. Syngenta's experiments
further demonstrate that both Vip3A and two Cry proteins (Cry1Ac and
Cry2Ab2) can be processed by either trypsin or gut juice extracts.
However, in Vip3A proteolysis occurs in susceptible as well as
non-susceptible insects and alone does not appear to be a key factor in
insect toxicity and specificity. Based on this information and published
literature stating that high levels of resistance have not been found to
correlate with the toxin activation step, Syngenta speculates that the
theoretical risk of cross-resistance is very small at this particular
step of the mode of action.

Second, Syngenta investigated whether Vip3A and Cry proteins (Cry1Ab,
Cry1Ac, and Cry2Ab) shared the same receptor sites in Lepidoptera
species (*M. sexta*, *H. virescens*, and *H. zea*) by conducting
receptor binding studies with Amino Peptidase N and cadherin-like
glycoproteins (identified as putative Cry1A protein receptors) as well
as others identified to be Cry1Ac and Cry2Ab2 binding sites. Those
studies show that the protease activated form Vip3A does not bind to
APN, the ectodomaine of the cadherin-like protein, or other putative
Cry1A toxin binding proteins. In yet another study with *H. zea* and *H.
virescens*, the non-specific binding of Cry2Ab was not inhibited by the
addition of unlabeled Vip3A indicating that Vip3A does not bind to the
Cry2Ab binding sites. Syngenta further demonstrated that activated Vip3A
bound to two proteins of ca. 80 and 110 kDa and not to APN and
cadherin-like proteins. These binding studies demonstrate that there is
little risk of cross-resistance between Vip3A and Cry1Ab, Cry1Ac, and
Cry2Ab2.

The last piece of analysis relates to pore forming properties in both Bt
toxin types. Syngenta states that Vip3A and Cry proteins share
symptomological manifestations of intoxication, and yet, the pore
forming properties of Vip3A are unique. The kinetics of Vip3A pore
formation are more than 8 times slower than for equimolar Cry1Ab; pore
channels were characterized by long open times and a predominant open
state; channels formed by Vip3A differ considerably in their conductance
state specificity from Cry1A protein. In addition, Domain I, modulated
by Domain III interactions, has been considered responsible for the pore
formation steps in the Bt Cry protein mode of action. Again, direct
structural information is not available for the Vip3A protein, yet, as
mentioned above, available information gives no indication of a similar
domain organization or α-helical bundle region within the polypeptide
sequence as exists for the Cry proteins.

BPPD agrees with Syngenta that the potential risk for cross-resistance
between Cry1Ab (and other Cry1A proteins as well as Cry2Ab2) and Vip3A
appears low considering that: 1) Vip3A does not bind to APN and
cadherin-like proteins and thus, the two types of Bt toxins do not share
binding sites; and 2) Vip3A pore channels formed in the midgut of
insects are structurally and functionally distinct from Cry1Ab (and
maybe other Cry proteins).

**5. Modeling**

EPA has used predictive models to compare IRM strategies for Bt crops.
Because models cannot be validated without actual field resistance,
models have limitations and the information gained from the use of
models is only a part of the weight of evidence used by EPA in assessing
the risks of resistance development. It was the consensus of the 2000
SAP Subpanel that models were an important tool in determining
appropriate Bt crop IRM strategies. They agreed that models were "the
only scientifically rigorous way to integrate all of the biological
information available, and that without these models, the Agency would
have little scientific basis for choosing among alternative resistance
management options." They also recommended that models must have an
agreed upon time frame for resistance protection. For example,
conventional growers may desire a maximum planning horizon of five
years, while organic growers may desire an indefinite planning horizon.
The Subpanel recommended that model design should be peer reviewed and
parameters validated. Models should also include such factors as level
of Bt crop adoption, level of compliance, economics, fitness costs of
resistance, alternate hosts, spatial components, stochasticity, and pest
population dynamics.

Syngenta commissioned Dr. Michael Caprio to evaluate the risk of
resistance evolving to VipCot cotton. In the next few paragraphs, BPPD
summarizes the most important features and assumptions of the model and
simulation results for CBW and TBW. Later, BPPD comments on the input
parameter assumptions and applicability of the simulation results.

Dr. Caprio used a spatially explicit, stochastic, two-locus population
genetic and landscape model simulating a heterogeneous environment (wild
hosts and other Bt crop hosts), incorporating pest biology/ecology,
parameter uncertainty (max/min value, most likely value, assuming normal
distribution), and allowing for parameter interactions. The first
modeling approach and simulations estimated the time to resistance in
CBW and TBW populations in the field when ≤100% of the Bt cotton planted
was assumed to be VipCot. The rate at which resistance evolved was
estimated by determining the amount of time required until the average
resistance allele frequency across all fields exceeded 0.5. The second
modeling approach explored the impact of VipCot on other single gene Bt
cotton events such as for Cry1Ac. These second simulations assumed
complete cross-resistance between Cry1A and VipCot (making Cry1Ac and
Cry1Ab functionally the same because of reported cross-resistance) and
no cross-resistance between Cry1A and Vip3A.

Simulation results for CBW indicate that there were few cases of
resistance (0.3%) for Cry1A toxin over 1000 simulations when 80% of all
cotton acres were assumed to be VipCot (with 10% of total corn acreage
planted to Cry1A hybrid); no resistance to Vip3A evolved in any of the
simulations. When 50% of the total corn acreage was planted to a
pyramided Vip3A x Cry1A hybrid, resistance to either protein did not
evolve after 400 model runs. In the first five years of the simulations,
the rate of increase for the Cry1A allele was lower than the rate of
increase for the Vip3A resistance allele; however, the rate measure was
strongly affected by the assumption of the initial resistance allele
frequency. The overall simulation results suggest that the introduction
of VipCot is not likely to select for resistance to Cry1A(b/c) and Vip3A
within 20-25 years.

The second set of simulations compared the impact of VipCot on a Cry1A
gene expressed in other Bt cotton products (mosaic ratio 1:1 vs. Cry1A
only). The simulation outcomes and sensitivity analysis support the
hypothesis that the introduction of VipCot delays to a small degree the
evolution of resistance to the Cry1A toxin. Specifically, the
sensitivity analysis determined that the dominance of the Cry1A
resistance gene and mortality due to Vip3A were the main parameters that
accounted for more than 83% of the unexplained variance, essentially
confirming that Vip3A could delay the resistance to the Cry1A trait. In
the simulations, 40% of the time, resistance evolved within 15 years in
the Cry1A simulations, and 13% of the time it evolved in the mosaic
(VipCot and Cry1A) within the same time frame. Under no circumstances
did evolution to Vip3A occur, which means, product failure (resistance
to both toxins) did not occur.

Simulation results for TBW indicate that there are few cases of
resistance evolving to the Vip3A toxin and that most values (number of
occurrences) clump around the frequency of 10^-3^. There is a 0.2%
chance of resistance evolving to Vip3A and Cry1Ab (product failure)
within a 25 year time. The most likely outcome for the resistance allele
was either 'no change' or 'slight decline' in frequency. Equilibrium for
Vip3A resistance allele at around 0.0032 except for when resistance
evolved for Cry1A allele leading to considerable variation in the final
resistance allele frequency for Vip3A. This suggests that the
equilibrium value for the Vip3A allele is dependent on interactions
between two loci which generate an effect similar to overdominance.
Whether this is an error in the model or an effect to multi-locus
overdominance still needs to be further investigated. Dr. Caprio
concludes that for the moment it appears that high-dose in combination
with fitness cost may lead to unusual results.

The second simulations again compared the impact of VipCot on a Cry1A
gene expressed in other Bt cotton products (mosaic ratio 1:1 vs. Cry1A
only) and indicate that there may be rapid evolution of resistance to a
Cry1A cotton product in absence of VipCot (despite high-dose against
TBW). Like in the case of CBW, the introduction of VipCot is expected to
decrease the risk of resistance in TBW to the Cry1A toxin in a single
trait cotton product. In the simulations, there was a 1% chance of
resistance evolving within 20 years and 4% chance that resistance would
evolve in 25 years to VipCot.

For CBW and TBW, comparison of VipCot simulations alone versus the
mosaic simulation results indicate that the presence of a Cry1A single
gene cotton product may seriously reduce the effectiveness of resistance
management strategies for dual gene products.

Syngenta provided Dr. Caprio with the following critical information
based on interpretations of laboratory and field results: Vip3A
mortality in CBW and TBW was assumed to have a maximum of 0.975 (near
high-dose assumption) and minimum of 0.875 with the most likely
mortality being at 0.92. Similarly, COT67B mortality in CBW and TBW was
assumed to have a maximum of 0.999 (high-dose assumption for both
species) and a minimum of 0.95 (for CBW). For CBW, the most likely
mortality was chosen to be 0.99, a near high-dose. For TBW the
assumption was that COT67B provides only a high-dose. These mortality
assumptions may be reasonable even though high-dose for Vip3A against
CBW was only demonstrated by verification method #5, but not by
verification method #1. The assumed actual and minimum mortality for CBW
may also be too high (see discussion below). In addition, the mortality
assumption (0.99) for COT67B in CBW may be somewhat high in light of the
data submitted. For high-dose verification method #1, the reported CBW
mortality ranged from 98 -100%, although for method #5, high-dose
expression of COT67B was demonstrated. Given what is known about
variation in susceptibility of CBW towards other Bt toxins, it may not
be realistic to assume that the actual CBW mortality due to COT 102 and
COT67B will be 97.5% and 99%, respectively, though the data show at
least a "near high dose" can be expected. Also, the COT67B high-dose
assumption for TBW has been completely verified by method #1 only. The
second verification method (#4) produced a *probable* high-dose for
COT67B. Therefore, a more conservative assumption for 'most likely'
mortality of 0.95 (instead of 0.999), together with a maximum and
minimum mortality value of 0.999 and 0.90 respectively, may have been
appropriate mortality input values for the model for TBW and COT67B.

[]{.mark}

Evolution of resistance to Cry1A toxin in CBW is predicted to occur well
beyond the life-time expectancy for any Bt product. Likewise, evolution
of resistance to Cry1A and Vip3A toxin in TBW (assuming high-dose and
near high-dose, respectively) is also predicted to occur well beyond the
life-time expectancy for any Bt product. As described above, BPPD has
some questions about the COT67B dose data submitted by Syngenta. If
COT67B is actually expressed at a level below those assumed in the
model, it is unclear how much or how little such a change in mortality
would affect the evolution of resistance in TBW and CBW.

BPPD reiterates from Dr. Caprio's report that the conclusion of delayed
resistance to Cry1A toxins following the introduction of VipCot hinges
greatly on the assumption that mortality caused by Vip3A (COT102) is
high (ranging between 0.875 -- 0.975 with most likely mortality being
0.92). BPPD notes that this particular assumption of high mortality was
supported by some (but not all) of the submitted dose and efficacy
studies. For CBW, the reported actual mortality on COT102 with
verification method #1 was 72.4% (rather than 0.92), although data from
method #5 indicated high dose and Syngenta supplied the modeler only
with the highest mortality data supported by method #5. For TBW, the
reported mortality on COT102 with verification method #1 ranged from
66.7 -- 95.0% and with method #5 was near high-dose. There are
indications that COT102 may not be very efficacious against TBW under
some conditions.

Since the uncertainties regarding dose/mortality are relatively minor,
BPPD does not request further modeling at this time and concludes that
the provided simulation results are sufficient to support the refuge
strategies requested for VipCot cotton. The refuge options proposed by
Syngenta match the presently in use cotton refuge strategies (see the
next section). While BPPD has some minor reservations about the
high-dose assumptions for some toxins against TBW and CBW, it appears
that VipCot falls into the existing paradigm for what constitutes an
'effective' high-dose pyramided product. However, since the conclusion
of delayed resistance in TBW to Cry1A toxins hinges greatly on the
assumption that Vip3A mortality is high, the evolution of resistance in
TBW following introduction of VipCot might be expected to occur in less
time than predicted by the model. Similarly for CBW, the evolution of
resistance to Cry1A toxins may not be delayed by the introduction of
VipCot when Vip3A mortality is low.

**6. Refuge Strategy**

The size, placement, and management of the refuge are critical to the
success of the high-dose/structured refuge strategy to mitigate insect
resistance to Bt proteins produced in cotton (as well as corn and
potatoes). The 1998 SAP Subpanel defined structured refuges to "include
all suitable non-Bt host plants for a targeted pest that are planted and
managed by people. These refuges could be planted to offer refuges at
the same time when the Bt crops are available to the pests or at times
when the Bt crops are not available." The 1998 Subpanel suggested that a
production of 500 susceptible adults in the refuge for every adult in
the transgenic crop area (assuming a resistance allele frequency of 5 x
10^-2^) would be a suitable goal. The placement and size of the
structured refuge employed should be based on the current understanding
of the pest biology data and the technology. The 2000 SAP Subpanel
echoed the 1998 SAP's recommendations that the refuge should produce
500:1 susceptible to resistant insects and that regional IRM working
groups would be helpful in developing policies (US EPA, 2001).

Under the established refuge strategy for cotton, growers can choose
from three structured refuge options, which are thoroughly described in
the Agency's 2001 Bt crop reassessment document and briefly listed here:

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

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

[Option 3]{.underline}: 95:5 embedded refuge; contiguous or within 1
mile^2^ of field and 150 ft wide.

According to their IRM plan (MRID 470176-34), Syngenta has requested
identical refuge requirements as for currently registered cotton PIPs.
In addition, Syngenta requests that VipCot be considered for the
community refuge plan that allows multiple growers to contribute to the
overall required refuge acres by planting 20% external, sprayed or 5%
external, unsprayed refuge.

BPPD notes that the simulations run by Dr. Caprio addressed refuge
option 2 only, the 20% external sprayed refuge (Appendix 5). BPPD would
like to expand on this apparent deficiency and clarify that the 20%
refuge option may actually be considered the least conservative approach
of all three options, and thus, the modeling assumptions could
potentially represent a worst case scenario for IRM because non-Bt
cotton refugia are often sprayed with multiple applications of
insecticides during a growing season. Shelton et al. (2000) indicate
that great care should be used to ensure that refuges sprayed with
highly efficacious insecticides produce adequate numbers of susceptible
alleles; thus, the 20% external sprayed refuge option for non-Bt cotton,
if over sprayed, may not produce a great amount of susceptible adults
that could potentially mate with resistant survivors from the Bt-field.
Gould & Tabashnik (1998) in their evaluation of Bt cotton IRM options
commented that a 20% external refuge that can be extensively treated
with insecticidal sprays may result in almost no refuge because all of
the susceptible target larvae would be killed. Therefore, the 5%
external unsprayed refuge as well as the embedded refuge can be expected
to generate the greatest number of susceptible insects that are able to
potentially mate with resistant survivors from adjacent Bt-fields in
comparison with the worst case scenario for the 20% external sprayed
cotton refuge. BPPD requests that in future reports Syngenta be clear as
to why certain assumptions were not included in the modeling efforts.

BPPD concludes that based on the modeling, dose, and efficacy studies,
the requested refuge options 1-3 and community refuge plan are
acceptable for VipCot cotton.

**7. Resistance Monitoring**

The need for proactive resistance detection and monitoring is critical
to the survival of Bt technology. The Agency mandates that registrants
monitor for insect resistance (measurement of resistance-conferring
alleles) to the Bt toxins as an important early warning sign to
developing resistance in the field and whether IRM strategies are
working. Grower participation (e.g., reports of unexpected damage) is
also important for monitoring. Resistance monitoring is also important
because it provides validation of biological parameters used in models.
However, resistance detection/monitoring is a difficult and imprecise
task. It requires both high sensitivity and accuracy. Good resistance
monitoring should have well-established baseline susceptibility data
prior to introduction of Bt crops. The chances of finding a resistant
larva in a Bt crop depend on the level of pest pressure, the frequency
of resistant individuals, the location and number of samples that are
collected, and the sensitivity of the detection technique. Therefore, as
the frequency of resistant individuals or the number of collected
samples increases, the likelihood of locating a resistant individual
increases (Roush & Miller 1986). If the phenotypic frequency of
resistance is one in 1,000, then more than 3,000 individuals must be
sampled to have a 95% probability of one resistant individual (Roush &
Miller 1986). Current sampling strategies have a target of 100 to 200
individuals per location. Previous experience with conventional
insecticides has shown than once resistant phenotypes are detected at a
frequency \>10%, control or crop failures are common (Roush & Miller
1986). Because of sampling limitations and monitoring technique
sensitivity, resistance could develop to Bt toxins prior to it being
easily detected in the field.
(<http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm>)

Syngenta is working with to develop appropriate assay methods for PBW
and as well as appropriate assay methods and baseline susceptibility
data for TBW and CBW. Collection of insects to be used in bioassays to
fulfill the Agency's annual monitoring requirement will focus on cotton
growing regions where VipCot cotton sales are expected to be the
highest.

Key features of Syngenta's monitoring plan include:

-   EPA receives monitoring plan for approval, revised monitoring plan
    within three months of the date of product registration, and
    detailed resistance monitoring

-   Development of diagnostic concentration assays by Jan 31 of the year
    after VipCot cotton is registered

-   Follow-up on grower, extension specialist, or consultant reports of
    unexpected damage or control failure of the three main target pests

BPPD considers the monitoring plan adequate for this step of the VipCot
registration process. In order to facilitate future communication
between BPPD and the registrant, the IRM team makes the following
recommendations for monitoring procedures: Syngenta should use the
diagnostic concentration (LC~99~) for both toxins and target pests where
the approach has proven successful, and pests are susceptible and
population variance is small. In addition, follow-up testing of larval
survivors needs to be conducted for all toxins where field population
survivorship (≥2 instar) is significantly different from lab/reference
colony's survivorship.

Specifically for CBW (but not only), BPPD has the following
recommendations for Syngenta: if a good amount of effort has been put
into developing a discriminating or diagnostic concentration for CRW and
FLCry1Ab and there is evidence that the diagnostic concentration cannot
be achieved due to i.e. high-variability in response to the toxin, then
a comparison in baseline susceptibility (i.e. LC~50~s) may be a feasible
approach to monitoring. Estimated LC~50~s may serve well as a baseline
monitoring tool for shifts in susceptibility to Bt toxins; however, the
LC~50~ approach is not useful in discriminating resistant from
susceptible individuals. Therefore, this approach must then be linked
with follow-up testing of populations with elevated LC~50~s relative to
previously established baseline susceptibility. Furthermore, BPPD
recommends that Syngenta consider head capsule width assay and DNA
markers in lieu of mortality based diagnostic concentrations.

**8. Grower Education**

Syngenta proposes to use the following methods to educate growers which
have already been established for other PIPs:

-   Signing of grower agreement with purchase of VipCot

-   Grower agreement and/or stewardship documents referenced in the
    grower agreement will set forth terms of current IRM program and
    contractually bind grower to comply with IRM requirements

-   Annual affirmation system for VipCot cotton growers to ensure they
    understand that they are contractually bound to comply to
    requirements

Syngenta proposes to 1) submit within 90 days from product registration
a copy of the grower agreement/stewardship documents and written
description of a system assuring that growers will sign grower
agreement; 2) revise and expand as necessary its education program to
take into account the information collected through the compliance
survey (discussed in Section 9); and

3\) maintain records of all signed VipCot cotton grower agreements for
three years.

BPPD concludes that the proposed grower education plan meets the
Agency's present requirement and is acceptable.

**9. Compliance**

Grower compliance with refuge and IRM requirements is a critical element
for resistance management. Significant non-compliance with IRM among
growers may increase the risk of resistance for Bt crops. To minimize
the effects of non-compliance, it is necessary to develop a broad
compliance program as part of the IRM strategy. Such a program has to
include 1) an understanding of the effect of non-compliance on IRM; 2)
identification of compliance mechanisms to maximize adoption of IRM
requirements; 3) measurement of the level of compliance; and 4)
establishment of an enforcement structure to ensure compliance and
penalize non-compliance.

Syngenta commits to implementing a compliance assurance program designed
to 1) evaluate the extent to which growers of VipCot cotton are
complying with the IRM requirements and 2) take reasonable actions
necessary to assure that non-compliant growers become compliant with
those requirements and submit within 90 days of the date of registration
a written description of their compliance assurance program. Consistent
with the registration of other cotton Bt PIPs, there are several key
elements to the CAP that Syngenta will employ:

-   Establish and publish a phased compliance approach that outlines
    instances of non-compliance to IRM terms and options of responding
    to non-compliant growers

-   Annual survey conducted by third party will measure degree of
    compliance by growers in different cotton growing regions and
    consider potential impact of non-response

-   Survey will obtain grower feedback on usefulness of educational
    tools and initiatives and provide understanding of any difficulties
    growers encounter with IRM requirements

-   Annual on-farm assessment followed by appropriate action consistent
    with the 'phased compliance approach' for non-compliant growers

-   'Tips and complaints' line with follow-up investigations and
    appropriate actions taken consistent with the 'phased compliance
    approach' for non-compliant growers

Syngenta proposes to revise and expand, as necessary, its compliance
assurance program to take into account information collected through the
compliance survey and allow a review of the compliance records by EPA or
by a State pesticide regulatory agency.

BPPD concludes that Syngenta has included the major requirements needed
by a compliance program and outlined by Agency in the first paragraph of
this section and the 2001 Bt crop reassessment document. Syngenta's
proposed CAP resembles CAPs for other introduced Bt PIPs and meets the
Agency's requirements at this time.

**10. Remedial Action Plan**

Remedial action plans are a potential response measure should resistance
develop to Bt crops.

Since resistance may develop in "localized" pest populations, it may be
possible to contain the resistance outbreak before it becomes
widespread. A specific remedial action plan should clearly indicate what
actions the registrant will take in cases of "suspected" resistance
(i.e., unexpected damage) and "confirmed" resistance. The remedial
action plan can also include appropriate adaptations for regional
variation and the inclusion of appropriate stakeholders. To fully
mitigate resistance, a critical element of any remedial action plan
should be that once pest resistance is confirmed, sales of all Bt cotton
hybrids that express a similar protein or a protein in which
cross-resistance potential has been demonstrated would be ceased in the
affected region
(<http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm>).

Syngenta states that it if resistance to any of the three major target
pests is suspected, growers will be informed to use alternate pest
control measures such as pesticide treatment, crop rotation the
following year, or use of soil or seed insecticides the following year.

Syngenta states that the following steps in order of events will be
taken if resistance to any of the three major target pests is confirmed:

-   Notify the Agency within 30 days of resistance confirmation

-   Notify affected customers and extension agents about confirmed
    resistance

-   Encourage affected customers and extension agents to employ
    alternative lepidopteran control measures

-   Cease sale and distribution of VipCot cotton in affected area

-   Devise long-term resistance management action plan according to
    characteristics of resistance event and local agronomic needs

BPPD concludes that the steps outlined in the remedial action plan and
the depth of detail are similar to remedial action plans for other Bt
PIP products. In addition, BPPD would prefer if Syngenta 'directed' and
'instructed' affected growers (or have a clause in grower agreements)
and extension agents to employ alternative control measures instead of
'encouraged' them when resistance to any of the target pests has been
confirmed. Since it is in the economic interest of all parties involved,
including the public, that the durability of VipCot cotton is preserved
for a long time, Syngenta will need to play a more aggressive and
supportive role in case of confirmed resistance to VipCot cotton. A
final remedial action plan (based on the steps described above) for
tobacco budworm and cotton bollworm will be needed as a term of the
registration. This remedial action plan should include definitions of
"suspected" and "confirmed" resistance and steps to take in the event of
confirmed resistance. The existing remedial action plan developed by the
Arizona Bt Cotton Working Group for pink bollworm should be used with
VipCot cotton.

**11. Reporting Requirements and IRM Conditions of Registration**

Syngenta commits to providing an initial report to EPA summarizing
activities carried out under the grower education program for the prior
year with annual updates thereafter of any substantive changes.

Syngenta will provide 1) a final written summary of the survey results
of the prior year to the Agency by January 31 of each year, beginning
the year after VipCot cotton is registered; 2) an annual report to the
Agency after January 31 of each year summarizing results of their CAP,
activities carried out under the CAP for the prior year, and plans for
the current year; 3) annual sales summed by state; 4) number of VipCot
cotton seeds shipped or sold and not returned, and number of such units
sold to persons who have signed grower agreements.

Syngenta will provide to the Agency an annual resistance monitoring
report (by August 31 of each year beginning with the year after VipCot
cotton is registered) conducted on populations collected the following
year.

At this point in the VipCot registration process, the Agency is
satisfied with Syngenta's commitment to fulfill the reporting
requirements.

In addition to the annual reporting requirements, the following items
will be required as terms and conditions of the VipCot registration:

1\) A detailed resistance monitoring plan to be submitted within 90 days
of the date of registration. The monitoring program description must
include sampling (number of locations and samples per location),
sampling methodology, bioassay methodology, standardization procedures,
detection technique and sensitivity, and the statistical analysis of the
probability of detecting resistance

2\) A report on baseline susceptibility and diagnostic concentrations
for TBW, CBW, and PBW to Vip3Aa19 and modified Cry1Ab by January 31,
2009. Syngenta must include the following testing scheme for survivors
of the diagnostic or discriminating concentrations (or identified
survivors of any resistance detection method): 1) Determine if the
observed effect is heritable; 2) Determine if the increased tolerance
can be observed in the field (i.e., survive on VipCot cotton plants); 3)
Determine if the effect is due to resistance, 4) Determine the nature of
resistance (dominant, recessive), 5) Determine the resistance allele
frequency, 6) Determine, in subsequent years, whether the resistance
allele frequency is increasing, and 7) Determine the geographic extent
of the resistance allele (or alleles) distribution. Should the
resistance allele frequency be increasing and spreading, the specific
remedial action plan should be implemented to mitigate the extent of Bt
resistance.

3\) A copy of the grower agreement/stewardship documents and written
description of a system assuring that growers will sign grower agreement
to be submitted within 90 days of the date of registration.

4\) A compliance assurance program (CAP) for VipCot to be submitted
within 90 days of the date of registration. The CAP must include a
"phased compliance approach" that outlines instances of non-compliance
to the IRM requirements and options of responding to non-compliant
growers. Options should include withdrawal of the right to purchase
VipCot cotton for an individual grower or for all growers in a specific
region. An individual grower found to be significantly out of compliance
two years in a row should be denied sales of the product the next year.
The CAP must also include a description of an annual survey conducted by
third party to measure (statistically representative) the degree of
compliance by growers in different cotton growing regions. In addition,
an annual on-farm survey must be included in the CAP. Non-compliant
growers identified in the on-farm survey should face actions consistent
with the "phased compliance approach" for non-compliant growers. A
program to investigate "tips and complaints" about non-compliant growers
must also be included with the CAP.

5\) A final remedial action plan (based on the steps described in
Syngenta's IRM submission) for tobacco budworm and cotton bollworm to be
submitted within 90 days of the date of registration. This remedial
action plan should include definitions of "suspected" and "confirmed"
resistance and steps to take in the event of confirmed resistance. The
existing remedial action plan developed by the Arizona Bt Cotton Working
Group for pink bollworm should be used with VipCot cotton (Dennehy,
2002).

**12. References**

Andow, D.A. and Hutchison, W.D., Corn resistance management, in *Now or
Never: Serious Plans to Save a Natural Pest Control*, Mellon, M. and
Rissler, J., Eds., Union of Concerned Scientists, Washington, D.C.,
1998, chap. 2.

BPPD, 2004. EPA Review of Syngenta Seed's Vip3A Cotton Insect Resistance
Management Plan for Section 3 Full Commercial Registration. S. Matten
memorandum to L. Cole, January 05, 2004.

Dennehy, T., 2002. A Remedial Action Plan for Responding to Pink
Bollworm Resistance to Bt Cotton in Arizona. Developed by the Arizona Bt
Cotton Working Group, University of Arizona Cooperative Extension. June
3, 2002.

Ferré, J. and J. Van Rie. 2002. Biochemistry and genetics of insect
resistance to *Bacillus* thuringiensis. Annu. Rev. Entomol. 47: 501-533.

Gould, F., Anderson, A., Reynolds, A., Bumgarner, L., and Moar, W. 1995.
Selection and genetic analysis of a *Heliothis virescens* (Lepidoptera:
Noctuidae) strain with high levels of resistance to *Bacillus
thuringiensis* toxins. J. Econ. Entomol. 88:1545-1559.

Gould, F. and B. Tabashnik, 1998. Bt-cotton resistance management. In
"Now or Never: Serious New Plans to Save a Natural Pest Control. Ed. M.
Mellon and J. Rissler. p. 65-105.

Kurtz, R.W., McCaffery, A., O'Reilly, D., Stone, T. 2006. Insect
resistance management considerations for VipCot™ Cotton. Report
submitted from Syngenta Biotechnology, Inc. MRID 470176-34.

Kurtz, R.W., 2006. Determining the dose of VipCot™ cotton and its
component events, COT67B and COT102, using US EPA Method #1 for
Helicoverpa zea and Heliothis virescens. Appendix 1 in report submitted
from Syngenta Biotechnology, Inc. MRID 470176-34.

Lee, M.K., Walters, F.S., Hart, H., Palekar, N., Chen, J.S. 2003. The
mode of action of the Bacillus thuringiensis vegetative insecticidal
protein Vip3A differs from that of Cry1Ab δ-endotoxin. Applied and
Environmental Microbiology, Vol. 69 (8): 4648-4657.

Martinez. J., 2008a. EPA Review of Syngenta Seed's VipCot Cotton Insect
Resistance Management Plan for Section 3 Full Commercial Registration.
Memorandum to A. Reynolds, dated February 11, 2008.

Martinez, J., 2008b. Addendum to EPA's Review of Syngenta's VipCot
Cotton Insect Resistance Management Plan for Section 3 Full Commercial
Registration. Memorandum to A. Reynolds, dated April 10, 2008.

Piggott, C.R. and D.J. Ellar. 2007. Role of receptors in *Bacillus
thuringiensis* crystal toxin activity. Micro. Molec. Bio. Reviews. 71:
255-281.

Reed, J., 2008. E-mail correspondence to A. Reynolds, dated February 10,
2008.

Roush, R.T. 1994. Managing pests and their resistance to *Bacillus
thuringiensis:* Can transgenic crops be better than sprays? Biocontrol
Science Technology, Vol. 4:501-516.

Roush, R. T., and G. L. Miller, 1986. Considerations for design of
insecticide resistance monitoring programs. Journal of Economic
Entomology, Vol. 79: 293-298.

Schnepf, E., Crickmore, N., Van Rie, D. Lereclus, D., Baum, J.,
Feitelson, J., Zeigler, D.R., Dean, D.H. 1998. *Bacillus thuringiensis*
and its pesticidal crystal proteins. Microbiology and Molecular Biology
Reviews, Vol. 62 (3): 775-806.

Shelton, A.M., J.D. Tang, R.T. Roush, T. D. Metz, and E.D. Earle, 2000.
Field tests on managing resistance to Bt-engineered plants. Nature
Biotechnology. 18: 339-342.

Tabashnik, B.E. 1994. Evolution of resistance to *Bacillus
thuringiensis.* Annu. Rev. Entomol. 39:47-79.

US EPA, 1998. FIFRA Scientific Advisory Panel Subpanel on *Bacillus
thuringiensis (Bt)* Plant-Pesticides and Resistance Management, February
9 and 10, 1998.

US EPA, 2001. Biopesticides Registration Action Document -- *Bacillus
thuringiensis* Plant Incorporated Protectants,
<http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm>.

II\. **E. Benefits and EPA Public Interest Finding**

Syngenta submitted two documents to support the benefits of VipCot which
will be summarized, and analyzed here: an efficacy study for the 2005
and 2006 season (O'Reilly et al. 2006, MRID 470176-33) and Public
Interest Document (Stone 2006, MRID 470347-01) with five sections, 1)
public interest finding, 2) grower benefits, 3) human health and
environmental benefits, 4) resistance management benefits, and 5) VipCot
cotton marketing plan. The IRM chapter submitted (Kurtz et al. 2006,
MRID 470176-34) is addressed in this review of the Public Interest
Document (PID) as applicable. A complete discussion of IRM for VipCot is
contained in section II.D. of this document.

**1. Public Interest Finding**

*a) Summary of Syngenta's Submission (MRID 470347-01):*

Syngenta believes that registration of the active ingredients in VipCot
meets the criteria for a conditional registration. The registration is
clearly in the public interest. Registration and market introduction of
Syngenta's VipCot product will result in agronomic, economic, human
health, environmental, and resistance management benefits that are
highlighted here and discussed in greater detail in later sections of
the PID (MRID 470347-01).

Syngenta's VipCot expresses high levels of the proteins, Vip3A and
FLCry1Ab, through the combination of transgenic cotton events, COT102
and COT67B, respectively. The Vip3A protein is characterized by a range
of properties that clearly distinguish it from the FLCry1Ab protein and
the Cry proteins expressed by the Bt cotton varieties currently
available to growers. The combination of the Vip3A and FLCry1Ab proteins
offers effective protection from the principal Lepidopteran pests of
cotton (TBW, CBW, and PBW). As discussed in the confidential marketing
plan, VipCot will eventually include an herbicide resistant trait.

Efficacy trials (see section 2 in this chapter) show effective control
of CBW, TBW, and PBW. Preliminary yield data demonstrates no negative
agronomic factors that will impact a variety development program. Strong
efficacy and yield potential combined with Syngenta marketing and field
expertise will result in varieties that are very competitive with those
varieties now on the market. Current *Bt*-based PIPs offer agronomic and
economic benefits compared to the use of chemical pesticides. The
introduction of VipCot will continue to enhance the agronomic and
economic benefit stream by offering growers a new choice in germplasm,
technology, and terms of use.

In addition, a comparison of human health and environmental factors
clearly demonstrates both the same low risk potential for VipCot as the
current *Bt* PIPs, and the strong reduced risk potential of VipCot
cotton compared to the use of alternative conventional chemical
pesticides. While VipCot will primarily replace other *Bt* products as
it gains market share, its presence in the marketplace will extend the
useful life of Bt-based cotton technology generally, and thus,
contribute to the continued human health and environmental benefits
resulting from the use of Bt cotton compared to chemical alternatives.

Finally, VipCot will introduce Vip3A, a vegetative insecticidal protein
that offers little chance for cross-resistance to its companion protein,
FLCry1Ab, or the other *Bt* Cry insecticidal proteins currently
marketed. Since VipCot expresses a protein with a unique mode of action,
its combination with a viable competitive market presence will offer
strong resistance management potential. As discussed in a later section
of the report, risk assessment modeling of VipCot cotton confirms the
low likelihood of cross-resistance and the potential to extend the
useful life of *Bt* cotton technology generally.

*b) BPPD's response*

BPPD concludes that VipCot cotton is expected to provide similar human
health, environmental, agronomic, economic, and IRM benefits also
provided by other cotton PIP products already registered by the Agency.
For a summary of these general *Bt* cotton benefits, refer to the 2001
Bt crop reassessment (U.S. EPA 2001,
<http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm>).

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 choice of *Bt* cotton product and the potential to extend the
useful life of *Bt* cotton technology generally due to the Vip3A
proteins novel mode of action and low likelihood of cross-resistance
with other Bt Cry proteins.

BPPD disagrees with Syngenta's claim that VipCot is expected to provide
equal or superior efficacy as Bollgard II because no comparative study
and data have been submitted in support of this claim. At this point,
this particular benefit is unverified.

Furthermore, Syngenta claims that efficacy trials show effective control
against PBW; however, no efficacy data for PBW were submitted as stated
in the efficacy section of BPPD's review. BPPD revises Syngenta's claim
by stating that high-dose results/data for VipCot may support the notion
that efficacy of VipCot against PBW may be similar to efficacy of VipCot
against TBW and CBW although no field data is available at this time.

**2. Efficacy Data**

*a) Summary of Syngenta's Efficacy Data and Review (MRIDs 470176-33):*

Vip3A is effective against the two most economically important
lepidopteran pests of cotton in the US (tobacco budworm and cotton
bollworm). Likewise, the FLCry1Ab protein provides control of TBW and
CBW as well as PBW (pink bollworm). The plant protection and insect
control provided by the combined effect of these proteins is expected to
be equivalent to or better than the cotton plant-incorporated
protectants (PIPs) currently registered by the EPA.

Syngenta's first field trial in 2005 was conducted with cotton plants
homozygous for both genes *vip3Aa19* and *flcrylab*. Syngenta planted
seeds for Coker 312 (control), COT102, COT67B, and heterozygous VipCot
(for both genes) cotton in three US locations (Leland - MS, Winnsboro -
LA, Louisburg - NC) with four replicates for each treatment in a
randomized complete block design. Field trials were conducted under
artificial and/or natural infestation for TBW and CBW, respectively.
Insect damage data was collected from each location and used in the
statistical analysis (Analysis of Variance, ANOVA).

The second field trial in 2006 was conducted with cotton plants
homozygous for both genes *vip3Aa19* and *flcrylab*. Data from trials
were collected from eight US locations (Leland -- MS, Bossier City --
LA, Winnsboro -- LA, Scott -- AR, Jackson -- TN, Seven Springs -- NC,
Beasley -- TX) with four replicates for each treatment plot (Coker 312,
COT102, COT67B, and VipCot) in a randomized complete block design. A
plot consisted of four 40-foot rows with approximately three plants per
foot. Duplicate plantings were made at six of the eight locations to
collect data from separate infestations of TBW and CBW. Field trials
were conducted under artificial and/or natural infestation for TBW and
CBW, respectively. Insect damage data were collected from each location
and used in the statistical analysis (ANOVA). While damage assessments
were made on a plot-by-plot basis, damage in transgenic plots was often
very low such that the data became unsuitable for statistical analysis
at the plot level. Consequently, data were often averaged across
replicates.

*i) Infestation results for 2005*

[TBW damage]{.underline} (Average % Fruiting Structure) was assessed
from artificially infested cotton plants at the three experimental
locations in 2005, and the data are presented in Table 14[^15]. At all
locations, there was negligible damage to heterozygous VipCot,
homozygous COT102, and homozygous COT67B cotton (no statistically
significant difference detectable). All three treatments had
significantly less damage than Coker 312 control plants at a
significance level of 5%.

Table 14. Average % Fruiting Damage Following Artificial infestation on
Bt Cotton and Control Plants with *H. virescens* in 2005

  -------------- -------------- --------------- --------------- ---------------
                 **Locations,                                   
                 average %                                      
                 damage**                                       

  **Genotype**   **L-MS**       **L --NC**      **W -- LA**     **Overall Mean
                                                                %**

  Coker 312      57.8           41.0            4.8             34.5

  COT102         3.5            1.0             0.7             1.7

  COT67B         0.6            1.0             0.7             0.8

  VipCot™        0.5            1.0             0               0.5
  -------------- -------------- --------------- --------------- ---------------

[CBW damage]{.underline} (Average % Fruiting Structure) was assessed
from naturally infested cotton plants at the experimental location in
North Carolina. The data are presented in Table 15. No damage was
reported on the heterozygous VipCot plants; low to moderate damage was
reported on homozygous COT102 and COT67B cotton plants, and great damage
on the control plants (Coker 312).

Table 15. Average % Fruiting Damage Following Natural Infestation on Bt
Cotton and Control Plants with *H. zea* in 2005

  ---------------- ------------------------------------------------------
                   **% damage^1^**

  **Genotype**     **L --NC**

  Coker 312        88.3

  COT102           8.3

  COT67B           1.3

  VipCot™          0
  ---------------- ------------------------------------------------------

^1^It was not possible to carry out any formal statistical analysis on
this dataset

[Combined CBW and TBW damage]{.underline} (% fruiting structure damage)
was assessed from naturally infested plots at the experimental location
in Louisiana. The data are presented in Table 16. Low level damage was
reported on heterozygous VipCot and both homozygous COT102 and COT67B
cotton plants; substantial damage was reported on the control plants
(Coker 312).

Table 16. Average % Fruiting Damage Following Natural Infestation of Bt
Cotton and Control Plants with Mixed *H. zea* and *H. virescens* in 2005

  ---------------- ------------------------------------------------------
                   **% damage^1^**

  **Genotype**     **W --LA**

  Coker 312        26.0

  COT102           5.1

  COT67B           1.7

  VipCot™          1.7
  ---------------- ------------------------------------------------------

^1^it was not possible to carry out any formal statistical analysis on
this dataset

*ii) Infestation results for 2006*

[CBW damage]{.underline} (Average % Fruiting Structure) was assessed
from infested cotton plants at seven experimental US locations in 2006,
and the data are presented in Table 17 -- 19. At all locations, there
was low or zero damage to homozygous VipCot, low to moderate damage to
homozygous COT102 and homozygous COT67B, and great damage to control
plants. Specifically, Table 17 shows that overall mean 'square damage'
results for homozygous VipCot cotton were significantly different from
individual event results on homozygous COT102 and homozygous COT67B
cotton at the 5% significance level. All three Bt treatments had
significantly less damage than Coker 312 control plants. Table 18 shows
that overall mean 'flower' damage for VipCot cotton was significantly
lower than for COT67B (but not for COT102 alone) and that all Bt
treatments had significantly less damage reported than for control
cotton plants. Table 19 shows no significant difference in overall mean
'boll' damage reported between all three Bt treatments but a significant
difference between Bt treatments and control cotton.

[]{.mark}

Table 17. Average % Square Damage Following Infestation of Bt Cotton and
Control Plants with *H. zea* in 2006

  -------------- ----------------- ------- ------ ------- ------- ------ ------- -----------
                 **Locations^1^,                                                 
                 average %                                                       
                 damage**                                                        

  **Genotype**   **BC -- LA**      **W --  **S -- **J --  **SS -- **J -- **B -   **Overall
                                   LA**    AR**   TN**    NC**    NC**   TX**    Mean %**

  Coker 312      12.4              13.6    12.8   11.5    60.3    67.5   18.5    28.1

  COT102         1.7               4.6     3.1    0       2.0     5.5    3.5     2.9

  COT67B         1.3               1.6     2.0    0       0.5     5.0    2.0     1.8

  VipCot™        0.4               0.6     0.4    0       0.8     0.5    0       0.4
  -------------- ----------------- ------- ------ ------- ------- ------ ------- -----------

^1^ No infestation occurred at L -- MS

Shaded number fields indicate locations where data are available for all
damage assessments in all locations

Table 18. Average % Flower Damage Following Infestation of Bt Cotton and
Control Plants with *H. zea* in 2006

  -------------- ----------------- ------- --------- --------- ---------- --------------
                 **Locations^1^,                                          
                 average %                                                
                 damage**                                                 

  **Genotype**   **BC -- LA**      **S --  **SS --   **J --    **B --     **Overall Mean
                                   AR**    NC**      NC**      TX**       %**

  Coker 312      10.2              20.5    35.5      43.0      16.3       25.1

  COT102         1.2               4.8     0         4.5       4.7        3.0

  COT67B         1.8               5.0     0.8       3.5       6.7        3.6

  VipCot™        0.4               1.5     0.8       1.0       0          0.7
  -------------- ----------------- ------- --------- --------- ---------- --------------

Shaded number fields indicate locations where data are available for all
types of damage assessments in all locations

^1^No infestation occurred at L -- MS; no data was collected from W-LA
and J-TN

Table 19. Average % Boll Damage Following Infestation of Bt Cotton and
Control Plants with *H. zea* in 2006

  -------------- ----------------- ------------ ----------- ----------- ---------------
                 **Locations^1^,                                        
                 average %                                              
                 damage**                                               

  **Genotype**   **BC -- LA**      **S -- AR**  **SS --     **J -- NC** **Overall Mean
                                                NC**                    %**

  Coker 312      15.3              21.0         34.5        67.2        34.5

  COT102         1.3               7.3          0           21.7        7.6

  COT67B         1.3               5.3          0           12.2        4.7

  VipCot™        0.3               1.3          0           7.8         2.4
  -------------- ----------------- ------------ ----------- ----------- ---------------

Shaded number fields indicate locations where data are available for all
types of damage assessments in all locations

^1^No infestation occurred at L -- MS; no data was collected from W-LA,
B-TX, and J-TN

[TBW damage]{.underline} was assessed by measuring square, flower, and
boll damage following artificial infestation. The trends observed for
TBW were similar to those reported above for CBW. At all locations,
there was low or zero damage to homozygous VipCot, low to moderate
damage to homozygous COT102 and homozygous COT67B, and great damage to
control plants. VipCot consistently showed the highest level of
protection against TBW damage, while COT102 and COT67B showed good
protection against damage. However, the difference between mean damage
from VipCot cotton and other Bt cotton plants was not statistically
significant (see Tables 20 - 22 for results).

Table 20. Average % Square Damage Following Artificial Infestation of Bt
Cotton and Control Plants *H. virescens* in 2006

  -------------- ----------------- --------- --------- --------- --------- ---------------
                 **Locations^1^, %                                         
                 damage**                                                  

  **Genotype**   **L-MS**          **BC --   **W --    **S --    **SS --   **Overall Mean
                                   LA**      LA**      AR**      NC**      %**

  Coker 312      29.0              21.5      32.5      20.0      44.5      29.5

  COT102         0.5               0.4       9.0       4.9       2.5       3.5

  COT67B         0                 0.1       4.5       3.8       0.5       1.8

  VipCot™        0                 0.3       1.0       2.0       0.5       0.8
  -------------- ----------------- --------- --------- --------- --------- ---------------

Shaded number field indicates location where data are available for all
types of damage assessments in 2006

^1^No infestations were carried out at J-NC. Not enough damage was
reported from J-TN and B-TX for results to be included in analysis

Table 21. Average % Flower Damage Following Artificial Infestation of Bt
Cotton and Control Plants with *H. virescens* in 2006

  ---------------- ------------------------------------------------------
                   **Average^1^ % damage**

  **Genotype**     **S -- AR**

  Coker 312        16.5

  COT102           5.8

  COT67B           5.9

  VipCot™          0.7
  ---------------- ------------------------------------------------------

Shaded number field indicates location where data are available for all
types of damage assessments in 2006

^1^Locations not included here did not get assessed for flower damage,
did not receive artificial infestation, or did not have enough damage
for inclusion in analysis.

Table 22. Average % Boll Damage Following Artificial Infestation of Bt
Cotton and Control Plants with *H. virescens* in 2006

  -------------- ----------------- -------------- ------------- ------------------
                 **Locations^1^,                                
                 average %                                      
                 damage**                                       

  **Genotype**   **L-MS**          **S -- AR**    **SS -- NC**  **Overall Mean %**

  Coker 312      33.0              12.5           74.0          39.8

  COT102         0.5               9.0            12.0          7.2

  COT67B         0                 5.0            7.0           4.0

  VipCot™        0                 0.3            1.0           0.4
  -------------- ----------------- -------------- ------------- ------------------

Shaded number field indicates location where data are available for all
types of damage assessments in 2006

^1^No infestation was carried out at J -- NC. Infestations at BC -- LA,
J -- TN, and B -- TX did not result in enough damage for results to be
included in analysis.

*iii) Syngenta's Efficacy Conclusions*

The efficacy trials performed in 2006 were designed to provide data on
the efficacy of Syngenta's VipCot cotton against the heliothine pests,
*H. virescens* and *H. zea*. Preliminary data obtained in 2005
demonstrated the efficacy of VipCot cotton against these pests (plants
were heterozygous for vip3A and flcry1Ab genes). In contrast, the VipCot
cotton tested in 2006 was homozygous for both traits. VipCot cotton
efficacy data against *H. virescens* were mainly derived from artificial
infested field trials, whereas, *H. zea* data were derived mostly from
natural infestations supplemented by artificial infestations at only one
location (Scott, Arizona).

Similar trends were observed following infestation by both *H.
virescens* and *H. zea* in 2006. COT102 and COT67B cotton showed good
levels of control under most circumstances, and results were consistent
with the data obtained in 2005. In every case, VipCot cotton provided as
good or better control of *H. virescens* and *H. zea* than either of the
component events. These data confirm the preliminary indications
obtained in 2005 that VipCot cotton has excellent activity to these key
heliothine cotton pests in the US.

*b) BPPD's response*

BPPD concludes that efficacy benefits of VipCot cotton are like the
efficacy benefits of other cotton PIPs already registered (i.e. a high
level of protection against the three major Bt cotton target pests).

Overall, 2005 and 2006 efficacy data show that VipCot cotton and its two
single event cotton plants provide good protection against TBW and CBW.
In 2005, efficacy results for CBW on VipCot were statistically different
from efficacy results of individual event cotton plants. It appears that
environmental variables may have the potential to affect efficacy of
VipCot as compared to the efficacy of the two single event plants.

Syngenta states that Vip3A is effective against tobacco budworm and
cotton bollworm, while FLCry1Ab protein provides control of TBW and CBW
as well as PBW (pink bollworm). BPPD clarifies here that efficacy data
were submitted for CBW and TBW and not for PBW. However, evidence of
high-dose was provided for VipCot and COT67B for PBW in the IRM
submission (MRID 470176-34).

**3. Grower Benefits**

*a) Summary of Syngenta's Submission and Expert Reports in the PID (MRID
470347-01):*

*i) Agronomic Benefits*

The introduction of Bt cotton has transformed cotton production in the
United States. The dramatic shift from conventional chemical
insecticides to Bt cotton during the last 10 years has occurred because
of the strongly positive agronomic and economic factors associated with
Bt cotton technology. VipCot will continue in this tradition.

Syngenta asked experts from three cotton growing regions, Dr. Bradley of
North Carolina University, Dr. Leonard of Louisiana State University and
Jack Hamilton, Regents chair in cotton production, as well as Dr. Rummel
of Texas A & M University, to comment on agronomics associated with Bt
cotton generally and on VipCot specifically. The following is a short
summary of the conclusions extracted by BPPD from the expert reports,
which were submitted by Syngenta as separated appendices in the PID
(MRID 470347-01):

The cotton growing region of southeastern US recovered from its demise
with the boll weevil eradication in the 1980s. The introduction of Bt
cotton in the 1990s was the second event that further boosted the cotton
industry and enabled farmers to further increase the cotton acreage, for
example, from 500 to 800 thousand acres annually (in NC) during the next
decade. Bt crops in general have produced great economic benefits to US
growers while leading to a reduced reliance on conventional
insecticides. For cotton alone, EPA estimated that there was a
7.5-million acre reduction in insecticide use due to planting of Bt
varieties in 1999. During the first several years of the Bt cotton
introduction, average (over years and locations) Bt cotton yields in the
US exceeded those of conventional cotton with a mean profit advantage
ranging from \$16 to less than \$173/acre. Aside from environmental
benefits mentioned above, other extended benefits of Bt cotton are fuel
savings and reduced labor expenditures. Specifically, in North Carolina,
Bt cotton is now planted in areas where cotton could not be grown before
because of insecticide drift concerns in school and housing areas, etc.

Preliminary field testing results suggest that Vip3A proteins may have
superior efficacy compared to Widestrike and equal efficacy to Bollgard
II. Dr. Bradley further writes that Cry1Ac resistant TBW have shown to
be susceptible to Vip3A toxins and Vip3A producing plants in the lab and
greenhouse. These data indicate that VipCot provides growers with viable
alternatives to other Bt cotton varieties presently registered. In
addition, VipCot offers a mechanism of resistance management.

In the mid-south states of the US (AR, LA, MS, TN), cotton is
historically one of four crops that makes up the largest amount of acres
planted (average range 530,000 -- 1.22 million acres). Since the
introduction of Bollgard (Cry1Ac toxin), losses to cotton growers have
been reduced from damage caused by TBW and CBW. Losses were further
reduced with the introduction of Bollgard II and Widestrike. Average
yield loss to cotton growers was 4.1% from 1990-1995. Since 1999, losses
from Bt and non-Bt varieties, dropped to \<1% and 2 - 5%, respectively.
Annual lint yields were the highest ever reported in these states from
2002 -- 2006. More than 80% of cotton acreage in 2005 was planted with
Bt varieties. Field trials across the mid-south States showed enhanced
efficacy to single Cry proteins against bollworms and satisfactory
control against other Lepidopteran pests. Cotton entomologists expect
VipCot to provide equal or better protection against these pests than
other currently registered Bt cotton varieties. Because Vip3A is the
first transgenic non-Cry protein introduction into cotton varieties,
VipCot is expected to have considerable value as a Bt resistance
management tool.

The boll weevil eradication and introduction of Bt cotton allowed cotton
production in Texas to rebound and also expand into previous non-cotton
areas of the Texas Pan handle. Texas cotton growing regions are as
diverse geographically (rainfall, soil type, length of growing season,
available irrigation, and production techniques) as they are numerous,
and production requirements from region to region vary greatly. Bt
cotton varieties that provide optimum performance in one region, may
exhibit marginal performance in another. Texas growers require access to
numerous insect resistant cotton varieties to meet the requirements of
the various production regions. Tests conducted across the cotton belt
show good efficacy of VipCot, and efficacy at least as good to Bollgard
II is expected. No cross-resistance between Cry1Ac and Vip3A was
detected in lab resistant CBW. The novel mode of action of Vip3A differs
in several respects compared to Cry1Ab, and its availability to growers
is expected to serve as a Bt resistance management tool.

Insect efficacy is only one factor that must be considered when
developing new transgenic technologies for use in commercial cotton.
Yield potential and fiber quality traits must also be considered. In
both cases, available data support the potential for viable VipCot
varieties.

Concerning yield data (Appendix 4 of PID, MRID 470347-01), Syngenta's
marketing partner evaluated VipCot in a multiple location trial series
representative of the US cotton growing region. The results showed no
significant differences in seed cotton yield between Cocker 312 and
VipCot (see Table 23). No significant location by variety interaction
occurred in the 2006 testing (p = 0.1031). The conclusion from this
preliminary work is that VipCot demonstrates no negative agronomic
characteristics that would impact a variety development program.
Agronomic, efficacy, yield, and fiber quality data and information all
support the strong potential for VipCot to become a quality competitive
product in the Bt cotton market. These factors coupled with an
aggressive marketing plan and strong market presence will provide
growers with additional choices and generate economic benefits.

Table 23. Seed Cotton per Acre generated for control and transgenic
cotton plants during 2006

+-----------------------+-----------------------+---+----------------------+
| **Location**          | **Cocker 312,**       | * |                      |
|                       |                       | * |                      |
|                       | **Seed cotton/acre**  | C |                      |
|                       |                       | O |                      |
|                       |                       | T |                      |
|                       |                       | 1 |                      |
|                       |                       | 0 |                      |
|                       |                       | 2 |                      |
|                       |                       | x |                      |
|                       |                       | 6 |                      |
|                       |                       | 7 |                      |
|                       |                       | B |                      |
|                       |                       | , |                      |
|                       |                       | * |                      |
|                       |                       | * |                      |
|                       |                       |   |                      |
|                       |                       | * |                      |
|                       |                       | * |                      |
|                       |                       | S |                      |
|                       |                       | e |                      |
|                       |                       | e |                      |
|                       |                       | d |                      |
|                       |                       | c |                      |
|                       |                       | o |                      |
|                       |                       | t |                      |
|                       |                       | t |                      |
|                       |                       | o |                      |
|                       |                       | n |                      |
|                       |                       | / |                      |
|                       |                       | a |                      |
|                       |                       | c |                      |
|                       |                       | r |                      |
|                       |                       | e |                      |
|                       |                       | * |                      |
|                       |                       | * |                      |
+=======================+=======================+===+======================+
| Belle Mina, AL        | 1387                  | 1 |                      |
|                       |                       | 3 |                      |
|                       |                       | 3 |                      |
|                       |                       | 0 |                      |
+-----------------------+-----------------------+---+----------------------+
| College Station, TX   | 1958                  | 2 |                      |
|                       |                       | 0 |                      |
|                       |                       | 3 |                      |
|                       |                       | 6 |                      |
+-----------------------+-----------------------+---+----------------------+
| Estill, SC            | 1833                  | 1 |                      |
|                       |                       | 7 |                      |
|                       |                       | 1 |                      |
|                       |                       | 3 |                      |
+-----------------------+-----------------------+---+----------------------+
| Hartsville, SC        | 2467                  | 2 |                      |
|                       |                       | 2 |                      |
|                       |                       | 2 |                      |
|                       |                       | 2 |                      |
+-----------------------+-----------------------+---+----------------------+
| Haskell, TX           | 2254                  | 1 |                      |
|                       |                       | 9 |                      |
|                       |                       | 9 |                      |
|                       |                       | 9 |                      |
+-----------------------+-----------------------+---+----------------------+
| Portageville, MO      | 2014                  | 2 |                      |
|                       |                       | 3 |                      |
|                       |                       | 0 |                      |
|                       |                       | 6 |                      |
+-----------------------+-----------------------+---+----------------------+
| Red Springs, NC       | 2300                  | 2 |                      |
|                       |                       | 0 |                      |
|                       |                       | 7 |                      |
|                       |                       | 2 |                      |
+-----------------------+-----------------------+---+----------------------+
| Tifton, GA            | 2879                  | 3 |                      |
|                       |                       | 3 |                      |
|                       |                       | 0 |                      |
|                       |                       | 3 |                      |
+-----------------------+-----------------------+---+----------------------+
| Winterville, MS       | 3060                  | 3 |                      |
|                       |                       | 3 |                      |
|                       |                       | 3 |                      |
|                       |                       | 9 |                      |
+-----------------------+-----------------------+---+----------------------+
| Variety x Location    | 0.1031                |   |                      |
| (p\< 0.05)            |                       |   |                      |
+-----------------------+-----------------------+---+----------------------+
|                       | **Cocker 312,**       |   | **COT 102 x 67B,**   |
|                       |                       |   |                      |
|                       | **Avg. seed           |   | **Avg. seed          |
|                       | cotton/acre**         |   | cotton/acre**        |
+-----------------------+-----------------------+---+----------------------+
|                       | 2239                  |   | 2257                 |
+-----------------------+-----------------------+---+----------------------+
| Variety (p\< 0.05)    | 0.7729                |   |                      |
+-----------------------+-----------------------+---+----------------------+

Concerning fiber quality, lint samples of the component events COT102
and COT67B (but not VipCot) have been analyzed for fiber characteristics
and quality cotton varieties. In work conducted to date, there were no
significant differences between both events and non-transgenic genotypes
for fiber micronaire, length, or uniformity and thus, none are expected
when these events are combined as VipCot.

*ii) Economic Benefits*

Syngenta considers the major benefits resulting from the introduction of
VipCot will be additional grower choice, increased competition, and
extended useful life of Bt cotton technology generally (resulting from
the unique mode of action of the Vip3A protein expressed by VipCot)
rather than a major shift from chemical insecticide treated acres to new
Bt planted acres. Syngenta projects additional economic benefits of \$83
million will accrue from the regulatory approval and use of VipCot
(report by Dr. Wailes submitted as Appendix 5 in PID, MRID 470347-01).
The \$83 million dollar value is the product of conservative estimates
to predict the net present value of VipCot and additional value that
VipCot will bring to the market in terms of added competition and grower
choice for the first eight years of sales. Furthermore, Dr. Wailes
estimates the regional distribution of these benefits based on existing
adoption levels of transgenic cotton varieties (Appendix 5 in PID).

*b) BPPD's response*

VipCot cotton has similar general grower benefits as
previously-registered cotton PIPs (i.e. yield, lint quality) as
described by the Agency in the 2001 Bt crop reassessment. The general
benefits for Bt cotton are summarized in U.S. EPA 2001
(<http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm>).

BPPD notes that the novel mode of action of Vip3A and lack of
cross-resistance observed in Cry1Ac-resistant lab colonies of CBW and
TBW may provide mechanisms by which the useful life of Bt technology
could be extended.

Syngenta states that preliminary field testing results suggest that
Vip3A proteins may have superior efficacy than Widestrike (registered by
Dow AgroSciences) against caterpillar pests and equal or superior
efficacy to Bollgard II (registered by Monsanto). No comparative data
were submitted to the Agency to back this claim. At this point, BPPD
will not consider this proposed benefit.

BPPD focused on the benefits from VipCot only and not on the potential
agronomic and economic benefits (i.e. additional economic benefits of
\$83 million dollars as cited in Dr. Wailes analysis, Appendix 5 of PID,
MRID 470347-01) of VipCot/RR Flex (herbicide tolerant trait). It may be
that additional benefits are derived from an herbicide tolerant trait in
VipCot. On the other hand, such a trait could also increase the risk of
weed resistance. Nonetheless, BPPD would like to note the following
regarding Dr. Wailes' analysis of expansion in Syngenta's share market.
The analysis is based on two major assumptions as stated in the
report: 1) increased productivity based on higher yields relative to
conventional cotton and potentially other transgenic varieties and 2)
increased productivity based on cost efficiencies available through more
competitive technology fees and services provided relative to other
transgenic varieties. As mentioned throughout BPPD's review of
Syngenta's PID, the higher yield assumption for VipCot as compared to
other transgenic varieties remains unsubstantiated at this time, and
therefore, should not be considered as an input in the model.
Furthermore, the 'new value extraction model of shared risk price
structures' described in the PID proposes to use the lower technology
fee approach for non-irrigated cotton regions as well as growers who
suffer reduced yield; however, the model is still awaiting final
approval by the Joint Syngenta/D & PL License Management Committee.
Therefore, the share market analysis predicting expansion in the
marketplace is based on two major assumptions that contain some
hypothetical (greater or equal yield than Bollgard II and greater yield
than WideStrike) and unverified information (lower technology fees still
awaiting approval). Once one of these assumptions or both are verified,
the benefit claims of Syngenta's share market analysis may be warranted.

With respect to the seed yield data and analysis submitted, BPPD notes
that the 'Variety x Location' interaction value is 0.1031 (variety
meaning Cocker 312 and VipCot). This value seems to indicate that there
is potential for interaction despite it not being statistically
significant at the 5% level. Nonetheless, it may be important for
farmers to realize that when planting VipCot across states in the cotton
belt, such as tested in the study (MS, AL, TX, SC, MO, NC, GA), 90 times
out of 100 times, they may see an effect on seed yield due to
geographic/environmental differences. BPPD notes that this frequency of
'Location x Variety' interaction is rather high and should be taken into
consideration when thinking about a variety development program even if
Syngenta concludes, based on statistical significance at the 5% level,
that no negative characteristics are exhibited.

**4. Human Health and Environmental Benefits**

*a) Summary of Syngenta's Submission* *(MRID 470347-01):*

EPA has consistently found that the registration of Bt PIPs is in the
public interest. These findings have largely been based on a
determination that Bt PIPs present less risk than conventional pesticide
alternatives. The Agency's view concerning Bt PIPs is well accepted and
supported by the work of others. Brookes and Barfoot (2005) presented
findings that the global introduction of genetically modified crops
resulted in the reduction in use of chemical pesticides by 172 million
kilograms, and reduced the environmental footprint associated with
pesticide use by 14% during the period 1996 to 2004.

Neither the Vip3A, nor the FLCry1Ab protein is likely to be a food
allergen, and toxicity studies indicate no hazard concern. The same
profile holds for the marker protein, hygromcyin B phosphotransferase,
which is exempt from the requirement for a tolerance. Syngenta studies
also provide the same solid evidence for the environmental safety of the
proteins expressed in VipCot. Extensive testing shows no real risk
concern.

*b) BPPD's response*

EPA reviewed product characterization, human health safety, and aquatic
and terrestrial wildlife studies submitted by Syngenta and agrees with
Syngenta's conclusion. There is no human health concern with respect to
toxicity or allergenicity and no environmental concern with respect to
toxicity of the two proteins expressed in VipCot, Vip3Aa and FLCry1Ab.
VipCot cotton has similar general human health and environmental
benefits of already registered cotton PIPs. For further information
regarding the Agency's summary of these benefits, refer to the 2001 Bt
crop reassessment (U.S. EPA 2001,
<http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm>).

**5. Resistance Management Benefits**

*a) Summary of Syngenta's Benefits Submission* *(MRID 470347-01)* *and
IRM Submission (470176-34):*

While COT102 alone does not express a high-dose against any of the three
key pests, it is active against CBW, TBW, and PBW at greater than
25-fold dilutions. COT67B alone expresses a high-dose for all three
pests, and both proteins together, as expressed in VipCot cotton, result
in a high-dose for CBW, TBW, and PBW.

Supporting information (i.e. lack of sequence homology between Vip3A and
Cry proteins, no indication of similar domain structure between Vip3A
and Bt Cry proteins, secretion of Vip and Bt Cry proteins at different
stage of bacterial growth, different binding sites) and study results
demonstrate the lack of cross-resistance between Vip3A and other Bt Cry
proteins including FLCry1Ab protein in VipCot as well as other Cry
proteins in currently sold Bt cotton PIPs.

Resistance risk assessment modeling by Dr. Caprio predicts that the risk
of resistance developing to VipCot is very low, and further, modeling
predicts that the use of VipCot can delay the development of resistance
to cotton varieties expressing Cry toxins.

*b)* *BPPD's response*

BPPD concurs with Syngenta's conclusion that VipCot has the following
benefits: lack of cross-resistance and potential to delay development of
resistance in other cotton varieties expressing Cry toxins (based on
i.e. unique mode of action of Vip3Aa and cross-resistance studies done
on Cry1Ac resistant lab colonies of CBW and TBW). As a unique mode of
action, VipCot will have similar general IRM benefits as other cotton
PIP products, which are thoroughly discussed and summarized in the
Agency's 2001 Bt crop reassessment (U.S. EPA 2001,
<http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm>).

**6. VipCot Cotton Marketing Plan**

Syngenta submitted a marketing plan for VipCot cotton as part of the
benefits submission. This plan included information on VipCot stacking
with herbicide tolerance traits, potential market share, pricing, and
models to determine marketing strategy. This portion of the benefits
submission was classified as Confidential Business Information and is
not included or summarized in this document.

BPPD notes that benefits from Syngenta's marketing plan seem numerous,
especially if the pricing models will get adopted as proposed in the
PID. Furthermore, the introduction of VipCot is expected to increase
competition and make the demand curve more elastic. Thus, VipCot may
lead to an increase in Bt use and a reduction in price of other
registered Bt cotton products. These are examples of benefits to
growers, the public, and environment. However, benefits based on
modeling are still hypothetical until the model has been vetted by
management of Syngenta and partners.

BPPD's comments regarding VipCot/herbicide tolerant trait are first
listed in section 3 (Grower's Benefits) and reiterated here. BPPD
focuses only on the benefits from VipCot and not on the potential
agronomic and economic benefits of VipCot/RR Flex. It may be that
additional benefits are derived from an herbicide tolerant trait in
VipCot. On the other hand, such a trait could also increase the risk of
weed resistance.

**7. References**

BPPD, 2003. EPA preliminary review of public interest document submitted
by Syngenta Seeds, Inc. for VIP3A cotton (event COT102) in support of a
conditional registration under FIFRA Section 3(c)(7)(C). E. Brandt and
S. Matten memorandum to L. Cole; Nov 13, 2003.

Kurtz, R., McCaffery, A., O'Reilly, D., Stone, T. 2006. Insect
resistance management considerations for VipCot™ Cotton. Report
submitted from Syngenta Biotechnology, Inc. MRID 470176-34.

O'Reilly, D., Dickerson, D., Negretto, D., Minton, B., Martin, S. 2006.
Field Efficacy Evaluation of component events COT102 and COT67B and
stacked COT102 x COT67B cotton (VipCot™ Cotton) in 2005 and 2006. Report
submitted from Syngenta Biotechnology, Inc. MRID 470176-33.

Schnepf, E., Crickmore, N., Van Rie, D. Lereclus, D., Baum, J.,
Feitelson, J., Zeigler, D.R., Dean, D.H. 1998. *Bacillus thuringiensis*
and its pesticidal crystal proteins. Microbiology and Molecular Biology
Reviews, Vol. 62 (3): 775-806.

Stone, T., Martin, T., and Tinsworth, E.F. 2006. Public Interest
Document in Support of Registration of the Plant Incorporated Protectant
Proteins Vip3A and FLCry1Ab as Expressed in the Combined Trait COT102 x
COT67B (VipCot™ Cotton). Report submitted from Syngenta Biotechnology,
Inc. MRID 470347-01.

US EPA, 1998. FIFRA Scientific Advisory Panel Subpanel on *Bacillus
thuringiensis (Bt)* Plant-Pesticides and Resistance Management, February
9 and 10, 1998.

US EPA, 2001. Biopesticides Registration Action Document -- *Bacillus
thuringiensis* Plant Incorporated Protectants,
<http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm>.

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

The following terms and/or conditions are required for the section
3(c)(7)(C) registration of VipCot cotton:

> 1\) The registration will automatically expire on midnight September
> 30, 2011.
>
> 2\) All data required for registration of the product under FIFRA §
> 3(c)(5) must be submitted when the Agency requires all registrants of
> similar products to submit such data.
>
> 3\) The following restrictions regarding gene flow are required:
>
> The following information regarding commercial production must be
> included in the grower guide for VipCot Cotton:
>
> a\) No planting of VipCot cotton is permitted south of Route 60 (near
> Tampa) in Florida.
>
> b\) Commercial culture of VipCot cotton is prohibited in Hawaii,
> Puerto Rico, and the U.S. Virgin Islands.
>
> The following information regarding test plots and seed production
> must be included on bags of VipCot cotton intended for these purposes:
>
> a\) Test plots or breeding nurseries, regardless of the plot size,
> established in Hawaii must not be planted within 3 miles of *Gossypium
> tomentosum*;
>
> b\) Experimental plots and breeding nurseries of Bt cotton are
> prohibited on the U.S. Virgin Islands; and
>
> c\) Test plots or breeding nurseries, regardless of the plot size,
> established on the island of Puerto Rico must not be planted within 3
> miles of feral cotton plants.
>
> 4\) The following restrictions regarding Insect Resistance Management
> are required:
>
> VipCot Bt cotton is not permitted to be planted in the following
> counties of the Texas panhandle: Dallam, Sherman, Hansford, Ochiltree,
> Lipscomb, Hartley, Moore, Hutchinson, Roberts, and Carson.
>
> 5\) The following data and/or information must be submitted in the
> time frames listed:

  -------------------------------------------------------------------------
  **Study Type**   **Required Data**                         **Due Date**
  ---------------- ----------------------------------------- --------------
  Residue          An independent lab validation of the      May 1, 2009
  Analytical       "SeedChek" analytical method for the      
  Method - Plants  detection of Vip3Aa19 and modified        
  (OPPTS 860.1340) Cry1Ab. You must also agree to provide to 
                   the EPA laboratory (Ft. Meade, MD)        
                   methodology and/or reagents necessary for 
                   validation of such analytical method      
                   within 6 months from the date that the    
                   Agency requests them.                     

  Aquatic          A 7-14 day *Daphnia* study as per the     May 1, 2009
  Invertebrate     OPPTS 885.4240 guideline must be          
  Toxicity (OPPTS  submitted as a condition of registration. 
  885.4240)        Alternatively, a dietary study of the     
                   effects on an aquatic invertebrate,       
                   representing the functional group of a    
                   leaf shredder in headwater streams, can   
                   be performed and submitted in lieu of the 
                   7-14 day *Daphnia* study. **Separate      
                   studies for Vip3Aa (COT102) and modified  
                   Cry1Ab (COT67B) must be preformed.**      

  Insect           A detailed resistance monitoring plan for Within 90 days
  Resistance       the major pests of VipCot cotton: tobacco of the date of
  Management -     budworm, cotton bollworm, and pink        registration
  Resistance       bollworm.                                 
  Monitoring                                                 

  Insect           Baseline susceptibility and diagnostic    January 31,
  Resistance       concentration determinations for tobacco  2009
  Management -     budworm, cotton bollworm, and pink        
  Resistance       bollworm to Vip3Aa19 and modified Cry1Ab. 
  Monitoring                                                 

  Insect           A compliance assurance program (CAP) for  Within 90 days
  Resistance       VipCot must be submitted and must include of the date of
  Management -     a "phased compliance approach" that       registration
  Compliance       outlines instances of non-compliance to   
                   the IRM requirements and options of       
                   responding to non-compliant growers.      

  Insect           A copy of the grower                      Within 90 days
  Resistance       agreement/stewardship documents and       of the date of
  Management -     written description of a system assuring  registration
  Compliance       that growers will sign grower agreement   
                   must be submitted.                        

  Insect           A final remedial action plan for tobacco  Within 90 days
  Resistance       budworm and cotton bollworm. The remedial of the date of
  Management --    action plan must include definitions of   registration
  Remedial Action  "suspected" and "confirmed" resistance    
  Plans            and steps to take in the event of         
                   confirmed resistance.                     
  -------------------------------------------------------------------------

6\) The following Insect Resistance Management Program is required for
VipCot:

> a\) The required IRM program for VipCot Bt cotton must have the
> following elements:

-   Requirements relating to creation of a non-Bt cotton refuge in
    conjunction with the planting of any acreage of VipCot Bt cotton.

-   Requirements for Syngenta Seeds to prepare and require VipCot Bt
    cotton users to sign "grower agreements" which impose binding
    contractual obligations on the grower to comply with the refuge
    requirements;

-   Requirements for Syngenta Seeds to develop, implement, and report to
    EPA on programs to educate growers about IRM requirements;

-   Requirements for Syngenta Seeds to develop, implement, and report to
    EPA on programs to evaluate and promote growers' compliance with IRM
    requirements;

-   Requirements for Syngenta Seeds to develop, implement, and report to
    EPA on programs to evaluate whether there are statistically
    significant and biologically relevant changes in susceptibility to
    the Vip3Aa19 and modified Cry1Ab proteins in the target insects;

-   Requirements for Syngenta Seeds to develop, and if triggered, to
    implement a "remedial action plan" which would contain measures
    Syngenta Seeds would take in the event that any insect resistance
    was detected as well as to report on activity under the plan to EPA;

-   Annual reports on or before January 31^st^ each year.

> b\) [Refuge Requirements]{.underline}
>
> All growers of VipCot cotton must employ one of the following
> structured refuge options:
>
> [1) External, Unsprayed Refuge]{.underline}
>
> Ensure that at least 5 acres of non-Bt cotton (refuge cotton) is
> planted for every 95 acres of VipCot cotton. The size of the refuge
> must be at least 150 feet wide, but preferably 300 feet wide. This
> refuge may not be treated with sterile insects, pheromone, or any
> insecticide (except listed below) labeled for the control of tobacco
> budworm, cotton bollworm, or pink bollworm. At the pre-squaring cotton
> stage only, the refuge may be treated with any lepidopteran
> insecticide to control foliage feeding caterpillars. The refuge may be
> treated with acephate or methyl parathion at rates which will not
> control tobacco budworm or the cotton bollworm (equal to or less than
> 0.5 lbs active ingredient per acre). The variety of cotton planted in
> the refuge must be comparable to VipCot cotton, especially in the
> maturity date, and the refuge must be managed (e.g., planting time,
> use of fertilizer, weed control, irrigation, termination, and
> management of other pests) similarly to VipCot cotton. Ensure that a
> non-Bt cotton refuge is maintained within at least ½ linear mile
> (preferably adjacent to or within 1/4 mile or closer) from the *Bt*
> cotton fields.
>
> [2) External, Sprayed Refuge]{.underline}
>
> Ensure that at least 20 acres of non-Bt cotton are planted as a refuge
> for every 80 acres of VipCot cotton (total of 100A). The variety of
> cotton planted in the refuge must be comparable to Bt cotton,
> especially in the maturity date, and the refuge must be managed (e.g.,
> planting time, use of fertilizer, weed control, irrigation,
> termination, and management of other pests) similarly to VipCot
> cotton. The non-Bt cotton may be treated with sterile insects,
> insecticides (excluding foliar Bt kurstaki products), or pheromones
> labeled for control of the tobacco budworm, cotton bollworm, or pink
> bollworm. Ensure that a non-Bt refuge is maintained within at least 1
> linear mile (preferably within ½ mile or closer) from the Bt cotton
> fields.
>
> [3) Embedded Refuge]{.underline}
>
> Ensure that at least 5 acres of non-Bt cotton (refuge cotton) are
> planted for every 95 acres of VipCot cotton (total of 100A). The
> refuge cotton must be embedded as a contiguous block within the VipCot
> field, but not at one edge of the field (i.e., refuge block(s)
> surrounded by Bt cotton). For very large fields, multiple blocks
> around the field may be used. For small or irregularly shaped fields,
> neighboring fields farmed by the same grower can be grouped into
> blocks to represent a larger field unit, provided the block exists
> within one mile squared of the Bt cotton and the block is at least 150
> feet wide, but preferably 300 feet wide. Within the larger field unit,
> one of the smaller fields planted to non-Bt cotton may be utilized as
> the embedded refuge. The variety of cotton planted in the refuge must
> be comparable to Bt cotton, especially in the maturity date, and the
> refuge must be managed (e.g., planting time, use of fertilizer, weed
> control, irrigation, termination, and management of other pests)
> similarly to VipCot cotton. The non-Bt cotton may be treated with
> sterile insects, insecticides (excluding foliar Bt kurstaki products),
> or pheromones labeled for control of the tobacco budworm, cotton
> bollworm, or pink bollworm whenever the entire field is treated. The
> refuge may not be treated independently of the surrounding VipCot
> field in which it is embedded (or fields within a field unit).
>
> [4) Embedded Refuge (for pink bollworm only)]{.underline}
>
> Refuge cotton must be planted as at least one single non-Bt cotton row
> for every six to ten rows of VipCot cotton. The refuge may be treated
> with sterile insects, any insecticide (excluding foliar Bt kurstaki
> products), or pheromone labeled for the control of pink bollworm
> whenever the entire field is treated. The in-field refuge rows may not
> be treated independently of the surrounding Bt cotton field in which
> it is embedded. The refuge must be managed (fertilizer, weed control,
> etc.) identically to the VipCot cotton. There is no field unit option.
>
> [5) Community Refuge Option]{.underline}
>
> This option allows for multiple growers to manage refuge for external,
> unsprayed and external, sprayed refuge options or both. This option is
> not allowed for the embedded/in-field refuge options. The community
> refuge for insect resistance management must meet the requirements of
> the 5% external, unsprayed and/or 20% sprayed option, or an
> appropriate combination of the two options. The community refuge
> program must consist of the following:
>
> There will be a community refuge coordinator for each community. Each
> community refuge coordinator must submit a signed community refuge
> form listing all of the participants in the community to Syngenta
> Seeds by July 1st annually. Syngenta Seeds must provide EPA, if
> requested, with a copy of the signed community refuge form. The
> community refuge coordinator will maintain a copy of the field map (to
> scale) or suitable scalar representation of the community refuge for
> review by Syngenta Seeds or EPA as part of the compliance program.
>
> On an annual basis, Syngenta Seeds must conduct at least one telephone
> audit of a statistically representative sample of community refuge
> coordinators from communities in all states participating in the
> community refuge. EPA shall review the questions annually prior to the
> start of the growing season.
>
> The community refuge program users must be included in the telephone
> compliance survey and the on-farm visits to be conducted by Syngenta
> Seeds under section d. below.
>
> Beginning January 31, 2010 and annually each January 31st, Syngenta
> Seeds must provide a written report to EPA annually on community
> refuge use and compliance. The community refuge report may be combined
> in a single report with other compliance activities.
>
> On an annual basis, Syngenta Seeds must conduct a review of the
> community refuge program and submit that review to the Agency as to
> any proposed changes by January 31st. An appropriate amendment for any
> proposed changes must be submitted to the Agency.
>
> [c) Grower Agreements]{.underline}
>
> The following provisions regarding grower agreements are required for
> VipCot:
>
> 1\) Persons purchasing the VipCot cotton product must sign a grower
> agreement. The term "grower agreement" refers to any grower purchase
> contract, license agreement, or similar legal document.
>
> 2\) The grower agreement and/or specific stewardship documents
> referenced in the grower agreement must clearly set forth the terms of
> the current IRM program. By signing the grower agreement, a grower
> must be contractually bound to comply with the requirements of the IRM
> program.
>
> 3\) Syngenta Seeds must implement a system which is reasonably likely
> to assure that persons purchasing the Bt cotton product will affirm
> annually that they are contractually bound to comply with the
> requirements of the IRM program. A description of the system must be
> submitted to EPA within 90 days from the date of registration.
>
> 4\) Syngenta Seeds must use an approved grower agreement and must
> submit to EPA within 90 days from the date of registration a copy of
> that agreement and any specific stewardship documents referenced in
> the grower agreement. If Syngenta Seeds wishes to change any part of
> the grower agreement that would affect either the content of the IRM
> program or the legal enforceability of the provisions of the agreement
> relating to the IRM program, thirty days prior to implementing a
> proposed change, Syngenta Seeds must submit to EPA the text of such
> changes to ensure the agreement is consistent with the terms and
> conditions of this amendment.
>
> 5\) Syngenta Seeds must implement an approved system which is
> reasonably likely to assure that persons purchasing VipCot cotton sign
> grower agreement(s). A description of the system must be submitted to
> EPA within 90 days from the date of registration.
>
> 6\) Syngenta Seeds shall maintain records of all VipCot cotton grower
> agreements for a period of three years from December 31 of the year in
> which the agreement was signed.
>
> 7\) Beginning on January 31, 2010 and annually thereafter, Syngenta
> Seeds shall provide EPA with a report on the number of units of the
> VipCot cotton seed shipped and not returned and the number of such
> units that were sold to persons who have signed grower agreements. The
> report shall cover the time frame of the twelve-month period covering
> the prior October through September.
>
> 8\) Syngenta Seeds must allow a review of the grower agreements and
> grower agreement records by EPA or by a State pesticide regulatory
> agency if the State agency can demonstrate that the names, personal
> information, and grower license number will be kept as confidential
> business information.
>
> [d) IRM Education and IRM Compliance Monitoring Programs]{.underline}
>
> The following IRM education and compliance monitoring programs must be
> implemented for VipCot:
>
> 1\) Syngenta Seeds must design and implement a comprehensive, ongoing
> IRM education program designed to convey to VipCot cotton users the
> importance of complying with the IRM program. The program shall
> include information encouraging Bt cotton users to pursue optional
> elements of the IRM program relating to refuge configuration and
> proximity to Bt cotton fields. The education program shall involve the
> use of multiple media, e.g. face-to-face meetings, mailing written
> materials, and electronic communications such as by internet or
> television commercials. Copies of the materials, including the Grower
> Guide or other technical bulletins, must be submitted to EPA for their
> records. The program shall involve at least one written communication
> annually to each VipCot cotton grower separate from the grower
> agreement. Syngenta Seeds shall coordinate its education program with
> educational efforts of other organizations, such as the National
> Cotton Council and state extension programs.
>
> 2\) Annually, Syngenta Seeds shall revise, and expand as necessary,
> its education program to take into account the information collected
> through the compliance survey required under paragraph 6 below and
> from other sources. The changes shall address aspects of grower
> compliance that are not sufficiently high.
>
> 3\) Beginning January 31, 2009 and annually thereafter, Syngenta Seeds
> shall provide a report to EPA summarizing the activities it carried
> out under its education program for the prior year and its plans for
> its education program during the current year.
>
> 4\) Syngenta Seeds shall design and implement an IRM compliance
> assurance program designed to evaluate the extent to which growers are
> complying with the IRM program and that takes such actions as are
> reasonably needed to assure that growers who have not complied with
> the program either do so in the future or lose their access to VipCot
> cotton. Syngenta Seeds must prepare and submit within 90 days of the
> date of registration a written description of the compliance assurance
> program. Other required features of the program are described in
> paragraphs 5 - 12 below.
>
> 5\) Syngenta Seeds shall establish and publicize a "phased compliance
> approach," i.e., a guidance document that indicates how Syngenta Seeds
> will address instances of non-compliance with the terms of the IRM
> program and general criteria for choosing among options for responding
> to any non-compliant growers. The options shall include withdrawal of
> the right to purchase VipCot cotton for an individual grower or for
> all growers in a specific region. An individual grower found to be
> significantly out of compliance two years in a row would be denied
> sales of the product the next year.
>
> 6\) The IRM compliance assurance program shall include an annual
> survey of a statistically representative sample of VipCot cotton
> growers conducted by an independent third party. The survey shall
> measure the degree of compliance with the IRM program by growers in
> different regions of the country and consider the potential impact of
> non-response. Syngenta Seeds shall provide a written summary of the
> results of the prior year's survey to EPA by January 31st of each
> year. Syngenta Seeds shall confer with EPA on the design and content
> of the survey prior to its implementation.
>
> 7\) Annually, Syngenta Seeds shall revise, and expand as necessary,
> its compliance assurance program to take into account the information
> collected through the compliance survey (required under paragraph 6)
> and from other sources. The changes shall address aspects of grower
> compliance that are not sufficiently high. Syngenta Seeds will confer
> with EPA prior to adopting any changes.
>
> 8\) Syngenta Seeds must conduct an annual on-farm assessment program.
> Syngenta Seeds shall train its representatives who make on-farm visits
> with VipCot cotton growers to perform assessments of compliance with
> IRM requirements. In the event that any of these visits results in the
> identification of a grower who is not in compliance with the IRM
> program, Syngenta Seeds shall take appropriate action, consistent with
> its "phased compliance approach," to promote compliance.
>
> 9\) Syngenta Seeds shall carry out a program for investigating "tips
> and complaints" that an individual grower or growers is/are not in
> compliance with the IRM program. Whenever an investigation results in
> the identification of a grower who is not in compliance with the IRM
> program, Syngenta Seeds shall take appropriate action, consistent with
> its "phased compliance approach."
>
> 10\) If a grower, who purchases VipCot cotton for planting, was
> specifically identified as not being in compliance during the previous
> year, Syngenta Seeds shall visit the grower and evaluate whether that
> the grower is in compliance with the IRM program for the current year.
>
> 11\) Beginning January 31, 2010 and annually thereafter, Syngenta
> Seeds shall provide a report to EPA summarizing the activities it
> carried out under its compliance assurance program for the prior year
> and its plans for its compliance assurance program during the current
> year. Included in that report will be the percent of growers using
> each refuge option (or combination of options) by region, the
> approximate number or percent of growers visited on farm by Syngenta
> Seeds and the results of these visits the number of tips investigated,
> the percent of growers not in compliance with each refuge option (both
> size and distance), and the follow-up actions taken.
>
> 12\) Syngenta Seeds must allow a review of the compliance records by
> EPA or by a State pesticide regulatory agency if the State agency can
> demonstrate that the names, personal information, and grower license
> number of the growers will be kept as confidential business
> information.
>
> [e. Insect Resistance Monitoring]{.underline}.
>
> The registration of Vip3Aa19 and modified Cry1Ab PIPs expressed in
> VipCot cotton is conditioned on Syngenta Seeds carrying out
> appropriate programs to detect the emergence of insect resistance as
> early as possible. Resistance monitoring programs include surveying
> insects for potential resistance and collection of information from
> growers about events that may indicate resistance. Syngenta Seeds
> should coordinate its monitoring efforts VipCot with the current
> resistance monitoring programs for other registered Bt cotton
> products. The following resistance monitoring terms are required for
> VipCot:
>
> 1\) Syngenta Seeds must submit a VipCot cotton (Vip3Aa19 and modified
> Cry1Ab toxins) resistance monitoring plan for *Heliothis virescens*
> (tobacco budworm), *Helicoverpa zea* (cotton bollworm), and
> *Pectinophora gossypiella* (pink bollworm) to EPA within 90 days of
> the date of registration. The monitoring program description must
> include sampling (number of locations and samples per location),
> sampling methodology, bioassay methodology, standardization
> procedures, detection technique and sensitivity, and the statistical
> analysis of the probability of detecting resistance. Collection sites
> must be focused in areas of high adoption of VipCot for tobacco
> budworm, cotton bollworm, and pink bollworm. Syngenta Seeds shall
> provide baseline susceptibility and diagnostic concentration
> determinations for tobacco budworm, cotton bollworm, and pink bollworm
> to Vip3Aa19 and modified Cry1Ab by January 31, 2009.
>
> 2\) The following testing scheme for survivors of the diagnostic or
> discriminating concentrations (or identified survivors of any
> resistance detection method) must be implemented: 1) Determine if the
> observed effect is heritable; 2) Determine if the increased tolerance
> can be observed in the field (i.e., survive on VipCot cotton plants);
> 3) Determine if the effect is due to resistance, 4) Determine the
> nature of resistance (dominant, recessive), 5) Determine the
> resistance allele frequency, 6) Determine, in subsequent years,
> whether the resistance allele frequency is increasing, and 7)
> Determine the geographic extent of the resistance allele (or alleles)
> distribution. Should the resistance allele frequency be increasing and
> spreading, a specific remedial action plan should be designed to
> mitigate the extent of Bt resistance. See section f ("Remedial Action
> Plans") below.
>
> 3\) Syngenta Seeds must also follow up on grower, extension specialist
> or consultant reports of less than expected results or control
> failures (such as increases in damaged squares or bolls) for the
> target lepidopteran pests (*Heliothis virescens* (TBW) and
> *Helicoverpa zea* (CBW), *Pectinophora gossypiella* (PBW)) as well as
> for cabbage looper, soybean looper, saltmarsh caterpillar, black
> cutworm, fall armyworm, southern armyworm, and European corn borer.
> Syngenta Seeds will instruct its customers (growers and seed
> distributors) to contact them (e.g., via a toll‑free customer service
> number) if incidents of unexpected levels of tobacco budworm, cotton
> bollworm, or pink bollworm damage occur. Syngenta Seeds will
> investigate all damage reports. See Remedial Action Plans (section f)
> below.
>
> 4\) Syngenta Seeds must provide to EPA for review and approval any
> revisions to the tobacco budworm, cotton bollworm, and pink bollworm
> resistance monitoring plans prior to their implementation.
>
> 5\) Beginning in 2009, a report on results of resistance monitoring
> and investigations of damage reports must be submitted to the Agency
> annually by September 1st each year for the duration of the
> conditional registration.
>
> [f. Remedial Action Plans]{.underline}
>
> Specific remedial action plans are required for VipCot cotton for the
> purpose of containing resistance and perhaps eliminating resistance if
> it develops. One remedial action plan is for the areas where pink
> bollworm is the predominate pest and the other is for the areas where
> tobacco budworm and cotton bollworm are the predominate pests.
>
> [1) Remedial Action Plan for Pink Bollworm]{.underline}
>
> If resistance involves the pink bollworm (*Pectinophora gossypiella*),
> Syngenta Seeds must implement the Arizona Bt Cotton Working Group's
> Remedial Action Plan. Syngenta Seeds must obtain approval from EPA
> before modifying the Arizona Bt Cotton Working Group's Remedial Action
> Strategy. The Arizona Bt Cotton Working Group's Remedial Action Plan
> can be found in Enclosure 1.
>
> [2) Remedial Action Plan for Tobacco Budworm and Cotton
> Bollworm]{.underline}
>
> If resistance involves the tobacco budworm (*Heliothis virescens*)
> and/or the cotton bollworm (*Helicoverpa zea*), Syngenta Seeds must
> implement a Remedial Action Plan approved by EPA. Once approved,
> Syngenta Seeds must obtain approval from EPA before modifying the
> Remedial Action Plan for tobacco budworm and cotton bollworm. A final
> remedial action plan for tobacco budworm and cotton bollworm must be
> submitted within 90 days of the date of registration. This remedial
> action plan must include definitions of "suspected" and "confirmed"
> resistance and steps to take in the event of confirmed resistance. The
> plan should be based on the steps described in Syngenta Seed's IRM
> submission, including:

-   Notification to the Agency within 30 days of resistance
    confirmation;

-   Notification to affected customers and extension agents about
    confirmed resistance;

-   Encourage affected customers and extension agents to employ
    alternative lepidopteran control measures;

-   Cease sale and distribution of VipCot cotton in affected area;

-   Devise long-term resistance management action plan according to
    characteristics of resistance event and local agronomic needs.

> [g. Annual Reporting]{.underline}
>
> The annual reporting requirements for VipCot are as follows:
>
> 1\) Annual Sales: reported and summed by state (county level data
> available by request), January 31st each year, beginning in 2010;
>
> 2\) Grower Agreements: number of units of Bt corn seeds shipped or
> sold and not returned, and the number of such units that were sold to
> persons who have signed grower agreements, January 31st each year,
> beginning in 2010;
>
> 3\) Grower Education: substantive changes to education program
> completed previous year, January 31st each year, beginning in 2009;
>
> 4\) Compliance Assurance Plan: Compliance Assurance Program activities
> and results, January 31st each year, beginning in 2010;
>
> 5\) Compliance Survey Results: to include annual survey results and
> plans for the next year; full report January 31st each year, beginning
> in 2010;
>
> 6\) Insect Resistance Monitoring Results: results of monitoring and
> investigations of damage reports, September 1 each year, beginning in
> 2009.

**IV. Regulatory Position** forNumber range CHAPTER *Bacillus
thuringiensis* modified Cry1Ab (OECD Unique Identifier SYN-IR67B-1) and
Vip3Aa19 (OECD Unique Identifier SYN-IR102-7) insecticidal proteins and
the genetic material necessary for their production in COT102 X COT67B
cotton (VipCot)

Pursuant to FIFRA section 3(c)(7)(C), EPA may conditionally register a
new pesticide active ingredient for a period of time reasonably
sufficient for the generation and submission of required data that are
lacking because insufficient time has elapsed since the imposition of
the data requirement for those data to be developed. EPA may grant such
conditional registration only if EPA determines that (1) the use of the
pesticide product during the period of the conditional registration will
not cause any unreasonable adverse effect on the environment, and (2)
the registration and use of the pesticide during the conditional
registration is in the public interest. EPA determines that all of these
criteria have been fulfilled.

The first criterion under FIFRA Section 3(c)(7)(C) mentioned above has
been met because insufficient time has elapsed since the imposition of
the data requirements for:

1\) A 7-14 day Daphnia study as per the OPPTS 885.4240 guideline
(Aquatic Invertebrate Testing) on Vip3Aa and modified Cry1Ab.
Alternatively, a dietary study of the effects on an aquatic
invertebrate, representing the functional group of a leaf shredder in
headwater streams, can be performed and submitted in lieu of the 7-14
day Daphnia study.

2\) An independent lab validation of the analytical method for the
detection of Vip3Aa19 and modified Cry1Ab.

3\) Insect resistant management data for Vip3Aa and modified Cry1Ab: a)
development of baseline susceptibility and diagnostic concentrations for
resistance monitoring of the major target pests; b) development of a
compliance assurance program for refuge requirements; c) completion of a
final remedial action plan in the event of pest resistance.

The applicants submitted or cited data sufficient for EPA to determine
that conditional registration of Vip3Aa19 and modified Cry1Ab protein
and the genetic material necessary for their production in COT102 X
COT67B cotton (VipCot) under FIFRA 3(c)(7)(C) will not result in
unreasonable adverse effects to the environment, as discussed above. The
applicants submitted and/or cited satisfactory data pertaining to the
proposed use. The human health effects data and non-target organism
effects data are considered sufficient for the period of the conditional
registration. These data demonstrate that no foreseeable human health
hazards or ecological effects are likely to arise from the use of the
product and that the risk of resistance developing to Vip3Aa19 and/or
modified Cry1Ab proteins during the time of the conditional registration
is not expected to be significant.

Registration of Vip3Aa19 and modified Cry1Ab proteins and the genetic
material necessary for their production in COT102 X COT67B cotton
(VipCot) is in the public interest because:

1.  Efficacy trials have demonstrated effective control of the major
    cotton target pests of Vip3Aa19 and modified Cry1Ab: cotton
    bollworm, tobacco budworm, and pink bollworm.

2.  Vip3Aa19 has a novel mode of action which may benefit insect
    resistance management for this and other cotton PIP products.

3.  If COT102 X COT67B (VipCot) cotton is registered, it will be the
    fourth *Bt* cotton product on the market for control of cotton
    bollworm, tobacco budworm, and pink bollworm. The availability of
    multiple Bt cotton products will increase grower choice and price
    competition, likely resulting in lower seed prices for consumers and
    higher adoption rates.

4.  The registration of VipCot cotton is expected to result in further
    reduction of chemical insecticide use by cotton growers. Lower
    insecticide use should result in benefits for both human health and
    the environment.

In view of these minimal risks and the clear benefits related to
Vip3Aa19 and modified Cry1Ab proteins and the genetic material necessary
for their production in COT102 X COT67B cotton (VipCot), EPA believes
that the use of the product during the limited period of the conditional
registration will not cause any unreasonable adverse effects.

Although the data with respect to this particular new active ingredient
are satisfactory, they are not sufficient to support an unconditional
registration under FIFRA 3(c)(5). Additional data are necessary to
evaluate the risk posed by the continued use of this product.
Consequently, EPA is imposing the data requirements specified earlier in
Section III.

EPA has determined, as explained in section II.E., that the third
criterion for a FIFRA 3(c)(7)(C) conditional registration has been
fulfilled because the use of Vip3Aa19 and modified Cry1Ab proteins and
the genetic material necessary for their production in COT102 X COT67B
cotton (VipCot) under this registration is in the public interest.

The data submitted in support of this registration under section
3(c)(7)(C) of the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) have been reviewed and determined to be adequate. Studies
mentioned above are included in the terms, conditions, and limitations
of this registration. This registration will not cause unreasonable
adverse effects to man or the environment and is in the public interest.

The expiration date of the registration has been set to September 30,
2011.

[^1]: Prior to receiving the Crickmore designation of Vip3Aa19, the
    protein produced in COT102 was referred to as Vip3A or Vip3Aa.

[^2]: Study submitted with EUP request and reviewed in memorandum from
    C. Wozniak to L. Cole dated March 24, 2004.

[^3]: Study submitted with EUP request and reviewed in memorandum from
    A. Waggoner to M. Mendelsohn dated February 8, 2007.

[^4]: Reviewed in a memorandum from S. Matten to A. Reynolds dated April
    4, 2007.

[^5]: Prior to receiving the Crickmore designation of Vip3Aa19, the
    protein produced in Events COT102 and Pacha were referred to as
    VIP3A, Vip3A or Vip3Aa.

[^6]: Non-target invertebrate hazard tests often are conducted at
    exposure concentrations several times higher than the maximum
    concentrations expected to occur under realistic exposure scenarios.
    This has customarily allowed an endpoint of 50% mortality to be used
    as a trigger for additional higher-tier testing. Lower levels of
    mortality under these conditions of extreme exposure suggest that
    population effects are likely to be negligible given realistic
    exposure scenarios. Thus, it follows that the observed proportion of
    responding individuals can be compared to a 50% effect to determine
    if the observed proportion is significantly lower than 50%. For
    example, using a binomial approach, a sample size of 30 individuals
    is sufficient to allow a treatment effect of 30% to be
    differentiated from a 50% effect with 95% confidence using a
    one-sided Z test. A one-sided test is appropriate because only
    effects of less than 50% indicate that further experiments are not
    needed to evaluate risk.

[^7]: OPPTS Testing Guidelines, Series 850 and 885 website:

    <http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/>[885Microbial_Pesticide_Test_Guidelines/Series]{.underline}

[^8]: The dose margin can be less than 10x where uncertainty in the
    system is low or where high concentrations of test material are not
    possible to achieve due to test organism feeding habits or other
    factors. High dose testing also may not be necessary where many
    species are tested or tests are very sensitive, although the test
    concentration used must exceed 1X EEC.

[^9]: It is notable that that the 10 X EEC MHD testing approach is not
    equivalent to what is commonly known as "testing at a 10X SAFETY
    FACTOR" where any adverse effect is considered significant. Tier I
    screen testing is not 'safety factor testing'. In a "10X safety
    factor" test any adverse effect noted is a "level of concern",
    whereas in the EPA environmental risk assessment scenario any
    adverse effect is viewed as a concern only at 1X the field exposure.

[^10]: The 1X EEC test dose is based on plant tissue content and is
    considered a high worst case dose (sometimes referred to as HEEC).
    This 1X EEC is still much greater than any amount which any given
    non-target organism may be ingesting in the field because most
    non-target organisms do not ingest plant tissue.

[^11]: The established peer and EPA Science Board reviewed guidance on
    screening test levels of concern is 50% mortality at 5X
    environmental concentration. The appropriate endpoints in high dose
    limit/screening testing are based on mortality of the treated, as
    compared to the untreated (control) non-target organisms. A single
    group of 30 test animals may be tested at the maximum hazard dose.

[^12]: This research was funded by Environmental Protection Agency grant
    CR-832147-01. The *Bt* crop non-target effects database can be found
    on the National Center for Ecological Analysis and Synthesis
    (NCEAS). Website. (<http://delphi.nceas.ucsb.edu/btcrops/>).

[^13]: Model hypothesis: if a certain amount of protein A alone kills x%
    of a sample, and a certain amount of protein B kills y%, the
    predicted percentage kill of a mixture of these amounts of protein
    is given by x + y -- (xy/100).

[^14]: Bridging of data between the variants of Vip3Aa as well as the
    Cry1Ab proteins was addressed in the Agency' reviews of the VipCot™
    Experimental Use Permit (see memoranda: Matten, 2006; Milofsky and
    Vaituzis, 2007b).

[^15]: All data tables contain results submitted by Syngenta (Public
    Interest Document - MRID 470347-01; Efficacy study - MRID
    470176-33).