Document ID: EPA-HQ-OPP-2007-0306-0044
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2008-10-29T04:00Z

SEQ CHAPTER \h \r 1 		

			

BIOPESTICIDES REGISTRATION ACTION DOCUMENT

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

	U.S. Environmental Protection Agency

	Office of Pesticide Programs

	Biopesticides and Pollution Prevention Division

						Table of Contents		

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

	A.  Executive Summary

	

	B.  Use Profile

	C.  Regulatory History

	  

	

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

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

        

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

											

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

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

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

		

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

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

Regulatory Action Team

Product Characterization and Human Health

John Kough, Ph.D.

Annabel Waggoner, B.S.

Rebbeca Edelstein, Ph.D.

Sharlene Matten, Ph.D.

Chris Wozniak, Ph.D.

Environmental Fate and Effects

Zigfridas Vaituzis, Ph.D.

Annabel Waggoner, B.S.

Tessa Milofsky, M.S.

Mika Hunter, B.S.

Insect Resistance Management

Jeannette Martinez, M.S.

Benefit Assessment

Jeannette Martinez, M.S.

Biopesticides Registration Action Document Team Leader

Alan Reynolds, M.S.

				

Office of General Council

Angela Huskey, Esq.

Chris Kaczmarek, Esq.

Keith Matthews, Esq.I.  Overview					

			

A.  Executive Summary

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

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

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

Benefits

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

Public Interest Finding

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

Product Characterization

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

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

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

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

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

Human Health Assessment

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

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

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

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

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

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

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

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

Environmental Assessment

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

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

Insect Resistance Management

 

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

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

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

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

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

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

				

B.  Use Profile

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

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

											

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

Basic Manufacturers:  Syngenta Seeds, Inc.

Type of Pesticide:  Plant-Incorporated Protectant

Uses:  Cotton

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

C.  Regulatory History

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

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

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

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

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

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

II. Science Assessment	

The classifications that are found for each data submission are assigned
by EPA science reviewers and are an indication of the usefulness of the
information contained in the documents for risk assessment.  A rating of
“ACCEPTABLE” indicates the study is scientifically sound and is
useful for risk assessment. A “SUPPLEMENTAL” rating indicates the
data provide some information that can be useful for risk assessment.
The studies may have certain aspects determined not to be scientifically
acceptable (“SUPPLEMENTAL: UPGRADABLE”).  If a study is rated as
“SUPPLEMENTAL: UPGRADABLE,” EPA always provides an indication of
what is lacking or what can be provided to change the rating to
“ACCEPTABLE.”  If there is simply a “SUPPLEMENTAL” rating, the
reviewer will often state that the study is not required by the current
40 CFR Part 158.  Both “ACCEPTABLE” and “SUPPLEMENTAL” studies
may be used in the risk assessment process as appropriate.  An
“UNACCEPTABLE” rating indicates that new data need to be submitted.

	II.A.  Product Characterization

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

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

Transformation System: 

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

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

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

Protein Characterization: 

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

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

Analytical Detection Methods:

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

Protein Expresson: 

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

 

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

Tissue Type	Vip3Aa19

(µg/g dry weight + standard deviation)	APH4

(µg/g dry weight + standard deviation)

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

Squares	116 + 22	2.2 + 0.4

Flowers	162	1.68

Pollen	3.47	64.3

Bolls	19 + 4	< 0.39

Whole Plants	25 + 4	< 0.37

Seed	7 + 2	1.4 + 0.3

Roots	16 + 2	0.53 + 0.11

*Ranges reflect means at different growth stages for leaves

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

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

Study Type/Title	

Summary	

MRID #

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

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

Classification:  ACCEPTABLE	

45835801

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

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

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

Superseded by MRID 47017603	

45835802

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

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

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

45766501

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

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

Classification:  ACCEPTABLE	

45835812

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

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

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

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

45835803

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

Classification: ACCEPTABLE  

	46880801

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

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

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

Classification: ACCEPTABLE	

47017602

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

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

Classification: ACCEPTABLE	

47017603

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

Classification: ACCEPTABLE	

47017609

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

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

 

Transformation System: 

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

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

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

Protein Characterization: 

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

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

Analytical Detection Methods:

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

Protein Expresson: 

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

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

Tissue Type	Cry1Ab

(µg/g dry weight + standard deviation)*

Leaves	65 + 9 – 158 + 40

Squares	93 + 13

Flowers	101

Pollen	12.1

Bolls	47 + 7

Whole Plants	26 + 2

Seed	29 + 5

Roots	17 + 1

*Range reflects means at different growth stages for leaves

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

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

Study Type/Title	

Summary	

MRID #

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

Report No. SSB-125-06.	

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

Classification:  ACCEPTABLE	

46885901

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

Report No.SSB-022-06d	

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

 

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

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

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

Superseded by MRID 47017607.	

46885902

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

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

Classification: ACCEPTABLE	

47017604

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

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

Classification: ACCEPTABLE	

47017605

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

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

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

Classification: ACCEPTABLE	

47017608

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

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

Classification: ACCEPTABLE	

47017610

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

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

Classification: ACCEPTABLE	

47017611

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

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

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

Analytical Detection Methods:

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

Protein Expresson: 

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

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

Tissue Type	Vip3Aa19

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

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

(µg/g dry weight + standard deviation)*

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

Squares	132 + 18	2.1 + 0.5	94 + 10

Flowers	148	1.80	121

Pollen	3.06	74.7	10.7

Bolls	21 + 4	< 0.43	42 + 7

Whole Plants	25 + 7	< 0.40	29 + 7

Seed	7 + 1	1.6 + 0.4	27 + 4

Roots	11 + 3	0.46 + 0.05	20 + 4

*Ranges reflect means at different growth stages for leaves

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

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

Study Type/Title	

Summary	

MRID #

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

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

Classification:  ACCEPTABLE	

47017606

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

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

Classification: ACCEPTABLE	

47017607

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

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

Classification: ACCEPTABLE	

47074101



II.B.  Human Health Assessment

II.B.1.  Human Health Assessment of Vip3Aa

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

A.  Mammalian Toxicity and Allergenicity Assessment

Consistent with section 408(b) (2) (D) of the FFDCA, EPA has reviewed
the available scientific data and other relevant information in support
of this action and considered its validity, completeness and reliability
and the relationship of this information to human risk. EPA has also
considered available information concerning the variability of the
sensitivities of major identifiable subgroups of consumers, including
infants and children. 

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

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

When proteins are toxic, they are known to act via acute mechanisms and
at very low dose levels (Sjoblad et al., 1992). Therefore, since no
acute effects were shown to be caused by the Vip3Aa19 and Vip3Aa20
proteins, even at relatively high dose levels, they are not considered
toxic.  (This is also true of the Vip3Aa1 protein that was tested.) 
Further, amino acid sequence comparisons showed no similarities between
Vip3Aa19 and Vip3Aa20, on the one hand, and known toxic proteins in
protein databases, on the other hand, that would raise a safety concern.

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

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

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

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

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

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

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

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

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

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

B.  Aggregate Exposures

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

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

C.  Cumulative Effects 

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

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

1) Toxicity and Allergenicity Conclusions 

The data submitted and cited regarding potential health effects for
Vip3Aa proteins includes the characterization of representative Vip3Aa
proteins, as well as the acute oral toxicity studies, amino acid
sequence comparisons to known allergens and toxins, and in vitro
digestibility of the representative Vip3Aa proteins. The results of
these studies were used to evaluate human risk, and the validity,
completeness, and reliability of the available data from the studies
were also considered.

 

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

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

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

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

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

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

 	

2) Infants and Children Risk Conclusions 

FFDCA section 408(b)(2)(C) provides that EPA shall assess the available
information about consumption patterns among infants and children,
special susceptibility of infants and children to pesticide chemical
residues and the cumulative effects on infants and children of the
residues and other substances with a common mechanism of toxicity. In
addition, FFDCA section 408(b)(2)(C) also provides that EPA shall apply
an additional tenfold margin of safety for infants and children in the
case of threshold effects to account for prenatal and postnatal toxicity
and the completeness of the database unless EPA determines that a
different margin of safety will be safe for infants and children. 

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

 

3) Overall Safety Conclusion 

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

E.  Other Considerations 

1) Endocrine Disruptors 

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

2) Analytical Method(s) 

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

3) Codex Maximum Residue Level 

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

F.  Tolerance Exemptions

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

G.  Supporting Data

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

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

Study Type/Title	

Summary	

MRID #

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

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

Classification: SUPPLEMENTAL.

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

45766502

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

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

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

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

45766503

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

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

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

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

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

Classification: Acceptable	

45766505

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

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

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

	45766506

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

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

Classification: ACCEPTABLE

	

45835805

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

Report No. SSB-122-064	

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

Classification:  Acceptable; Supersedes MRID 457665-02

	

46885903

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

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

Classification: Acceptable; Supersedes MRID 457665-02

	46885906

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

Classification: ACCEPTABLE

	46864808

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

Classification: ACCEPTABLE

	46864809

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

Classification: ACCEPTABLE 	47017613

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

Classification: ACCEPTABLE	47017617

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

A. Mammalian Toxicity and Allergenicity Assessment

Consistent with section 408(b)(2)(D) of the FFDCA, EPA has reviewed the
available scientific data and other relevant information in support of
this action and considered its validity, completeness and reliability
and the relationship of this information to human risk. EPA has also
considered available information concerning the variability of the
sensitivities of major identifiable subgroups of consumers, including
infants and children.

Syngenta has submitted acute oral toxicity data demonstrating the lack
of mammalian toxicity at high levels of exposure to the pure modified
Cry1Ab protein containing the additional 26 amino acid ‘Geiser
motif’.   The 26 amino acid sequence is found at the C-terminus of the
pro-toxin portion of the modified Cry1Ab protein.  The pro-toxin is
enzymatically cleaved in the insect gut to produce active Cry1Ab.  These
toxicity data demonstrate the safety of the product at a level well
above maximum possible exposure levels that are reasonably anticipated
in the crop.  Basing this conclusion on acute oral toxicity data without
requiring further toxicity testing and residue data is similar to the
Agency position regarding toxicity testing and the requirement of
residue data for the microbial Bacillus thuringiensis products from
which this plant-incorporated protectant was derived (See 40 CFR
158.2140). For microbial products, further toxicity testing (Tiers II &
III) and residue data are triggered by significant adverse acute effects
in studies such as the acute oral toxicity study, to verify the observed
adverse effects and clarify the source of these effects. 

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

When proteins are toxic, they are known to act via acute mechanisms and
at very low dose levels (Sjoblad et al., 1992). Therefore, since no
acute effects were shown to be caused by modified Cry1Ab, even at
relatively high dose levels, the modified Cry1Ab protein is not
considered toxic.    

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

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

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

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

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

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

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

B. Aggregate Exposures 

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

The Agency has considered available information on the aggregate
exposure levels of consumers (and major identifiable subgroups of
consumers) to the pesticide chemical residue and to other related
substances. These considerations include dietary exposure under the
tolerance exemption and all other tolerances or exemptions in effect for
the plant-incorporated protectants chemical residue, and exposure from
non-occupational sources. Exposure via the skin or inhalation is not
likely since the plant-incorporated protectant is contained within plant
cells, which essentially eliminates these exposure routes or reduces
these exposure routes to negligible. In addition, even if exposure can
occur through inhalation, the potential for modified Cry1Ab to be an
allergen is low, as discussed above.  Although the allergenicity
assessment focuses on potential to be a food allergen, the data also
indicate a low potential for modified Cry1Ab to be an inhalation
allergen.  Exposure via residential or lawn use to infants and children
is also not expected because the use sites for the modified Cry1Ab
protein is agricultural.  Dietary exposure may occur from ingestion of
processed cotton products but is expected to be very low because the
already low expression levels in the seed and would be reduced further
by the heat and pressure used for processing.  Also, dietary exposure
may theoretically occur through exposure in drinking water because plant
stubble may release modified Cry1Ab protein into ground water upon
decay.  This protein would not be expected to survive in the soil due to
microbial degradation, adherence to soil components and removal upon
drinking water treatment procedures.  In addition, oral toxicity testing
showed no adverse effects. 

C. Cumulative Effects 

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

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

1) Toxicity and Allergenicity Conclusions

The data submitted and cited regarding potential health effects for the
modified Cry1Ab protein includes the characterization of the expressed
modified Cry1Ab protein in cotton, as well as the acute oral toxicity
study, amino acid sequence comparisons to known allergens, and in vitro
digestibility of the protein. The results of these studies were used to
evaluate human risk, and the validity, completeness, and reliability of
the available data from the studies were also considered.

 

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

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

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

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

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

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

 	

2) Infants and Children Risk Conclusions 

FFDCA section 408(b)(2)(C) provides that EPA shall assess the available
information about consumption patterns among infants and children,
special susceptibility of infants and children to pesticide chemical
residues and the cumulative effects on infants and children of the
residues and other substances with a common mechanism of toxicity. In
addition, FFDCA section 408(b)(2)(C) also provides that EPA shall apply
an additional tenfold margin of safety for infants and children in the
case of threshold effects to account for prenatal and postnatal toxicity
and the completeness of the database unless EPA determines that a
different margin of safety will be safe for infants and children. 

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

 

3) Overall Safety Conclusion 

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

E. Other Considerations 

1) Endocrine Disruptors 

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

2) Analytical Method(s) 

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

3) Codex Maximum Residue Level 

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

F.  Tolerance Exemptions

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

G.  Supporting Data

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

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

Study Type/Title	

Summary	

MRID #

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

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

Classification: ACCEPTABLE	

47017614

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

Classification: ACCEPTABLE	47017615

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

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

Classification: ACCEPTABLE	

47017616

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

Classification: ACCEPTABLE 	47017619

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

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

Summary of new data submitted for APH4

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

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

Classification: ACCEPTABLE

II.B.4.  References

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

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

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

Food and Agriculture Organization of the United Nations and World Health

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

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

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

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

protein components of biological pesticide products.  Regulatory
Toxicol. 

Pharmacol. 15:  3-9.

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

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

II. C.	Environmental Hazard Assessment 

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

Background			

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

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

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

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

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

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

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

A.  Tiered Testing and Risk Assessment Process 

To minimize data requirements and avoid unnecessary tests, risk
assessments are structured such that risk is determined first from
estimates of hazard under “worst-case” exposure conditions.  A lack
of adverse effects under these conditions would provide enough
confidence that there is no risk and no further data would be needed. 
Hence, such screening tests conducted early in an investigation tend to
be broad in scope but relatively simple in design, and can be used to
demonstrate acceptable risk under most conceivable conditions.  When
screening studies suggest potentially unacceptable risk additional
studies are designed to assess risk under more realistic field exposure
conditions.  These later tests are more complex than earlier screening
studies. Use of this “tiered” testing framework saves valuable time
and resources by organizing the studies in a cohesive and coherent
manner and eliminating unnecessary lines of investigation.  Lower tier,
high dose screening studies also allow tighter control over experimental
variables and exposure conditions, resulting in a greater ability to
produce statistically reliable results at relatively low cost.  

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

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

Testing methods which utilize the tiered approach were last published by
the EPA as Harmonized OPPTS Testing Guidelines, Series 850 and 885 (EPA
712-C-96-280, February 1996). These guidelines, as defined in 40 CFR
152.20, apply to microbes and microbial toxins when used as pesticides,
including those that are naturally occurring, and those that are
strain-improved, either by natural selection or by deliberate genetic
manipulation.  Therefore, PIPs containing microbial toxins are also
covered by these testing guidelines. 

The Tier I screening maximum hazard dose (MHD) approach to environmental
hazard assessment is based on some factor (whenever possible >10) times
the maximum amount of active ingredient expected to be available to
terrestrial and aquatic non-target organisms in the environment (EEC).
Tier I tests serve to identify potential hazards and are conducted in
the laboratory at high dose levels which increase the statistical power
to test the hypotheses.  Elevated test doses, therefore, add certainty
to the assessment, and such tests can be well standardized. The
Guidelines call for initial screening testing of a single group or
several groups of test animals at the maximum hazard dose level. The
Guidelines call for testing of one treatment group of at least 30
animals or three groups of 10 test animals at the screening test
concentration. The Guidelines further state that the duration of all
Tier I tests should be approximately 30 days. Some test species, notably
non-target insects, may be difficult to culture and the suggested test
duration has been adjusted accordingly. Control and treated insects
should be observed for at least 30 days, or in cases where an insect
species cannot be cultured for 30 days, until negative control mortality
rises above 20 percent. 

Failing the Tier I (10 X EEC) screening at the MHD dose does not
necessarily indicate the presence of an unacceptable risk in the field
but it triggers the need for additional testing. A less than 50%
mortality effect at the MHD is taken to indicate minimal risk.  However,
greater than 50% mortality does not necessarily indicate the existence
of unacceptable risk in the field, but it does trigger the need to
collect additional dose-response information and a refinement of the
exposure estimation before deciding if the risk is acceptable or
unacceptable. Where potential hazards are detected in Tier I testing
(i.e. mortality is greater than  50%), additional information at lower
test doses is required which can serve to confirm whether any effect
might still be detected at more realistic field [1X EEC] concentrations
and routes of exposure.  

When screening tests indicate a need for additional data, the OPPTS
Harmonized Guidelines call for testing at incrementally lower doses in
order to establish a definitive LD50 and to quantify the hazard.  In the
definitive testing, the number of doses and test organisms evaluated
must be sufficient to determine an LD50 value and, when necessary, the
Lowest Observed Effect Concentration (LOEC), No Observed Adverse Effect
Level (NOAEL) , or reproductive and behavioral effects such as feeding
inhibition,  weight loss, etc.  In the final analysis, a risk assessment
is made by comparing the LOAEC to the EEC; when the EEC is lower than
the LOAEC, a no risk conclusion is made. These tests offer greater
environmental realism, but they may have lower statistical power.
Appropriate statistical methods, and appropriate statistical power, must
be employed to evaluate the data from the definitive tests. Higher
levels of replication, the number of test species, and/or repetition are
needed to enhance statistical power in these circumstances. 

Data that shows less than 50 % mortality at the maximum hazard dosage
level – (i.e. LC50, ED50, or LD50 >10 X EEC) is sufficient to evaluate
adverse effects, making lower field exposure dose definitive testing
unnecessary.   It is also notable that the recommended >10X EEC maximum
hazard dose level is a highly conservative factor.  The published EPA
Level of Concern [LOC] is 50% mortality at 5X EEC  (US EPA, 1998).  

Validation:  The tiered hazard assessment approach was developed for the
EPA by the American Institute of Biological Sciences (AIBS) and
confirmed in 1996 as an acceptable method of environmental hazard
assessment by a FIFRA Scientific Advisory Panel (SAP) on microbial
pesticides and microbial toxins. The December 9, 1999 SAP agreed that
the Tiered approach was suitable for use with Plant-Incorporated
Protectants (PIPs); however, this panel recommended that, for PIPs with
insecticidal properties, additional testing of beneficial invertebrates
closely related to target species and/or likely to be present in GM crop
fields should be conducted. Testing of Bt Cry proteins on species not
closely related to the target insect pest was not recommended, although
it is still performed to fulfill the published EPA non-target species
data requirements.  In October 2000, another SAP also recommended that
field testing should be used to evaluate population-level effects on
non-target organisms. The August 2002 SAP, and some public comments,
generally agreed with this approach, with the additional recommendation
that indicator organisms should be selected on the basis of potential
for field exposure to the subject protein (US EPA, 2000, 2001a, 2002,
and 2004). 

Chronic studies: Since delayed adverse effects and/or accumulation of
toxins through the food chain are not expected to result from exposure
to proteins, protein toxins are not routinely tested for chronic effects
on non-target organisms.  However, the 30 day test duration requirement
does amount to subchronic testing when performed at field exposure test
doses. Proteins do not bioaccumulate. The biological nature of proteins
makes them readily susceptible to metabolic, microbial, and abiotic
degradation once they are ingested or excreted into the environment. 
Although there are reports that some proteins (Cry proteins) bind to
soil particles, it has also been shown that these proteins are degraded
rapidly by soil microbial flora upon elution from soil particles.  

Conclusion:  The tiered approach to test guidelines ensures, to the
greatest extent possible, that the Agency requires the minimum amount of
data needed to make scientifically sound regulatory decisions. The EPA
believes that maximum hazard dose Tier I screening testing presents a
reasonable approach for evaluating hazards related to the use of
biological pesticides and for identifying negative results with a high
degree of confidence. The Agency expects that Tier 1 testing for
short-term hazard assessment will be sufficient for most studies
submitted in support of PIP registrations. However, if long range
adverse effects must be ascertained, then higher-tier longer-term field
testing will be required  As noted above, the October 2000 SAP and the
National Academy of Sciences (NAS, 2000) recommended testing non-target
organisms directly in the field. This approach, with an emphasis on
testing invertebrates found in corn fields, was also recommended by the
August 2002 SAP and was supported by several public comments. Based on
these recommendations, the Agency has required field studies on long
term invertebrate population/community and Cry protein accumulation in
soils as a condition of registration due to the lack of baseline data on
the potential for long-term environmental effects from the cultivation
of PIP-producing plants.

Since the commercialization of Bt crops, the number of field studies
published in scientific literature in combination with the
post-registration field studies submitted to the Agency has accumulated
to a level where empirical conclusions can be made.  As a result, the
issue of long range effects of cultivation of these Cry proteins on the
invertebrate community structure in Bt crop fields has since been
adequately addressed.  Specifically, a meta-analysis of the data
collected from 42 field studies indicated that non-target invertebrates
are generally more abundant in Bt cotton and Bt maize fields than in
non-transgenic fields managed with insecticides (Marvier, et al., 2007).
 In addition, a comprehensive review of short and long term field
studies on the effects of invertebrate populations in Bt corn and cotton
fields indicated that no unreasonable adverse effects are taking place
as a result of wide scale Bt crop cultivation (Sanvido, et al. 2007). 
Another review of field tests published to date concluded that the
large-scale studies in commercial Bt cotton have not revealed any
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.   SEQ CHAPTER \h \r 1 Despite 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 Requirement 	OPPTS

Guideline	Test Substance	Results Summary and Classification	MRID No. 

Avian dietary testing, 

broiler chicken, Gallus domesticus 

	885.4050	Vip3Aa19 maize grain

 (Event Pacha)  	A 49-day dietary study showed no adverse affects to
broiler chickens when fed a 50% diet composed of Event Pacha maize grain
(containing VIP3A).  Therefore, the NOEC was 0.588 µg VIP3A/g corn feed
and the LC50 was > 0.588 µg VIP3A/g feed corn grain.

Classification:  Acceptable	470176-23

Avian injection testing	885.4100

	N/A	Acceptable waiver rationale	N/A

Avian oral testing, bobwhite quail,

Colinus virginianus	850.2100	Microbial Vip3Aa1 (VIP3A-0198)	A 14-day
study showed no adverse effects to bobwhite quail from VIP3A-0198, after
a single oral dose via gavage.  The NOEL was 400 mg VIP3A/kg and the
LD50 was > 400 mg VIP3A/kg bird body weight.

Classification:  Acceptable	457665-08

Wild mammal testing	885.4150

	N/A	Acceptable bridging rationale to acute oral  toxicity test on mice
(MRID No. 457665-05).	N/A

Freshwater fish testing, 

channel catfish, Ictalurus punctatus	885.4200

	Vip3Aa19 maize grain (FFPACHA-0100)	A 30-day study showed no adverse
effects on juvenile catfish after exposure to Vip3Aa protein from Event
Pacha corn grain.  Therefore, the NOEC was 7.10 µg Vip3Aa19/g fish feed
and the LC50 was > 7.10 µg Vip3Aa19/g

Classification:  Acceptable	470176-24

Freshwater aquatic invertebrate testing, 

water flea, Daphnia  magna 	885.4240

 	Vip3Aa19 maize pollen 

(PHOPACHA-0199)	In a 48-hour static renewal limit bioassay, VIP3A maize
pollen (containing 83.8 µg VIP3A protein/g) had no adverse effects on
the survival of Daphnia magna, when suspended in 120 mg pollen/L.  The
LC50 was > 83.8 µg VIP3A protein/g.

Classification:  Unacceptable. The 885 Series Guidelines call for a 21
day study. The submitted 48 hour acute study is inadequate.	457921-01

Estuarine and marine animal testing 	885.4280

	N/A	Acceptable waiver rationale	N/A

Non-target plant testing	885.4300

	N/A	Acceptable waiver rationale	N/A

Non-target insect testing, minute pirate/insidious flower  bug , Orius
insidiosus	885.4340

	Microbial Vip3Aa19

(VIP3A-0104)	Orius insidiosus nymphs fed a meat-based diet containing
microbial-derived 7.25 mg Vip3Aa19 protein/ g diet showed no adverse
effects after 21 days.  The NOEC was 7.25 mg Vip3Aa19 protein/ g and the
LC50  was > 7.25 mg Vip3Aa19 protein/ g.

Classification:  Acceptable	468648-14

Non-target insect testing, pink-spotted lady beetle, Coleomegilla
maculata 	885.4340

	Vip3Aa19 maize pollen (PHOPACHA-0100)	Coleomegilla maculata adults were
fed a diet containing 5.0% VIP3A maize pollen (containing 144.8 µg
VIP3A protein/g pollen) for 21 days with no adverse effects observed. 
The NOEC was 7.24 µg VIP3A protein/g pollen and the LC50  was > 7.24
µg/g VIP3A/g pollen.

Classification:  Acceptable	457665-09

Non-target insect testing, seven-spotted ladybird beetle, Coccinella
septempunctata 	885.4340

	Microbial Vip3Aa19 (VIP3A-0204)	C. septempunctata adults fed a 50%
sucrose diet containing 7250 µg Vip3Aa19 protein/g diet for showed no
adverse effects after 15 days.  The NOEC was 7250 µg Vip3Aa19 protein/g
diet and the LC50  was > 7250 µg/g Vip3Aa19 protein/g diet.

Classification:  Acceptable	468848-02

Non-target insect testing, green lacewing,  Chyrsoperla carnea	885.4340
Microbial Vip3Aa19 (VIP3A-0104)	Chyrsoperla carnea larvae fed a
meat-based diet containing 7250 µg Vip3Aa19 protein/g diet showed no
adverse effects.  The NOEC of 7250 µg Vip3Aa19 protein/g diet and the
LC50 was > 7250 µg Vip3Aa19 protein/g diet 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	468848-15

Non-target insect testing, collembolan, Folsomia candida	885.4340

	Vip3Aa19 maize leaves 

(LLPACHA-0100)	Collembola were fed a diet containing 50% yeast and 50%
leaf tissue for 28 days.  No statistically significant effects on
survival or reproduction were found among the test and negative control
groups.  The NOEC was 43.2 µg Vip3Aa19 protein/g diet and the LC50 was 
 > 43.2 µg Vip3Aa19 protein/g diet.

Classification:  Acceptable	458358-10

Honeybee testing, Honeybee larvae,

Apis mellifera	885.4380

	Vip3Aa19 maize pollen 

(PHOPACHA-0199)	Three-to-five day old honeybee larvae were administered
a single dose of ca.2 mg of pollen moistened with a drop of 30% sucrose
solution (containing the equivalent 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 LD50  was  > 83.8 µg Vip3Aa19 protein/g diet. 

Classification:  Acceptable	458358-09

Earthworm toxicity, 

Eisenia foetida	OECD Guideline 207, 850.6200	Vip3Aa19 maize leaves

(LPPACHA-0199)	Adult earthworms were exposed to artificial soil
containing 3.60 µg VIP3A protein/g soil for 14 days.  No mortality or
differences in body weights were observed in the test group.  The NOEC
was 3.60 µg VIP3A protein/g soil and the LC50  > 3.60 µg VIP3A
protein/g soil.

Classification:  Acceptable	457921-02

Soil fate and degradation	885.5200	Vip3Aa19 maize leaves 

(LPPACHA-0199)	Results of this degradation study indicate that the DT50
of 16 mg/g concentration of the Vip3Aa19 test material 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	470176-30

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 Requirement 	OPPTS

Guideline	Test Substance	Results Summary and Classification	MRID No. 

Avian dietary testing, 

broiler chicken, Gallus domesticus 	885.4050	Bt11 maize grain	A 42-day
dietary study showed no deleterious effects on broiler chicken survival
or carcass yield when fed a 50% diet composed of Bt11 maize grain
(containing Cry1Ab). 

Classification:   Acceptable	4565251-01

Avian injection testing	885.4100

	N/A	Acceptable waiver rationale	N/A

Avian oral testing, bobwhite quail,

Colinus virginianus	850.2100	Bt176 Maize leaf protein  

(LP176-0194)	A 14-day study on bobwhite quail showed no adverse effects
after a single oral dose of Bt176 grain, containing Cry1Ab.  The NOEL
was 140 mg Cry1Ab/kg bodyweight and the LD50 was  > 140 mg Cry1Ab/kg
bodyweight.

Classification:   Acceptable	433236-09

Wild mammal testing	885.4150

	N/A	Acceptable bridging rationale to acute oral toxicity test on mice
(MRID No. 47017614)	N/A

Freshwater fish testing, 

channel catfish, Ictalurus punctatus 	885.4200

	Microbial FLCry1Ab

(FLCRY1AB-0103) 	A 30-day study showed no adverse effects to juvenile
channel catfish.  The NOAEC was 7.10 µg  FLCry1Ab/g fish feed and  the
LC50  was > 7.10 µg  FLCry1Ab/g fish feed.

Classification:   Acceptable 	470176-25

Freshwater aquatic invertebrate, water flea neonate, Daphnia magna 
885.4240

 	Bt176 maize pollen (PHO176-0194)	In a 48-hour static renewal limit
bioassay, Event 176 maize pollen containing 12.36 µg Cry1Ab protein/g
had no adverse effects on the survival of Daphnia  magna, when suspended
in 150 mg pollen/L.  The LC50  was >  12.36 µg Cry1Ab protein/L.

Classification:   Unacceptable. The 885 Series Guidelines call for a 21
day study. The submitted 48 hour acute study is inadequate	433236-10 

Estuarine and marine animal  testing	885.4280

	N/A	Acceptable waiver rationale	N/A

Non-target plant testing 	885.4300

	N/A	Acceptable waiver rationale	N/A

Non-target insect testing, predatory bug, Orius laevigatus	885.4340

	Microbial FLCry1Ab

(FLCRY1AB-0103) and 

Microbial Vip3Aa19 

(VIP3A-0204)	Orius laevigatus had no adverse effects after fed
meat-based artificial diets, containing either 1.0039 mg  FLCry1Ab/g
diet or 1.0039 mg  FLCry1Ab + 0.1950 mg Vip3Aa19/g diet  for 14 days.
Only the results from the first study were valid.  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 and Event COT102 x COT67B
cotton leaves, respectively.  Furthermore, the LC50 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)
	470176-28

Non-target insect testing, pink-spotted lady beetle, Coleomegilla
maculata 	885.4340

	Microbial FLCry1Ab

(FLCRY1AB-0103) and 

Microbial Vip3Aa19 

(VIP3A-0204)	Coleomegilla maculata larvae were fed prepared diets
containing bee pollen, Ephestia eggs, and either FLCRY1AB-0103
(containing 1000 µg FLCry1Ab protein/g diet) or FLCRY1AB-0103 and
VIP3A-0204 (containing 1000 µg FLCry1Ab and 250 µg Vip3Aa protein/g
diet) for 21 days.  No adverse effects were observed on larval, pupal,
or adult survival from either test material diet.  The NOAEC for
FLCry1Ab was 1000 µg FLCry1Ab/g diet and the LC50 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 LC50 was greater than 1000 µg FLCry1Ab/g diet + 250 µg Vip3Aa19/g
diet.

Classification:  Acceptable	470176-26

Non-target insect testing, rove beetle, Aleochara bilineata 	885.4340

	Microbial FLCry1Ab

(FLCRY1AB-0103)	A. bilineata adults were fed a meat diet containing
1298.7 g FLCry1Ab protein/g diet for 35 days with a LC50  >  1298.7 g
FLCry1Ab /g.  Reproductive effects were also assessed by counting the
number of second-generation adult beetles emerging from parasitized
pupae of the onion fly (Delia antique).  There were no differences noted
between the treatment and negative control groups.

Classification:  Acceptable	470176-27

Non-target insect testing, collembolan, Folsomia candida	885.4340

	Lyophilized Bt11 maize leaf 

(LLBt11-0100)	Collembola were fed a diet containing 50% yeast and 50%
Bt11 leaf tissue for 28 days.  No statistically significant effects on
survival or reproduction were found among the test and negative control
groups.  The NOEC for the survival and reproduction of F. candida was
17.1 µg Cry1Ab protein/g diet and the LC50  was  > 17.1 µg Cry1Ab
protein/g diet.

Classification:  Acceptable	458358-10

Honeybee  testing,  

Apis mellifera larvae, adults, and whole hive conditions 	885.4380

	Microbial FLCry1Ab

(FLCRY1AB-0103)	Honeybees were exposed via oral ingestion to
microbial-derived FLCry1Ab test material in a sucrose solution, 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 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 LD50 was greater than 92.4 µg FLCry1Ab/mL.

Classification:  Acceptable-for the purposes of the environmental risk
assessment	470176-29

Soil fate and degradation	885.5200	Microbial FLCry1Ab

(FLCRY1AB-0103)	The degradation of FLCry1Ab protein (incorporated at a
rate equivalent to 80 µg FLCry1Ab/g dry wt soil) in a 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	470176-31

3-year Soil Degradation 	885.5200	Soil from Bt11 corn cultivated fields
Soil samples were collected from five fields, representing four
different soil types, in five 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	460224-01

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.

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 LC50  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 LC50 for broilers is greater than 0.588
µg VIP3A /g feed. Based on the information presented, this study is
acceptable.

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 LD50 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 LD50 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.

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 LC50 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 LC50 was greater than 7.1 µg Vip3Aa19/g diet.   Based on the
information presented, this study is acceptable.

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 LC50 was greater than 120 mg VIP3A pollen/L.

Conclusions/Recommendations: Results of the 48-hour limit test showed
the LC50 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 (-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.  

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 LC50 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 LC50
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 LC50 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 LC50 was > 7.25 mg Vip3Aa19
protein/g diet.  This study was previously reviewed and found acceptable
(Milofsky and Vaituzis, 2007).

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 LC50 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 LC50 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 LC50 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
LC50 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 LC50 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 LC50 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 LC50 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 LC50 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 LC50 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 LC50 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.

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).

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 LD50 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 LD50 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 LD50 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 LD50 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 LC50 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 LC50 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.  

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 LC50 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 LC50 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 LC50 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 LC50 was greater than
1000 µg FLCry1Ab/g diet + 250 µg Vip3Aa19/g diet.  Based on the
information presented, this study is acceptable.

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 LC50 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 LC50 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 Bt crops on insect predators showing no significant differences in
the density of beneficial insects, including  Orius insidiosus (Pilcher
et al., 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 LC50 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 LC50 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 LC50 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 LD50 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 LD50 was greater than 92.4 µg
FLCry1Ab/mL.Therefore, the NOEL was 92.4 µg FLCry1Ab/mL and the LD50
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 DT50 or degradation time to Cry
proteins and these proteins are both Bt toxins.

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
al. 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 (-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 

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 (-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 (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 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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Bacillus thuringiensis on 

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Colby, S.R. (1967). Calculating synergistic and antagonistic responses
of herbicide 

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DeMaagd, et al. (2003) Structure, diversity and evolution of protein
toxins from spore-forming entomopathogenic bacteria.  Annual Review of
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Dively, G.P. (2005).  Impact of transgenic VIP3A x Cry1Ab
lepidopteran-resistant field corn on the nontarget arthropod community. 
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Duan J.J., Marvier M., Huesing J., Dively G., Huang Z.Y. (2008). A
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e1415.doi:10.1371/journal.pone.0001415

Edelstein, R. (2008). Review of Human Health and Product
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in Event COT102 x COT67B. U.S. Environmental Protection Agency.
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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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insecticidal protein with a wide spectrum of activities against
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Geiser, M. Schweizer, S. & Grimm, C. (1986). The hypervariable region in
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Graser, et al. (2006).  Analysis of Vip3A or Vip3A-like Proteins in Six
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Hanley ,et al. (2003) Effects of dietary transgenic Bt corn pollen on
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Hunter, M. and Z. Vaituzis. (2007). Environmental Risk Assessment for
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Division. U.S. Environmental Protection Agency. Washington, D.C.
Memorandum dated January 22, 2007.

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.

Marvier, M., McCreedy, C., Regetz, J. & Kareiva, P. (2007). A
meta-analysis of effects of Bt 

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

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,
D.C. Memorandum dated April 4, 2007.

Milofsky, T. and Z. Vaituzis (2006). Review the soil fate study
submitted in support of ABSTC’s Cry1Ab corn registrations.
Biopesticides and Pollution Prevention Division. U.S. Environmental
Protection Agency. Washington, D.C. Memorandum dated March 29, 2006.

Milofsky, T. and Z. Vaituzis (2007a). Environmental effects risk
assessment for Syngenta’s MIR162 Bt corn EUP. Biopesticides and
Pollution Prevention Division. U.S. Environmental Protection Agency.
Washington, D.C. Memorandum dated January 3, 2007.

Milofsky, T. and Z. Vaituzis (2007b). Environmental Risk Assessment for
Syngenta’s COT102 x COT67B Bacillus thuringiensis Cotton Experimental
Use Permit. U.S. Environmental Protection Agency. Washington, D.C.
Memorandum dated March 21, 2007.	

Naranjo, S.E., Head, G. and Dively, G.P. (2005). Field Studies assessing
arthropod nontarget effects in Bt transgenic crops:  Introduction. 
Environmental Entomology 34:  1178-1180.

National Academy of Science. (2000). Environmental Effects of Transgenic
Plants: The Scope and Adequacy of Regulation is available from the
National Academy Press, 2101 Constitution Avenue, N.W., Lockbox 285,
Washington, DC 20055; (800) 624-6242 or (202) 334-3313 (in the
Washington metropolitan area);   HYPERLINK "http://www.nap.edu" 
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insect growth-

regulating insecticides. OEPP/EPPO Bulletin, 22:  613-616.

Pilcher et al. (1997a). Preimaginal development, survival, and field
abundance of insect predators on transgenic Bacillus thuringiensis corn.
Environmental Entomology 26(2): 446-454.

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. Entomol.
90: 669-678.

Pilcher et al. (2005) Impact of transgenic Bacillus thuringiensis corn
and crop phenology on five nontarget arthropods. Environmental
Entomology 34(5): 1302-1316.

Raybould, A. (2007) Environmental Risk Assessment of Genetically
Modified Crops:  

General Principles and Risks to Non-target Organisms BioAssay 2:8, pg.
1-15

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
Effects of Proteinaceous Insecticidal Plant-Incorporated Protectants on
Non-Target Arthropods for Regulatory Risk Assessments. U.S.
Environmental Protection Agency. Washington, D.C.

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

thuringiensis toxins and biological control. Nature Biotechnology 24: 
63-71. 

Rosi-Marshall E. J., J. L. Tank, T. V. Royer, M. R. Whiles, M.
Evans-White, C. Chambers, N. A. Griffiths, J. Pokelsek, and M. L.
Stephen. (2007). Toxins in transgenic crop byproducts may affect
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Sanvido,O., Romeis, J., Bigler, F. (2007). Ecological Impacts of
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Cultivation. Adv Biochem Engin/Biotechnol 107: 235–278.

Saxena, D. and Stotzky, G. (2001) Bacillus thuringiensis (Bt) toxin
released from root exudates and biomass of Bt corn has no apparent
effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil.
Soil Biol. Biochem. 33: 1225–1230. 

Schnepf, E., Crickmore, N., van Rie, J., Lereclus, D., Baum, J.,
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Schur A., Tornier I., and Neumann C. (2000). Bt-Mais und non Bt-Mais:
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II. D. Insect Resistance Management 

1.  Background

etermined 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 LD99.9 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 LD50 value similar to field
strains; and 5) Determine if a later larval instar of the targeted pest
could be found with an LD50 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 LD99 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 LD50 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 LC50 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 LC50 estimates for CBW neonates range from 504.6ng/cm2 to
2669ng/cm2. 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 LC50 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 LC50s 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 LC50 estimates were greater than neonate
LC50 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 2nd instar larvae show a 36-fold difference in
susceptibility/ LC50 estimates. BPPD would like to add that there were
two estimated LC50s 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 LD99 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 1st 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 high-dose	***	Probable
high-dose	---	---

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

t 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. 

 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.

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

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

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

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. (  HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm" 
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 (LC99) 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. LC50s) may be a feasible
approach to monitoring.  Estimated LC50s may serve well as a baseline
monitoring tool for shifts in susceptibility to Bt toxins; however, the
LC50 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 LC50s 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 (  HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm" 
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,   HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm" 
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,   HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm" 
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 (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. 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

VipCot™	0.5	1.0	0	0.5

CBW damage (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

VipCot™	0

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

Combined CBW and TBW damage (% 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

VipCot™	1.7

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

ii) Infestation results for 2006

		

CBW damage (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.

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

	Locations1, average % damage

	Genotype	BC – LA	W – LA	S – AR

 	J – TN	SS – NC	J – NC

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

	Locations1, average % damage

	Genotype	BC – LA	S – AR

	SS – NC

	J – NC

	B – TX	Overall Mean %

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

1No 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

	Locations1, average % damage

	Genotype	BC – LA	S – AR

 	SS – NC

	J – NC

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

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

TBW damage 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

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

1No 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

	Average1 % 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

1Locations 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

	Locations1, 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

1No 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	COT 102 x 67B,

Seed cotton/acre

Belle Mina, AL	1387	1330

College Station, TX	1958	2036

Estill, SC	1833	1713

Hartsville, SC	2467	2222

Haskell, TX	2254	1999

Portageville, MO	2014	2306

Red Springs, NC	2300	2072

Tifton, GA	2879	3303

Winterville, MS	3060	3339

Variety x Location (p< 0.05)	0.1031

	Cocker 312,

Avg. seed cotton/acre	COT 102 x 67B,

Avg. seed 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 (  HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm" 
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,   HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm" 
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,   HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm" 
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.

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.

tant 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,   HYPERLINK
"http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm" 
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 Analytical  Method - Plants (OPPTS 860.1340)  	An independent
lab validation of the “SeedChek” analytical method for the detection
of Vip3Aa19 and modified 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.	May 1, 2009

Aquatic Invertebrate Toxicity (OPPTS 885.4240)	A 7-14 day Daphnia study
as per the OPPTS 885.4240 guideline must 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.  Separate studies for Vip3Aa (COT102) and
modified Cry1Ab (COT67B) must be preformed. 	May 1, 2009

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

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

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

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

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

 	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 31st each year.  

b) Refuge Requirements

All growers of VipCot cotton must employ one of the following structured
refuge options:

1)  External, Unsprayed Refuge

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

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

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) 

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

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

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

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.  

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

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

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

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

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;

	

	

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萎ː萏ࡰ葝ː葞ࡰ摧䡢ºഀ3) 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 for  SEQ CHAPTER \h \r 1  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:

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

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

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.

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. 

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

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

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

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

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

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

 OPPTS Testing Guidelines, Series 850 and 885 website: 

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

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

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

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

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

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

 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).  

VipCot™ Experimental Use Permit (see memoranda:  Matten, 2006;
Milofsky and Vaituzis, 2007b).   

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

Vip3Aa19 and Modified Cry1Ab Cotton

Biopesticide Registration Action Document (BRAD)      			June 2008

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