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**UNITED STATES ENVIRONMENTAL PROTECTION AGENCY**

WASHINGTON, D.C. 20460

> OFFICE OF PESTICIDES AND TOXIC
>
> SUBSTANCES
>
> **[MEMORANDUM]{.underline}** FEB 07 2008
>
> **SUBJECT:** 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
>
> **TO:** Alan Reynolds
>
> Regulatory Action Leader
>
> Microbial Pesticides Branch, Biopesticides and
>
> Pollution Prevention Division (7511P)
>
> **FROM:** Rebecca L. Edelstein, Ph.D., Chemist /s/
>
> Microbial Pesticides Branch, Biopesticides and
>
> Pollution Prevention Division (7511P)
>
> **THROUGH:** John L. Kough, Ph.D., Biologist /s/
>
> Microbial Pesticides Branch, Biopesticides and
>
> Pollution Prevention Division (7511P)
>
> **ACTION REQUESTED:** To review the human health and product
> characterization data for registration of COT67B x COT102 cotton and
> conduct a risk assessment
>
> **RECOMMENDATION:**

Syngenta has submitted a registration application request for event
COT67B x COT102 cotton, as well as petitions for permanent tolerance
exemptions for modified Cry1Ab protein containing an additional 26 amino
acids and Vip3Aa in or on all crops. The product characterization and
human health data submitted on event COT67B x COT102 cotton are
sufficient to support the requests. An independent lab validation of the
analytical detection method for Vip3Aa and modified Cry1Ab is still
needed. []{.mark}

**DATA REVIEW RECORD**

Active Ingredient: *Bacillus thuringiensis* modified Cry1Ab containing
an additional 26 amino acids and Vip3Aa19 insecticidal proteins and the
genetic material necessary for their production in cotton

Product Name: COT67B X COT102 Cotton

Company Name: Syngenta Seeds, Inc.---Field Crops---NAFTA

ID No: 67969-O

Chemical Number: 006499 and 006529

Decision Number: 373333

DP Barcode: 337037

MRID/Study Titles: 47017602---Re-Characterization of Vip3A Protein Test
Substance (Vip3A-0204)

47017603---Additional Molecular Characterization of Event COT102 Cotton
by Southern Analyses

47017604---Characterization of Cry1Ab Test Substance FLCRY1AB-0103 and
Certificate of Analysis

47017605---Re-Characterization of Cry1Ab Test Substance FLCRY1AB-0103

47017606---Comparative Southern Analyses of Stacked COT102 x COT67B
Cotton

47017607---Comparison of Transgenic Protein Expression in Event COT102
and COT67B Cotton and Stacked COT102 x COT67B Cotton Lines

47017608---Characterization of the Cry1Ab Protein Produced in Event
COT67B-Derived Cotton Plants and Comparison with Cry1Ab Protein Produced
in Recombinant *Escherichia coli*

47017609---Stability of Vip3Aa19 and APH4 Protein Expression Across
Multiple Generations of Event COT102 Cotton

47017610---Stability of Cry1Ab Protein Expression Across Multiple
Generations of Event COT67B Cotton

47017611---Analysis for the Presence of Cry1Ab Protein in Linters,
Toasted Cottonseed Oil from Processed Seed of Event COT67B Cotton
Expressing Full-Length Cry1Ab Protein

47017613---Analysis of Vip3A or Vip3A-Like Proteins in Six Different
Commercial Microbial *Bacillus thuringiensis* Products

47017614---FLCRY1AB-0103: Single Dose Oral Toxicity Study in the Mouse

47017615---*In vitro* Digestibility of Full-Length Cry1Ab Protein (Test
Substances FLCRY1AB-0103 and IAPCOT67B-0106) Under Simulated Mammalian
Gastric Conditions

47017616---Effect of Temperature on the Stability of Full-Length Cry1Ab
Protein

47017617---Vip3Aa19: Assessment of Amino Acid Sequence Homology with
Known Allergens

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

47017619---Full-Length Cry1Ab as Expressed in Event COT67B Cotton:
Assessment of Amino Acid Sequence Homology with Known Allergens

47074101---Analytical Method for the Detection of Vip3A and FL Cry1Ab
Protein in Cotton Tissues Derived from COT102 x COT67B Cotton (VipCot
Cotton)

**[BACKGROUND:]{.underline}**

Syngenta Seeds, Inc. previously submitted EUP requests to conduct field
tests on Event COT102, Event COT67B, and Event COT102 x Event COT67B.
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, for
the control of several lepidopteran pests of cotton. COT102 cotton also
expresses a selectable marker, hygromycin B phosphotransferase (APH4).
Event COT67B cotton, which was developed by *Agrobacterium*-mediated
transformation of cotton using two binary vectors, pNOV4641 and
pNOV1914, expresses a full-length Cry1Ab protein containing an
additional 26 amino acids (referred to by Syngenta as the 'Geiser
motif'). This protein is intended to control a number of lepidopteran
pests. *Bacillus thuringiensis* VIP3Aa19 protein in cotton has a
temporary exemption from the requirement of a tolerance, which will
expire on May 1, 2008 (40 CFR 174.501). Hygromycin B phosphotransferase
(APH4) marker protein in all plants is exempt from the requirement of a
tolerance when used as a plant-incorporated protectant inert ingredient
(40 CFR 174.526). *Bacillus thuringiensis* Cry1Ab protein in all plants
is exempt from the requirement of a tolerance when used as a
plant-incorporated protectant in all food commodities (40 CFR 174.511).
It was previously determined that this tolerance exemption was
sufficient to cover the small amounts of modified Cry1Ab protein present
in food as a result of the EUPs for COT67B and COT102 x COT67B. However,
because the Cry1Ab protein produced from these events has an additional
26 amino acids that are not present in any naturally occurring Cry1Ab
protein, EPA has determined that a new tolerance exemption, specific for
the modified protein, is required for registration.

For registration of Event COT102 x Event COT67B cotton, Syngenta has
submitted petitions for permanent tolerance exemptions for Vip3Aa in or
on all crops and modified Cry1Ab containing an additional 26 amino acids
in or on all crops. Syngenta is relying on previously submitted data
(reviewed in memoranda dated March 24, 2004 from C. Wozniak to L. Cole,
February 8, 2007 from A. Waggoner to M. Mendelsohn, and April 4, 2007
from S. Matten to A. Reynolds) to support the tolerance exemption for
Vip3Aa but has submitted new data, reviewed in this memorandum, to
support the tolerance exemption for modified Cry1Ab. The tolerance
exemption for APH4 discussed above covers the APH4 protein produced in
Event COT102 x Event COT67B.

Product characterization and human health data submitted for
registration of COT102 x Event COT67B and for establishing permanent
tolerance exemptions for Vip3Aa and modified Cry1Ab containing an
additional 26 amino acids are reviewed in this memorandum. Studies
relevant to the risk assessment that were reviewed previously are also
discussed and included in the data tables, and footnotes are provided
with citations to the previous reviews.

**[RISK ASSESSMENT]{.underline}**

**[PRODUCT CHARACTERIZATION]{.underline}**

**[Event COT102 Cotton (OECD Unique Identifier:SYN-IR102-7) Expressing
Vip3Aa19]{.underline}**

[]{.mark}

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

**Transformation System**:

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

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

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

**Protein Characterization**:

The insecticidal protein produced in event COT102 cotton, designated as
Vip3Aa19[^1], is a variant of the naturally occurring Vip3Aa1 protein
isolated from *Bacillus thuringiensis* strain AB88, differing from the
Vip3Aa1 protein by one amino acid (Vip3Aa19 contains a glutamine at
position 284, while Vip3Aa1 contains a lysine). Both proteins are 789
amino acids in length and have a molecular weight of approximately 89
kDa. Syngenta has also developed a transgenic corn variety, MIR162, that
produces another variant, designated as Vip3Aa20, differing from the
naturally occurring Vip3Aa1 protein by two amino acids; at position 284,
Vip3Aa20 has the same amino acid substitution as Vip3Aa19 (i.e., K284Q),
and in addition, at position 129, Vip3Aa20 contains an isoleucine, while
Vip3Aa1 contains a methionine (M129I).

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

**Analytical Detection Methods**:

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

**Protein Expresson**:

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

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

+-----------------------+-----------------------+-----------------------+
| Tissue Type           | Vip3Aa19              | APH4                  |
|                       |                       |                       |
|                       | (µg/g dry weight      | (µg/g dry weight      |
|                       | [+]{.underline}       | [+]{.underline}       |
|                       | standard deviation)   | standard deviation)   |
+-----------------------+-----------------------+-----------------------+
| Leaves\*              | 44 [+]{.underline}    | \< 0.42 -- 8.2        |
|                       | 10 - 277              | [+]{.underline} 1.4   |
|                       | [+]{.underline} 41    |                       |
+-----------------------+-----------------------+-----------------------+
| Squares               | 116 [+]{.underline}   | 2.2 [+]{.underline}   |
|                       | 22                    | 0.4                   |
+-----------------------+-----------------------+-----------------------+
| Flowers               | 162                   | 1.68                  |
+-----------------------+-----------------------+-----------------------+
| Pollen                | 3.47                  | 64.3                  |
+-----------------------+-----------------------+-----------------------+
| Bolls                 | 19 [+]{.underline} 4  | \< 0.39               |
+-----------------------+-----------------------+-----------------------+
| Whole Plants          | 25 [+]{.underline} 4  | \< 0.37               |
+-----------------------+-----------------------+-----------------------+
| Seed                  | 7 + 2                 | 1.4 [+]{.underline}   |
|                       |                       | 0.3                   |
+-----------------------+-----------------------+-----------------------+
| Roots                 | 16 [+]{.underline} 2  | 0.53 [+]{.underline}  |
|                       |                       | 0.11                  |
+-----------------------+-----------------------+-----------------------+

\*Ranges reflect means at different growth stages for leaves

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

Table 2. Product Characterization Data Submitted for Event COT102 Cotton

+---------------+----------------------------------------------+------+
| **Study       | **Summary**                                  | **   |
| Type/Title**  |                                              | MRID |
|               |                                              | \#** |
+---------------+----------------------------------------------+------+
| Expression    | Transgenic cotton plants (COT102) and a      | 4583 |
| Levels/       | non-transgenic isoline (Coker 312) were      | 5801 |
| Quantitation  | grown concurrently in 2001 in Camilla, GA;   |      |
| of VIP3A and  | Maricopa, AZ; and Idalou, TX. Ten whole      |      |
| APH4 Protein  | transgenic plants (including roots) and two  |      |
| in Cotton     | control plants were harvested approximately  |      |
| Tissues and   | 2, 4, 9, 13, 15, and 22 week post-emergence  |      |
| Whole Plants  | (stages: four-leaf, squaring, first white    |      |
| Derived from  | bloom, peak bloom, first open boll,          |      |
| T             | pre-harvest, respectively). Tissue extracts  |      |
| ransformation | were analyzed for VIP3A and APH4 by ELISA.   |      |
| Event         | VIP3A protein was detected in COT102 whole   |      |
| COT102[^2]    | plants, leaves, roots, squares, and bolls at |      |
|               | all six developmental stages examined. VIP3A |      |
|               | levels varied in all plant tissues,          |      |
|               | generally declined with time, but stayed     |      |
|               | constant in the roots. The highest levels    |      |
|               | were found in leaves at the squaring stage   |      |
|               | (mean of 8.56 to 10.78 μg VIP3A/g fresh      |      |
|               | tissue). Low VIP3A levels were found in seed |      |
|               | (mean of 2.51 to 3.23 μg VIP3A/g) and in     |      |
|               | pollen (1.09 μg VIP3A/g). VIP3A was not      |      |
|               | detected in cotton fiber or nectar. The      |      |
|               | protein marker, APH4, was detected in COT102 |      |
|               | plants at low, non-quantifiable levels at    |      |
|               | some developmental stages in leaves, roots,  |      |
|               | bolls, squares, and whole plants and at      |      |
|               | quantifiable levels in pollen (2.25 μg       |      |
|               | APH4/g air-dried pollen). APH4 was not       |      |
|               | detected in cotton fiber or nectar.          |      |
|               | Geographic location appeared not to have a   |      |
|               | significant effect on VIP3A levels, but no   |      |
|               | statistical analysis was done. APH4 levels   |      |
|               | appears to be similar across locations, but  |      |
|               | the lack of data points in many instances    |      |
|               | and the detectable levels falling below the  |      |
|               | level of quantitation (LOQ) do not allow for |      |
|               | any definitive conclusions to be made. The   |      |
|               | estimated amount of VIP3A/acre cotton varied |      |
|               | considerably among the developmental stages  |      |
|               | with the greatest mean level found at the    |      |
|               | peak bloom stage (105.80 g VIP3A/acre based  |      |
|               | on whole plant VIP3A levels).                |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Cha           | Southern blot analysis and DNA sequencing    | 4583 |
| racterization | suggest that event COT102 has one transgene  | 5802 |
| of Inserted   | insertion site with a single copy of intact  |      |
| DNA/Molecular | *vip3A(a)* and *aph4* expression cassettes   |      |
| Cha           | (containing one copy of the *vip3A(a)* gene, |      |
| racterization | *aph4* gene, actin-2 promoter, and ubq3      |      |
| and Genetic   | promoter). DNA sequence alignment revealed   |      |
| Stability of  | an exact sequence match between the pCOT-1   |      |
| Event         | vector and event COT102, and showed the lack |      |
| COT102^2^     | of *Agrobacterium* sequence beyond the T-DNA |      |
|               | borders. VIP3 protein expression measurement |      |
|               | (by ELISA) of five generations of COT102     |      |
|               | seedlings (F1, BC1F2, BC2F1, BC2F2, and      |      |
|               | BC3F1) showed that the *vip3A(a)* gene was   |      |
|               | stable across generations and segregated in  |      |
|               | a Mendelian fashion, consistent with a       |      |
|               | single transgene insertion site. MRID        |      |
|               | 458358-02 provided very scant experimental   |      |
|               | details. Insufficient experimental methods   |      |
|               | details were provided for the Southern       |      |
|               | blots, DNA cloning and sequencing, PCR       |      |
|               | analysis, and protein detection and          |      |
|               | segregation analysis by ELISA, precluding    |      |
|               | confirmation of their appropriateness by an  |      |
|               | independent reviewer. Sample Southern blots  |      |
|               | demonstrating the integration copy number    |      |
|               | and lack of rearrangements through           |      |
|               | appropriate restriction analyses must be     |      |
|               | provided in order to assess the results of   |      |
|               | this study. Further information is required  |      |
|               | regarding the number of plants utilized in   |      |
|               | the segregation and heritability analysis.   |      |
|               |                                              |      |
|               | **Classification: SUPPLEMENTAL,**            |      |
|               | upgradeable to acceptable pending submission |      |
|               | of additional methods details and            |      |
|               | correction/clarification of typographic      |      |
|               | errors in Figure 1, Figure 2, and/or the     |      |
|               | text of MRID 458358-02.                      |      |
|               |                                              |      |
|               | **Superseded by MRID 47017603**              |      |
+---------------+----------------------------------------------+------+
| Ch            | The *Bacillus thuringiensis* (*Bt*) VIP3A    | 4576 |
| aracteristics | insect control protein as expressed in       | 6501 |
| of *Bacillus  | transgenic cotton seed confers protection    |      |
| t             | against the bollworm complex and other       |      |
| huringiensis* | lepidopteran cotton pests. The seeds are     |      |
| VIP3A Protein | derived from transgenic cotton event COT102, |      |
| and VIP3A     | which contains the insecticidal gene via     |      |
| Cotton Plants | plasmid vector pCOT1. The product active     |      |
| Derived from  | ingredient is ≤0.0015 % dry weight *Bacillus |      |
| Event         | thuringiensis* VIP3A Protein and the genetic |      |
| COT102^2^     | material necessary for its production (pCOT1 |      |
|               | in cotton). The product also contains        |      |
|               | ≤0.0001% dry weight marker protein and the   |      |
|               | genetic material necessary for its           |      |
|               | production (pCOT1 in cotton). VIP3A protein  |      |
|               | in transgenic cotton plants derived from     |      |
|               | Event COT102, is produced by a synthetic     |      |
|               | *vip3A(a)* gene, which encodes a polypeptide |      |
|               | of 789 amino acids. The VIP3A toxin is       |      |
|               | proteolytically activated to a toxin core in |      |
|               | the lepidopteran larval midgut and forms     |      |
|               | pores in the gut membranes of sensitive      |      |
|               | species. Several formulated microbial *Bt*   |      |
|               | products containing VIP3A-like proteins and  |      |
|               | the genetic components in plasmid pCOT1, as  |      |
|               | well as its expression analysis, are         |      |
|               | described in MRID 457665-01.                 |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE.** The wide     |      |
|               | certified limits of the active ingredient    |      |
|               | need to be explained, although they are      |      |
|               | within the bounds covered by the acute oral  |      |
|               | toxicity studies submitted for review.       |      |
+---------------+----------------------------------------------+------+
| Cha           | VIP3A protein produced in cotton plants      | 4583 |
| racterization | derived from transgenic cotton event         | 5812 |
| of the active | "COT102\" was characterized for its          |      |
| i             | biochemical and functional similarity with   |      |
| ngredient/Cha | VIP3A expressed in recombinant *Escherichia  |      |
| racterization | coli* and "Pacha" derived transgenic maize   |      |
| of VIP3A      | plants. Samples of purified VIP3A protein    |      |
| Protein       | from *E. coli* and maize were dissolved in   |      |
| Produced in   | buffer for analysis by SDS-PAGE and Western  |      |
| C             | blotting. VIP3A from cotton leaves was       |      |
| OT102-Derived | extracted following published procedures and |      |
| Cotton and    | prepared for SDS-PAGE and Western blotting.  |      |
| Comparison    | VIP3A proteins from all three sources were   |      |
| with VIP3A    | determined to have the predicted molecular   |      |
| Protein       | weight of ca. 89,000 and cross-reacted       |      |
| Expressed in  | immunologically with the same anti-VIP3A     |      |
| Both Maize    | antibody. No evidence of any                 |      |
| (Corn)        | post-translational modification of VIP3A was |      |
| Derived from  | observed in any of the three Vip3A protein   |      |
| Event PACHA   | sources. Peptides representing *ca*. 85%     |      |
| and           | (673/789) of the complete VIP3A amino acid   |      |
| Recombinant   | sequence were identified by mass spectral    |      |
| *Escherichia  | analysis of cotton produced VIP3A protein.   |      |
| coli*^2^      | Amino acid sequences corresponded            |      |
|               | identically to the predicted amino acid      |      |
|               | sequence of the VIP3A protein. Comparisons   |      |
|               | of bioactivity of *E.* *coli*-expressed and  |      |
|               | cotton-expressed VIP3A protein in larvae of  |      |
|               | four lepidopteran species demonstrated       |      |
|               | comparable activities, with the exception of |      |
|               | the tobacco budworm bioassays (TBW). A 35%   |      |
|               | difference in mortality was noted in TBW     |      |
|               | assays comparing these two sources of test   |      |
|               | substance. In the absence of an in-depth     |      |
|               | statistical analysis, it is not possible to  |      |
|               | assign a particular factor as the causal     |      |
|               | agent in delimiting this result. Given that  |      |
|               | both test substances contain other           |      |
|               | constituents, it is difficult to assess the  |      |
|               | reason for this observation. TBW is          |      |
|               | considered as one of the least sensitive     |      |
|               | species of lepidopteran insects evaluated. A |      |
|               | similar rank order of species sensitivity    |      |
|               | was found for both test solutions; FAW was   |      |
|               | the most sensitive to VIP3A, while CBW and   |      |
|               | TBW were the least sensitive. These data     |      |
|               | indicate that VIP3A proteins from            |      |
|               | recombinant *E. coli*, Pacha-derived maize   |      |
|               | and event COT102-derived cotton are          |      |
|               | substantially equivalent.                    |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| []{.mark}     | []{.mark}                                    | [    |
|               |                                              | ]{.m |
| Expression    | Processing transgenic COT102 and control     | ark} |
| Level/        | Coker 312 cotton seeds resulted in similar   |      |
| Analysis of   | yields for the hulls, lint, kernels, refined | 4583 |
| Processed     | oil, and de-fatted meal. Analysis of the     | 5803 |
| COT102        | refined oil and de-fatted meal (non-toasted  |      |
| Cottonseed    | and toasted) by ELISA detected VIP3A protein |      |
| Products for  | in COT102 meal but not in oil, and not in    |      |
| Yield and     | meal or oil from control seeds. Analysis of  |      |
| Presence of   | both COT102 and Coker 312 de-fatted meal for |      |
| Gossypol and  | the plant toxin gossypol detected free       |      |
| Vip3A         | gossypol (HPLC method) and total gossypol    |      |
| Protein^2^    | (free + protein-bound; spectrophotometric    |      |
|               | method). Refined oil had \>100-fold lower    |      |
|               | levels of total gossypol than meal. MRID     |      |
|               | 45835803 provided inadequate and/or          |      |
|               | conflicting details for some experimental    |      |
|               | methods and results.                         |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE.** **Submission |      |
|               | of additional methods details and correction |      |
|               | and/or clarification of the MRID 458358-03   |      |
|               | text as listed under "Deficiencies" is,      |      |
|               | however, recommended to ensure adequate      |      |
|               | recording in the official record.**          |      |
|               |                                              |      |
|               | **The additional information was             |      |
|               | subsequently determined to be unnecessary    |      |
|               | because no adverse effects were observed in  |      |
|               | the nontarget studies.**                     |      |
+---------------+----------------------------------------------+------+
| The mode of   | This publication (Lee *et al.,* 2003), which | 4688 |
| action of the | examined the differences in the mechanism of | 0801 |
| *Bacillus     | insecticidal activity of Cry1Ab and Vip3A,   |      |
| t             | was submitted by the registrant to provide   |      |
| huringiensis* | additional product characterization data,    |      |
| vegetative    | specifically Vip3A's mode of action. The     |      |
| insecticidal  | submitted publication examined differences   |      |
| protein Vip3A | in the mechanism of insecticidal activity of |      |
| differs from  | Cry1Ab and Vip3A proteins. Ligand blotting   |      |
| that of       | showed that activated Cry1Ab and Vip3A-G     |      |
| Cry1Ab        | (Vip3A proteolytically cleaved with          |      |
| delta-        | lepidopteran gut juice) bound different      |      |
| endotoxin[^3] | receptor molecules in midgut of Tobacco      |      |
|               | hornworm (*Manducta sexta*, Linnaeus) and    |      |
|               | that Vip3A-G did not bind Cry1A receptors.   |      |
|               | Voltage clamping assays showed that Vip3A-G  |      |
|               | formed distinct pores in dissected midgut    |      |
|               | from *M. sexta* but not in the monarch       |      |
|               | butterfly (*Danaus plexippus*, Linnaeus).    |      |
|               | Cry1Ab and Vip3A both formed                 |      |
|               | voltage-independent and cation-selective     |      |
|               | stable ion channels in planar lipid          |      |
|               | bilayers, but their primary conductance      |      |
|               | state and cation specificity differed.       |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
|               |                                              |      |
|               | []{.mark}                                    |      |
+---------------+----------------------------------------------+------+
| []{.mark}     | []{.mark}                                    | [    |
|               |                                              | ]{.m |
| Cha           | The purpose of this study was to             | ark} |
| racterization | re-characterize the microbially produced     |      |
| of Test       | test substance, VIP3A-0204. The purity,      | 4701 |
| Sub           | integrity, and bioactivity of the test       | 7602 |
| stance/Re-Cha | substance were determined and compared with  |      |
| racterization | previous analyses after being stored *ca.*   |      |
| of Vip3A      | 15 months under desiccation at -20 °C. Total |      |
| Protein Test  | protein in VIP3A-0204 was quantified         |      |
| Substance     | spectrophotometrically, and the purity was   |      |
| (Vip3A-0204)  | determined using SDS-PAGE followed by        |      |
|               | densitometric analysis. The integrity of the |      |
|               | Vip3Aa19 protein in test substance           |      |
|               | VIP3A-0204 was determined using Western blot |      |
|               | analysis, and bioactivity was assessed in    |      |
|               | insect feeding assays using freshly hatched  |      |
|               | first-instar *S. frugiperda* (fall army      |      |
|               | worm) larvae.                                |      |
|               |                                              |      |
|               | This re-characterization study demonstrated  |      |
|               | that VIP3A-0204 largely retained its         |      |
|               | insecticidal activity (LC~50~ of 34 ng       |      |
|               | Vip3A/cm^2^ diet surface vs. 45.1 initially) |      |
|               | after storage for 15 months. The purity of   |      |
|               | test substance VIP3A-0204 was determined to  |      |
|               | be *ca.* 92% Vip3Aa19 by weight. Western     |      |
|               | blot analysis revealed a dominant            |      |
|               | immunoreactive band corresponding to the     |      |
|               | predicted molecular weight of Vip3Aa19 of    |      |
|               | *ca.* 89 kDa. These results are similar to   |      |
|               | those obtained in previous analyses,         |      |
|               | demonstrating that the test substance is     |      |
|               | stable when stored desiccated at -20 °C for  |      |
|               | approximately 15 months.                     |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Cha           | Molecular analysis of event COT 102 was      | 4701 |
| racterization | performed using restriction enzyme digestion | 7603 |
| of the        | and Southern blot analysis to determine the  |      |
| inserted DNA/ | number of inserts, copy number of functional |      |
| Additional    | elements, and the presence or absence of     |      |
| Molecular     | plasmid backbone sequences. This study also  |      |
| Cha           | assessed the inheritance and stability of    |      |
| racterization | the insert. Data from the Southern analyses  |      |
| of Event      | demonstrated that the BC4F1 generation of    |      |
| COT102 Cotton | COT102 cotton: (1) contains a single intact  |      |
| by Southern   | insert; (2) contains a single copy of the    |      |
| Analysis      | *vip3Aa19* gene and the *aph4* gene; (3)     |      |
|               | contains a single copy of the Act2 promoter; |      |
|               | (4) contains a single copy of the Ubq3       |      |
|               | promoter; (5) does not contain any           |      |
|               | detectable backbone sequences from the       |      |
|               | transformation plasmid pCOT1; and (6) the    |      |
|               | insert is stably integrated into the cotton  |      |
|               | genome. These results are consistent with    |      |
|               | results from previous molecular analysis     |      |
|               | studies on event COT 102.                    |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Inheritance   | The purpose of this study was to use ELISA   | 4701 |
| and           | to analyze the levels of expression of the   | 7609 |
| Stability/    | Vip3Aa19 and hygromycin B phosphotransferase |      |
| Stability of  | (APH4) proteins in leaves (collected at the  |      |
| Vip3Aa19 and  | 1^st^ white bloom stage) of three            |      |
| APH4 Protein  | generations (F1, BC1F1, and BC4F1) of Event  |      |
| Expression    | COT102 cotton. The levels of Vip3Aa19        |      |
| Across        | protein measured were comparable (ca. 60     |      |
| Multiple      | µg/g dry weight) in all three generations    |      |
| Generations   | analyzed. APH4 protein was detectable in all |      |
| of Event      | three generations analyzed, but the          |      |
| COT102 Cotton | concentrations were below the limit of       |      |
|               | quantification (LOQ). The consistency of     |      |
|               | Vip3Aa19 and APH4 protein concentrations     |      |
|               | demonstrate the stability of transgenic      |      |
|               | protein expression across multiple           |      |
|               | generations of COT102 cotton at the 1^st^    |      |
|               | white bloom stage.                           |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+

**[Event COT67B Cotton (OECD Unique Identifier:SYN-IR67B-1) Expressing
Modified Cry1Ab]{.underline}**

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 containing
an additional 26 amino acids. The modified Cry1Ab protein is intended to
control several lepidopteran pests of cotton including, but not limited
to, *Helicoverpa zea* (cotton bollworm/corn earworm), *Heliothis
virescens* (tobacco budworm), *Spodoptera frugiperda* (fall armyworm),
*Spodoptera exigua* (beet armyworm), and *Trichoplusia ni* (cabbage
looper).

**Transformation System**:

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

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

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

**Protein Characterization**:

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

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

**Analytical Detection Methods**:

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

**Protein Expresson**:

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

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

+-----------------------------------+-----------------------------------+
| Tissue Type                       | Cry1Ab                            |
|                                   |                                   |
|                                   | (µg/g dry weight [+]{.underline}  |
|                                   | standard deviation)\*             |
+-----------------------------------+-----------------------------------+
| Leaves                            | 65 [+]{.underline} 9 -- 158       |
|                                   | [+]{.underline} 40                |
+-----------------------------------+-----------------------------------+
| Squares                           | 93 [+]{.underline} 13             |
+-----------------------------------+-----------------------------------+
| Flowers                           | 101                               |
+-----------------------------------+-----------------------------------+
| Pollen                            | 12.1                              |
+-----------------------------------+-----------------------------------+
| Bolls                             | 47 [+]{.underline} 7              |
+-----------------------------------+-----------------------------------+
| Whole Plants                      | 26 [+]{.underline} 2              |
+-----------------------------------+-----------------------------------+
| Seed                              | 29 [+]{.underline} 5              |
+-----------------------------------+-----------------------------------+
| Roots                             | 17 [+]{.underline} 1              |
+-----------------------------------+-----------------------------------+

\*Range reflects means at different growth stages for leaves

Table 1. Mean Expression Levels of Cry1A.105 and Cry2Ab2 from MON 89034
Plant Tissues

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

Table 4. Product Characterization Data Submitted for Event COT67B

+---------------+----------------------------------------------+------+
| **Study       | **Summary**                                  | **   |
| Type/Title**  |                                              | MRID |
|               |                                              | \#** |
+---------------+----------------------------------------------+------+
| Cha           | The purpose of this study was to determine   | 4688 |
| racterization | the DNA sequence and contiguousness of the   | 5901 |
| of Inserted   | full length *cry1Ab* (*flcry1Ab*) gene       |      |
| DNA/ Harper,  | present in Syngenta's COT67B cotton and its  |      |
| B. (2006).    | inheritance ratio across generations. COT67B |      |
| Molecular     | cotton plants express a modified full length |      |
| cha           | Cry1Ab *Bacillus thuringiensis* protein      |      |
| racterization | (FLCry1Ab) that contain an additional 26     |      |
| of Event      | amino acids in the C-terminal portion of the |      |
| COT67B        | protein described as the "Geiser motif."     |      |
| cotton.       | FLCry1Ab confers resistance to certain       |      |
|               | lepidopteran insects in cotton. The T-DNA    |      |
| Report No.    | insert (via the pNOV4641 plasmid) in COT67B  |      |
| SS            | cotton was analyzed by Southern blots and    |      |
| B-125-06.[^4] | DNA sequencing. These analyses confirmed     |      |
|               | that there was a single, contiguous copy of  |      |
|               | the *flcry1Ab* gene present in COT67B. No    |      |
|               | backbone sequences from the transformation   |      |
|               | plasmid pNOV4641 were found in COT67B. The   |      |
|               | left border (LB) and the adjacent 13 bp of   |      |
|               | the insert along with 24 bp of the right     |      |
|               | border (RB) were deleted during the          |      |
|               | insertion of the T-DNA. However, such        |      |
|               | deletions are common during transformation   |      |
|               | and do not affect the functioning of the     |      |
|               | T-DNA itself. Additionally, COT67B cotton    |      |
|               | did not contain the selectable marker gene,  |      |
|               | hygromycin B phosphotransferase (*aph4*), or |      |
|               | the Ubq3 promoter from the transformation    |      |
|               | plasmid pNOV1914 and was also free of any    |      |
|               | backbone sequences from pNOV1914.            |      |
|               | Inheritance studies of the *flcry1Ab* gene   |      |
|               | in COT67B verified that it is segregating in |      |
|               | an expected Mendelian fashion of 1:1.        |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Expression    | The purpose of this study was to quantify    | 4688 |
| Levels/ Hill, | expression of Cry1Ab protein in Event        | 5902 |
| K. (2006).    | COT67B-derived cotton plants. Quantifiable   |      |
| Q             | levels of Cry1Ab protein in Event            |      |
| uantification | COT67B-derived cotton plants were determined |      |
| of Cry1Ab     | by enzyme-linked immunosorbent assay (ELISA) |      |
| protein in    | for various plant tissues and whole plants   |      |
| Event COT67B  | at five developmental stages in four         |      |
| cotton        | locations. Corresponding, near-isogenic,     |      |
| tissues and   | non-transgenic control cotton plants were    |      |
| whole plants. | analyzed in parallel. As expected, Cry1Ab    |      |
|               | protein was detected in all COT67B plant     |      |
| Report        | tissues (i.e., young leaves, old leaves,     |      |
| No.           | roots, flowers, pollen, bolls) except fiber  |      |
| SSB-022-06^4^ | and nectar. The concentrations of Cry1Ab in  |      |
|               | COT67B were similar between the four         |      |
|               | locations for each tissue type at each time  |      |
|               | point, although no specific conclusions      |      |
|               | about differences between locations can be   |      |
|               | made from the data. Where the concentrations |      |
|               | of Cry1Ab appeared variable, there were no   |      |
|               | consistent trends to indicate that the       |      |
|               | plants grown in a given location had higher  |      |
|               | or lower Cry1Ab concentrations. No           |      |
|               | statistical analysis was performed. Cry1Ab   |      |
|               | concentrations in most of the near-isogenic, |      |
|               | nontransgenic control samples were either    |      |
|               | below the limit of detection (LOD) or below  |      |
|               | the limit of quantification (LOQ). The       |      |
|               | negative control seed from Quitman, GA was   |      |
|               | determined to have a low level of Cry1Ab     |      |
|               | (0.24 μg/g dw) that was likely due to        |      |
|               | contamination during processing or           |      |
|               | extraction. The average relative extraction  |      |
|               | efficiency for the various plant tissues     |      |
|               | analyzed varied between 70.7% for whole      |      |
|               | plants to 78.5% for pollen. The absolute     |      |
|               | amount of Cry1Ab in the cotton tissue        |      |
|               | samples is unknown and some Cry1Ab may be    |      |
|               | unextractable with the methods used.         |      |
|               | Extraction efficiency for the purposes of    |      |
|               | satisfying the analytical method would need  |      |
|               | to use a spike-recovery method. Several      |      |
|               | deviations from the protocol were noted by   |      |
|               | the study authors, but none of these         |      |
|               | affected the overall conclusions of the      |      |
|               | study.                                       |      |
|               |                                              |      |
|               | Across all growth stages, mean Cry1Ab        |      |
|               | concentrations (averaged across locations)   |      |
|               | measured in young leaves, old leaves and     |      |
|               | roots of COT67B cotton ranged from 87.70 -   |      |
|               | 323.84, 194.02 - 255.74, and 12.61 - 56.56   |      |
|               | μg/g dry weight (dw), respectively. Mean     |      |
|               | Cry1Ab concentrations measured in bolls      |      |
|               | (collected at 1^st^ open boll), whole plants |      |
|               | (collected at pre-harvest), and seed         |      |
|               | (collected at pre-harvest) averaged 45.24,   |      |
|               | 42.87, and 25.17 μg/g dw across locations,   |      |
|               | respectively. Cry1Ab concentrations in       |      |
|               | flowers and pollen collected at the          |      |
|               | Winnsboro, LA site at peak-bloom averaged    |      |
|               | 161.74 and 5.45 μg/g dw, respectively.       |      |
|               | Cry1Ab concentrations in nectar taken from   |      |
|               | the same cotton plants was not detectable    |      |
|               | (limit of detection = 0.0002 μg/mL). Cry1Ab  |      |
|               | concentrations in fiber samples collected at |      |
|               | this site at pre-harvest was \<0.02 μg/g dw  |      |
|               |                                              |      |
|               | The average Cry1Ab protein per acre and per  |      |
|               | hectare in pre-harvest COT67B plants         |      |
|               | collected from 4 sites was determined        |      |
|               | assuming a planting density of 50,000        |      |
|               | plants/acre (123,500 plants/hectare). The    |      |
|               | average Cry1Ab protein concentration ranged  |      |
|               | from 46 to 183 g/acre (115 to 451            |      |
|               | g/hectare).                                  |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE for the         |      |
|               | purposes of supporting the Experimental Use  |      |
|               | Permit**. Statistically-valid trends in the  |      |
|               | data (e.g., expression level differences     |      |
|               | between tissue types, across developmental   |      |
|               | stages, between locations) cannot be made.   |      |
|               | For a quantitative analysis, it is           |      |
|               | recommended that the expression data         |      |
|               | submitted to support the Section 3           |      |
|               | registration include an appropriate          |      |
|               | statistical analysis.                        |      |
|               |                                              |      |
|               | **Superseded by MRID 47017607.**             |      |
+---------------+----------------------------------------------+------+
| Cha           | The purity, integrity, and bioactivity of    | 4701 |
| racterization | *E. coli*-produced test substance            | 7604 |
| of test       | FLCRY1AB-0103, containing modified           |      |
| substance/Cha | full-length Cry1Ab, were determined          |      |
| racterization | initially and after *ca.* 5 months of being  |      |
| of Cry1Ab     | stored under desiccation at -20 °C. The      |      |
| Test          | purity of test substance FLCRY1AB-0103 was   |      |
| Substance     | determined to be *ca.* 86%, both before and  |      |
| FLCRY1AB-0103 | after storage, and Western blot analysis of  |      |
| and           | the test substance showed a dominant         |      |
| Certificate   | immunoreactive band corresponding to the     |      |
| of Analysis   | predicted molecular weight of *ca.* 133.5    |      |
|               | kDa before and after storage. N-terminal     |      |
|               | sequencing confirmed that the first 12 amino |      |
|               | acids of the test protein corresponded to    |      |
|               | the predicted N-terminal sequence of Cry1Ab. |      |
|               | The test substance was insecticidally active |      |
|               | and had a 72-hour LC~50~ of 3.7 ng           |      |
|               | Cry1Ab/cm^2^ diet surface against first      |      |
|               | instar larvae of the European corn borer.    |      |
|               | Re-analysis of FLCRY1AB-0103 *ca*. 5 months  |      |
|               | after the initial analysis demonstrated that |      |
|               | the test substance retained insecticidal     |      |
|               | activity when stored desiccated at -20 °C.   |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Cha           | The purpose of this study was to             | 4701 |
| racterization | re-characterize the purity, integrity, and   | 7605 |
| of test       | bioactivity of microbially produced test     |      |
| sub           | substance FLCRY1AB-0103 (containing modified |      |
| stance/Re-Cha | full-length Cry1Ab) after storage at -20 °C  |      |
| racterization | for *ca.* 14 months. Total protein in test   |      |
| of Cry1Ab     | substance FLCRY1AB-0103 was quantified       |      |
| Test          | spectrophotometrically by measuring its      |      |
| Substance     | absorption at 280 nm (A~280~ method). The    |      |
| FLCRY1AB-0103 | purity of test substance FLCRY1AB-0103 was   |      |
|               | calculated from the total sample weight and  |      |
|               | the total protein as determined by the       |      |
|               | A~280~ method in conjunction with            |      |
|               | densitometry data after electrophoretic      |      |
|               | separation. The integrity of the Cry1Ab      |      |
|               | protein in test substance FLCRY1AB-0103 was  |      |
|               | determined using Western blot analysis.      |      |
|               | Bioactivity of the Cry1Ab protein in         |      |
|               | FLCRY1AB-0103 was assessed in insect feeding |      |
|               | assays using freshly hatched first-instar    |      |
|               | *O. nubilalis* (European corn borer) larvae. |      |
|               | The results demonstrated that the test       |      |
|               | substance remained intact and retained       |      |
|               | insecticidal activity during this storage    |      |
|               | period.                                      |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| []{.mark}     | []{.mark}                                    | [    |
|               |                                              | ]{.m |
| Cha           | The purpose of this study was to use various | ark} |
| racterization | biochemical and functional parameters to     |      |
| of Expressed  | demonstrate the biochemical equivalence      | 4701 |
| Substance/    | between the Cry1Ab protein expressed in      | 7608 |
| Cha           | transgenic Event COT67B cotton and the       |      |
| racterization | Cry1Ab protein contained in test substance   |      |
| of the Cry1Ab | FLCRY1AB-0103 prepared from an *E. coli*     |      |
| Protein       | over-expression system. Cry1Ab protein was   |      |
| Produced in   | extracted from COT67B cotton plant tissue    |      |
| Event         | and its apparent molecular weight,           |      |
| C             | immunoreactivity, glycosylation status, and  |      |
| OT67B-Derived | bioactivity were compared to the Cry1Ab      |      |
| Cotton Plants | protein from test substance FLCRY1AB-0103.   |      |
| and           | In addition, the microbial- and              |      |
| Comparison    | plant-derived Cry1Ab proteins were analyzed  |      |
| with Cry1Ab   | by peptide mass mapping and the N-terminal   |      |
| Protein       | amino acid sequence of Cry1Ab from test      |      |
| Produced in   | substance FLCRY1AB-0103 was determined.      |      |
| Recombinant   |                                              |      |
| *Escherichia  | The Cry1Ab proteins from COT67B and from     |      |
| coli*         | microbially-derived test substance           |      |
|               | FLCRY1AB-0103 both had an apparent molecular |      |
|               | weight of *ca*. 133.5 kDa, and both reacted  |      |
|               | with anti-Cry1Ab antibodies, as shown by     |      |
|               | Western blot analysis. Also, both the        |      |
|               | protein extract from COT67B and              |      |
|               | FLCRY1AB-0103 showed strong insecticidal     |      |
|               | activity against *O. nubilalis* (European    |      |
|               | corn borer). There was no evidence of        |      |
|               | post-translational glycosylation of Cry1Ab   |      |
|               | protein from COT67B or from                  |      |
|               | microbially-derived test substance           |      |
|               | FLCRY1AB-0103. Peptide mass mapping analysis |      |
|               | provided additional evidence of the identity |      |
|               | of the insecticidal protein expressed in     |      |
|               | COT67B cotton and in test substance          |      |
|               | FLCRY1AB-0103. Based on the results of this  |      |
|               | study it can be concluded that Cry1Ab        |      |
|               | protein produced in recombinant *E. coli*    |      |
|               | (test substance FLCRY1AB-0103) is a suitable |      |
|               | surrogate for Cry1Ab expressed in COT67B     |      |
|               | cotton.                                      |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| []{.mark}     | []{.mark}                                    | [    |
|               |                                              | ]{.m |
| Expression    | The purpose of this study was to use ELISA   | ark} |
| lev           | to analyze the levels of expression of the   |      |
| els/Stability | modified Cry1Ab protein in leaves (collected | 4701 |
| of Cry1Ab     | at open boll stage) of the F1, BC1F1, and    | 7610 |
| Protein       | BC4F1 generations of Event COT67B cotton.    |      |
| Expression    | Identical plant tissues from two             |      |
| Across        | near-isogenic, nontransgenic cotton plants   |      |
| Multiple      | (cotton line 2429) from the BC1F1 and BC4F1  |      |
| Generations   | generations were concurrently sampled and    |      |
| of Event      | analyzed to identify any potential           |      |
| COT67B Cotton | background effects of the plant matrix on    |      |
|               | the ELISA. The levels of Cry1Ab protein      |      |
|               | measured in the three generations of COT67B  |      |
|               | cotton were comparable (\~60 μg/g dry        |      |
|               | weight). The consistency of the Cry1Ab       |      |
|               | protein concentrations demonstrates the      |      |
|               | stability of transgenic protein expression   |      |
|               | across multiple generations of COT67B cotton |      |
|               | at the open boll stage.                      |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Expression    | The purpose of this study was to quantify    | 4701 |
| Levels/       | Cry1Ab protein in linters, defatted toasted  | 7611 |
| Analysis for  | cottonseed meal, and once-refined cottonseed |      |
| the Presence  | oil derived from COT67B, and to determine    |      |
| of Cry1Ab     | Cry1Ab protein concentrations in the fuzzy   |      |
| Protein in    | seed used to generate these processed        |      |
| Linters,      | fractions. Quantification was carried out    |      |
| Toasted       | using an enzyme-linked immunosorbent assay   |      |
| Cottonseed    | (ELISA). The Cry1Ab extraction efficiencies  |      |
| Oil from      | were \>69% for fuzzy seed, linters, and      |      |
| Processed     | defatted toasted cottonseed meal from        |      |
| Seed of Event | COT67B. The mean concentrations of Cry1Ab    |      |
| COT67B Cotton | protein (corrected for extraction            |      |
| Expressing    | efficiency) in fuzzy seed, linters, and      |      |
| Full-Length   | defatted toasted cottonseed meal from COT67B |      |
| Cry1Ab        | were 25.1, 9.6, and 47.5 µg Cry1Ab/g,        |      |
| Protein       | respectively. Cry1Ab was not detectable in   |      |
|               | the once-refined oil from COT67B (limit of   |      |
|               | detection = 0.003 µg Cry1Ab/ml). Cry1Ab      |      |
|               | concentrations in all cottonseed samples     |      |
|               | from Coker 312 (negative control) were below |      |
|               | the limit of detection.                      |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+

[]{.mark}

[]{.mark}

**[Event COT102 x COT67B Cotton (OECD ID No SYN-IR102-7 x OECD ID No.
SYN-IR67B-1) Expressing Vip3Aa19, APH4, and Modified
Cry1Ab]{.underline}**

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

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

**Analytical Detection Methods**:

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

**Protein Expresson**:

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

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

+-----------------+-----------------+-----------------+---------------+
| Tissue Type     | Vip3Aa19        | APH4            | Cry1Ab        |
|                 |                 |                 |               |
|                 | (µg/g dry       | (µg/g dry       | (µg/g dry     |
|                 | weight          | weight          | weight        |
|                 | [+]{.underline} | [+]{.underline} | [+            |
|                 | standard        | standard        | ]{.underline} |
|                 | deviation)\*    | deviation)\*    | standard      |
|                 |                 |                 | deviation)\*  |
+-----------------+-----------------+-----------------+---------------+
| Leaves          | 55              | \<0.41 -- 6.3   | 70            |
|                 | [+]{.underline} | [+]{.underline} | [+            |
|                 | 7 -- 239        | 1.3             | ]{.underline} |
|                 | [+]{.underline} |                 | 14 -- 185     |
|                 | 46              |                 | [+            |
|                 |                 |                 | ]{.underline} |
|                 |                 |                 | 63            |
+-----------------+-----------------+-----------------+---------------+
| Squares         | 132             | 2.1             | 94            |
|                 | [+]{.underline} | [+]{.underline} | [+            |
|                 | 18              | 0.5             | ]{.underline} |
|                 |                 |                 | 10            |
+-----------------+-----------------+-----------------+---------------+
| Flowers         | 148             | 1.80            | 121           |
+-----------------+-----------------+-----------------+---------------+
| Pollen          | 3.06            | 74.7            | 10.7          |
+-----------------+-----------------+-----------------+---------------+
| Bolls           | 21              | \< 0.43         | 42            |
|                 | [+]{.underline} |                 | [+            |
|                 | 4               |                 | ]{.underline} |
|                 |                 |                 | 7             |
+-----------------+-----------------+-----------------+---------------+
| Whole Plants    | 25              | \< 0.40         | 29            |
|                 | [+]{.underline} |                 | [+            |
|                 | 7               |                 | ]{.underline} |
|                 |                 |                 | 7             |
+-----------------+-----------------+-----------------+---------------+
| Seed            | 7               | 1.6             | 27            |
|                 | [+]{.underline} | [+]{.underline} | [+            |
|                 | 1               | 4               | ]{.underline} |
|                 |                 |                 | 4             |
+-----------------+-----------------+-----------------+---------------+
| Roots           | 11              | 0.46            | 20            |
|                 | [+]{.underline} | [+]{.underline} | [+            |
|                 | 3               | 0.05            | ]{.underline} |
|                 |                 |                 | 4             |
+-----------------+-----------------+-----------------+---------------+

\*Ranges reflect means at different growth stages for leaves

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

Table 6. Product Characterization Data Submitted for Event COT102 x
COT67B

+---------------+----------------------------------------------+------+
| **Study       | **Summary**                                  | **   |
| Type/Title**  |                                              | MRID |
|               |                                              | \#** |
+---------------+----------------------------------------------+------+
| Cha           | Molecular analyses (restriction enzyme       | 4701 |
| racterization | digests and Southern blots) were performed   | 7606 |
| of Inserted   | to compare the integrity of the transgenic   |      |
| DNA/          | inserts in the cotton lines Event COT102     |      |
| Comparative   | cotton and Event COT67B cotton with the      |      |
| Southern      | transgenic inserts in stacked COT102 x       |      |
| Analysis of   | COT67B cotton, which was produced by         |      |
| Stacked       | conventional plant breeding of COT102 and    |      |
| COT102 x      | COT67B. The Southern blot data demonstrated  |      |
| COT67B        | the predicted molecular organization of the  |      |
|               | *vip3Aa19 and aph4* genes from COT102 cotton |      |
|               | and the *cry1Ab* gene from COT67B cotton.    |      |
|               | The DNA hybridization patterns from each     |      |
|               | single event cotton line were identical to   |      |
|               | those in stacked COT102 x COT67B cotton,     |      |
|               | demonstrating that the integrity of the      |      |
|               | transgenic inserts was retained when the     |      |
|               | component lines were combined into the       |      |
|               | COT102 x COT67B cotton.                      |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Expression    | The purpose of this study was to use an      | 4701 |
| Levels/       | enzyme-linked immunosorbent assay (ELISA) to | 7607 |
| Comparison of | analyze tissues from cotton plants derived   |      |
| Transgenic    | from transformation Event COT102, Event      |      |
| Protein       | COT67B and from COT102 x COT67B in order to  |      |
| Expression in | compare the concentrations of Vip3Aa19,      |      |
| Event COT102, | hygromycin B phosphotransferase (APH4), and  |      |
| Event COT67B, | Cry1Ab proteins produced in the transgenic   |      |
| and Stacked   | plants. For the Vip3Aa19 and APH4 proteins,  |      |
| COT102 x      | the concentrations and patterns of           |      |
| COT67B Cotton | expression were generally similar between    |      |
| Lines         | the COT102 line and the COT102 x COT67B      |      |
|               | line. Likewise, for the modified Cry1Ab      |      |
|               | protein, the concentrations and patterns of  |      |
|               | expression were generally similar between    |      |
|               | the COT67B line and the COT102 x COT67B      |      |
|               | line. Some statistically significant         |      |
|               | differences were seen in some tissues at     |      |
|               | certain sampling stages, but these           |      |
|               | differences were not consistent by genotype  |      |
|               | and/or were not consistent across the        |      |
|               | growing season.                              |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Analytical    | The SeedChek Vip3A/FLCry1Ab lateral flow     | 4707 |
| Detection     | test kit was tested for the qualitative      | 4101 |
| Meth          | detection of modified Cry1Ab and Vip3A       |      |
| od/Analytical | proteins in cotton seed and cotton leaf. The |      |
| Detection     | study showed that the SeedChek kit is able   |      |
| Method for    | to detect Vip3A and Cry1Ab in both cotton    |      |
| the Detection | seed and cotton leaf, and no unexpected      |      |
| of Vip3A and  | cross reactivity with other transgenic       |      |
| FLCry1Ab      | varieties was observed.                      |      |
| Protein in    |                                              |      |
| Cotton        | **Classification: ACCEPTABLE**               |      |
| Tissues       |                                              |      |
| Derived from  |                                              |      |
| COT102 x      |                                              |      |
| COT67B Cotton |                                              |      |
| (VipCot       |                                              |      |
| Cotton)       |                                              |      |
+---------------+----------------------------------------------+------+

**[Human Health Assessment of Vip3Aa]{.underline}**

**Background**

Vip3Aa is a vegetative (i.e., produced during the vegetative stage of
bacterial growth) insecticidal protein from *Bacillus thuringiensis*
(*Bt*) with insecticidal activity to several lepidopteran pests. 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; K284Q). 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 *Bt* delta endotoxin nomenclature committee assigns a quaternary
numerical rank (e.g., 1, 19, or 20) to each independently sequenced
gene; therefore, some toxins with different quaternary ranks may be
identical. All proteins designated as Vip3Aa are more than 95%
identical. EPA has determined that 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.

Section 408(c)(2)(A)(i) of the FFDCA allows EPA to establish an
exemption from the requirement for a tolerance (the legal limit for a
pesticide chemical residue in or on a food) only if EPA determines that
the exemption is "safe." Section 408(c)(2)(A)(ii) of the FFDCA defines
"safe" to mean that "there is a reasonable certainty that no harm will
result from aggregate exposure to the pesticide chemical residue,
including all anticipated dietary exposures and all other exposures for
which there is reliable information." This includes exposure through
drinking water and in residential settings, but does not include
occupational exposure. Pursuant to section 408(c)(2)(B), in establishing
or maintaining in effect an exemption from the requirement of a
tolerance, EPA must take into account the factors set forth in section
408(b)(2)(C), which require EPA to give special consideration to
exposure of infants and children to the pesticide chemical residue in
establishing a tolerance and to "ensure that there is a reasonable
certainty that no harm will result to infants and children from
aggregate exposure to the pesticide chemical residue\...."

Additionally, section 408(b)(2)(D) of the FFDCA requires that the Agency
consider "available information concerning the cumulative effects of a
particular pesticide's residues" and "other substances that have a
common mechanism of toxicity." EPA performs a number of analyses to
determine the risks from aggregate exposure to pesticide residues.
First, EPA determines the toxicity of pesticides. Second, EPA examines
exposure to the pesticide through food, drinking water, and through
other exposures that occur as a result of pesticide use in residential
settings.

**1. Toxicological Profile**

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

**Mammalian Toxicity and Allergenicity Assessment**

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,
which all indicated that Vip3Aa is non-toxic to humans. Three of the
studies were conducted with microbially-produced Vip3Aa proteins with
slight variations in amino acid sequence (1-2 amino acid differences),
and one study was conduced with transgenic corn leaf tissue as the test
material. No treatment-related adverse effects were observed in any of
the studies. The oral LD~50~ for mice (males, females, and combined) was
greater than 3675 mg Vip3Aa/kg body weight (the highest dose tested).

When proteins are toxic, they are known to act via acute mechanisms and
at very low dose levels (Sjoblad, Roy D., et al., "Toxicological
Considerations for Protein Components of Biological Pesticide Products,"
Regulatory Toxicology and Pharmacology 15, 3-9 (1992)). Therefore, since
no acute effects were shown to be caused by Vip3Aa proteins, even at
relatively high dose levels, the Vip3Aa is not considered toxic.
Further, amino acid sequence comparisons showed no similarities between
Vip3Aa and known toxic proteins in protein databases that would raise a
safety concern.

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

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

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

3.  Digestibility. The Vip3Aa protein was digested rapidly in simulated
    gastric fluid containing pepsin.

4.  Glycosylation. Vip3Aa was shown not to be glycosylated.

5.  Conclusion. Considering all of the available information, EPA has
    concluded that the potential for Vip3Aa to be a food allergen is
    minimal.

The amino acid sequence analysis was only performed on two Vip3Aa
proteins, Vip3Aa19 and Vip3Aa20. The amino acid sequence of Vip3Aa
proteins can vary up to 5%, and although very unlikely, it is possible
that another Vip3Aa protein could have sequence identity with an
allergen at the level of eight contiguous amino acids. Therefore, EPA is
including the restriction on this tolerance exemption that it only
applies to Vip3Aa proteins with no significant amino acid similarity
with known allergens.

Although Vip3Aa was only shown not to be glycosylated in cotton and
corn, 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, P. Cabanes-Macheteau, M., Rayon, C., Fichette-Lainé, A-C.,
Gomord, V., and Faye, L., "N-Glycoprotein biosynthesis in plants: recent
developments and future trends," *Plant Molecular Biology* **38**:
31-48, 1998).

**2. 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 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 the Vip3Aa protein is 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.

**3. 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, we conclude that there are no cumulative
effects for the Vip3Aa protein.

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

**a) Toxicity and Allergenicity Conclusions**

The data submitted and cited regarding potential health effects for the
Vip3Aa protein includes the characterization of the Vip3Aa protein, as
well as the acute oral toxicity studies, amino acid sequence comparisons
to known allergens and toxins, 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 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 support the prediction that the
Vip3Aa 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, Roy D., et al., "Toxicological
Considerations for Protein Components of Biological Pesticide Products,"
Regulatory Toxicology and Pharmacology 15, 3-9 (1992)). Since no
treatment-related adverse effects were shown to be caused by the Vip3Aa
protein, even at relatively high dose levels, the Vip3Aa protein is 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 Vip3Aa in corn and cotton tissues.

Since Vip3Aa is a protein, potential allergenicity is also considered as
part of the toxicity assessment. Considering all of the available
information (1) Vip3Aa originates from a non-allergenic source; (2)
Vip3Aa19 and Vip3Aa20 have no sequence similarities with known
allergens; (3) Vip3Aa is not glycosylated; and (4) Vip3Aa is rapidly
digested in simulated gastric fluid; 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 the Vip3Aa protein, as well as the minimal potential to be a
food 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 Vip3Aa
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.

**b) 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 Vip3Aa 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.

**c) 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
Vip3Aa protein, provided it has no significant amino acid similarity
with known allergens. 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.

**5. Other Considerations**

**a) 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.

**b) Analytical Method(s)**

A validated lateral flow enzyme-linked immubsorbent 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.

**c) Codex Maximum Residue Level**

No Codex maximum residue level exists for the plant-incorporated
protectant *Bacillus thuringiensis* Vip3Aa protein and the genetic
material necessary for its production in corn.

**Proposed Language for Tolerance Exemption**:

**Bacillus thuringiensis Vip3Aa proteins in all plants; exemption from
the**

**requirement of a tolerance.**

Residues of *Bacillus thuringiensis* Vip3Aa proteins in all plants are
exempt from the requirement of a tolerance when used as
plant-incorporated protectants in all food commodities provided that the
Vip3Aa protein does not have any significant amino acid similarity with
any known allergens.

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

+---------------+----------------------------------------------+------+
| **Study       | **Summary**                                  | **   |
| Type/Title**  |                                              | MRID |
|               |                                              | \#** |
+---------------+----------------------------------------------+------+
| Summary of    | No significant adverse effects were observed | 4576 |
| Mammalian     | in male and female mice dose by gavage at    | 6502 |
| Toxicology    | approximately 3675 mg VIP3A/kg body weight   |      |
| Data for the  | (the highest dose tested) and the LD~50~ for |      |
| VIP3A and     | pure VIP3A protein was \>3675 mg/kg body     |      |
| APH4 Proteins | weight. The LD~50~ for pure APH4 protein in  |      |
| Produced by   | male and female mice was \>774 mg/kg body    |      |
| Transgenic    | weight. The allergen database compiled by    |      |
| VIP3A Cotton  | Syngenta needs to be better defined or       |      |
| Event         | described in order to ascertain the number   |      |
| COT102^2^     | and types of allergens searched for          |      |
|               | homology.                                    |      |
|               |                                              |      |
|               | **Classification: SUPPLEMENTAL**, upgradable |      |
|               | to Acceptable with the submission of further |      |
|               | information on the SBI allergen database.    |      |
|               |                                              |      |
|               | **Note: this is a summary of multiple        |      |
|               | studies and is therefore superseded by the   |      |
|               | individual studies summarized below, which   |      |
|               | provide additional information, including    |      |
|               | the requested information on the SBI         |      |
|               | allergen database.**                         |      |
+---------------+----------------------------------------------+------+
| Acute Oral    | Eleven male and 11 female HSD:ICR albino     | 4576 |
| Toxicity/     | mice were dosed with VIP3A protein (Lot no.  | 6503 |
| Acute Oral    | VIP3A-0196 containing \~ 32% by weight VIP3A |      |
| Toxicity of   | protein). The mice were quarantined for 5    |      |
| Vip3A Protein | days and fasted approximately 16 hours prior |      |
| in Mice^2^    | to dosing. The test material (5050 mg/kg     |      |
|               | body weight) was dosed as a 12.5 % w/v       |      |
|               | suspension in 2 % w/v carboxymethyl          |      |
|               | cellulose (CMC) in distilled water by gavage |      |
|               | (Table 1). The dose volume was 40.4 mL/kg    |      |
|               | and was divided into 2 parts administered    |      |
|               | approximately one hour apart. The control    |      |
|               | group was treated with 2 % w/v CMC in the    |      |
|               | same manner as the test animals. Body        |      |
|               | weights were recorded prior to dosing, on    |      |
|               | days 7 and 14 or at death. The test animals  |      |
|               | were observed for clinical signs of toxicity |      |
|               | at least three times post-dosing and at      |      |
|               | least daily thereafter for 14 days. All      |      |
|               | decedent or euthanized animals were          |      |
|               | necropsied. One control male (No. 17-M) was  |      |
|               | found dead on day 2. All other mice survived |      |
|               | the study. With the exception of one female  |      |
|               | (No. 10-F) that failed to gain weight during |      |
|               | the first week, all surviving animals gained |      |
|               | weight during the study. In the vehicle      |      |
|               | control group (*i.e*., CMC treated), there   |      |
|               | was no affect on weight gain. The oral       |      |
|               | LD~50~ for males, females, and combined was  |      |
|               | greater than 5050 mg/kg (or \> 1616 mg VIP3A |      |
|               | protein/kg body weight).                     |      |
|               |                                              |      |
|               | **Classification: SUPPLEMENTAL.** The VIP3A  |      |
|               | protein used in this study differs from the  |      |
|               | VIP3A protein present in COT102 cotton by a  |      |
|               | single amino acid at position 2 (aspartate   |      |
|               | replaces asparagine).                        |      |
|               |                                              |      |
|               | **Note: this study provides additional       |      |
|               | support for the conclusion that Vip3Aa       |      |
|               | proteins are non-toxic to mammals.**         |      |
+---------------+----------------------------------------------+------+
| Acute Oral    | Twenty-seven male and 27 female CD-1^®^      | 4576 |
| Toxicity/     | (ICR)BR mice were dosed with VIP3A protein   | 6504 |
| Single Dose   | (Batch VIP3A-0199 containing \~ 54% by       |      |
| Oral Toxicity | weight VIP3A protein), produced in an *E.    |      |
| Study with    | coli* over-expression system. The VIP3A      |      |
| VIP3A-0199 in | protein used as the test material differs    |      |
| Mice^2^       | from that present in cotton Event COT102 by  |      |
|               | a single amino acid; glutamine substituted   |      |
|               | for lysine (Q284K). The mice were            |      |
|               | quarantined for 16 days and fasted           |      |
|               | approximately 4 hours prior to dosing. The   |      |
|               | test material (5000 mg/kg body weight) was   |      |
|               | dosed as a suspension of 200 mg/mL in 0.5%   |      |
|               | w/v carboxymethyl cellulose (CMC) in         |      |
|               | deionized water by gavage (Table 1). The     |      |
|               | dose volume was 25 mL/kg. The control group  |      |
|               | was treated with 0.5 % w/v CMC in the same   |      |
|               | manner and volume as the test animals. Body  |      |
|               | weights were recorded prior to dosing, and   |      |
|               | on day 8 for animals designated to be        |      |
|               | sacrificed on day 15, and on each animals's  |      |
|               | respective day of necropsy (days 1, 2, or    |      |
|               | 15). The animals were observed for clinical  |      |
|               | signs of toxicity approximately 1, 2.5, 4,   |      |
|               | and 6 hours post dosing and at least daily   |      |
|               | until sacrifice. Animals were observed for   |      |
|               | any abnormal behavior, changes in posture or |      |
|               | clonic / tonic movements. Mortality was      |      |
|               | observed twice daily. All animals were       |      |
|               | necropsied after sacrifice. The organ weight |      |
|               | of the brain, kidneys, liver with drained    |      |
|               | gallbladder, and stomach were recorded and   |      |
|               | organ to body weight and organ to brain      |      |
|               | weight were calculated. Histopathology was   |      |
|               | performed on brain, gallbladder, heart,      |      |
|               | intestines (cecum, colon, duodenum, ileum,   |      |
|               | jejunum, and rectum), kidneys, lesions,      |      |
|               | liver, lung, and stomach. All animals        |      |
|               | survived prior to the scheduled sacrifice.   |      |
|               | All animals sacrificed on day 15 had normal  |      |
|               | body weight gains. All control and a few     |      |
|               | test animals sacrificed on day 1 and one     |      |
|               | male test and some control animals           |      |
|               | sacrificed on day 2 lost weight prior to     |      |
|               | sacrifice. No significant differences        |      |
|               | considered to be test material related in    |      |
|               | organ/body weight or organ/brain weight      |      |
|               | between control and test animals were found. |      |
|               | The oral LD~50~ for males, females, and      |      |
|               | combined was greater than 5000 mg/kg (or \>  |      |
|               | 2700 mg VIP3A protein/kg body weight).       |      |
|               |                                              |      |
|               | **Classification: SUPPLEMENTAL -** The test  |      |
|               | material for this study, VIP3A-0199, differs |      |
|               | in sequence by one amino acid (Q284K) from   |      |
|               | that form of the protein which is present in |      |
|               | COT102.                                      |      |
|               |                                              |      |
|               | **Note: this study provides additional       |      |
|               | support for the conclusion that Vip3Aa       |      |
|               | proteins are non-toxic to mammals.**         |      |
+---------------+----------------------------------------------+------+
| Acute Oral    | The test animals (Sixteen male and 16 female | 4576 |
| Toxicity/     | Crl-1^®^ (ICR)BR mice) were quarantined for  | 6505 |
| Acute Oral    | 9 days and fasted approximately 4 hours      |      |
| Toxicity      | prior to dosing. The test material (5000     |      |
| Study with    | mg/kg body weight) was dosed as a suspension |      |
| Test          | of 196 mg/mL in 0.5% w/v carboxymethyl       |      |
| Substance     | cellulose (CMC) in deionized water by        |      |
| VIP3A-0100    | gavage. The dose volume was 25.5 mL/kg. The  |      |
| Protein in    | control group was treated with 0.5% w/v CMC  |      |
| Mice^2^       | in the same manner as the test animals. Body |      |
|               | weights were recorded prior to dosing and on |      |
|               | days 8 and 15 for animals designated to be   |      |
|               | sacrificed on day 15. The animals were       |      |
|               | observed for clinical signs of toxicity      |      |
|               | approximately 1, 2.5, 4, and 6 hours post    |      |
|               | dosing and at least daily until sacrifice.   |      |
|               | Mortality was observed twice daily. All      |      |
|               | animals were necropsied after sacrifice. The |      |
|               | organ weight of the brain, kidneys, liver    |      |
|               | with drained gallbladder, and stomach were   |      |
|               | recorded and organ to body weight and organ  |      |
|               | to brain weight were calculated.             |      |
|               | Histopathology was performed on brain,       |      |
|               | gallbladder, heart, intestines (cecum,       |      |
|               | colon, duodenum, ileum, jejunum, and         |      |
|               | rectum), kidneys, lesions, liver, lung, and  |      |
|               | stomach. All animals sacrificed on day 15    |      |
|               | had normal body weight gains. No test        |      |
|               | material related macroscopic alterations     |      |
|               | were noted. In addition, no significant      |      |
|               | differences related to the test material in  |      |
|               | organ/body weight or organ/brain weight      |      |
|               | between control and test animals were found. |      |
|               | The oral LD~50~ for males, females, and      |      |
|               | combined was greater than 5000 mg/kg (or \>  |      |
|               | 3675 mg VIP3A protein/kg body weight).       |      |
|               |                                              |      |
|               | **Classification: Acceptable**               |      |
+---------------+----------------------------------------------+------+
| Acute Oral    | VIP3A-Enriched Maize (Corn) Leaf Protein     | 4576 |
| Toxicity/     | (Sample Lot. No. LPPACHA-0199 containing \~  | 6506 |
| Single Dose   | 0.36% by weight VIP3A protein) was prepared  |      |
| Oral Toxicity | from transgenic VIP3A maize (corn) leaves.   |      |
| Study with    | The mice were quarantined for at least 7     |      |
| V             | days and fasted approximately 4 hours prior  |      |
| IP3A-Enriched | to dosing. The test material (5000 mg/kg     |      |
| Maize (Corn)  | body weight) was dosed as a suspension of    |      |
| Leaf Protein  | 250 mg/mL in 0.5% w/v carboxymethyl          |      |
| (             | cellulose (CMC) in deionized water by gavage |      |
| LPPACHA-0199) | (Table 1). The dose volume was 20 mL/kg. The |      |
| in Mice^2^    | control group was treated with Control Maize |      |
|               | (Corn) Leaf Protein, Batch LPPACHA-0199C in  |      |
|               | 0.5% w/v CMC in deionized water at a         |      |
|               | concentration of 250 mg/mL in the same       |      |
|               | manner as the test animals. Body weights     |      |
|               | were recorded prior to dosing, and on days   |      |
|               | 7, 14, or at death. The test animals were    |      |
|               | observed for clinical signs of toxicity at   |      |
|               | least three times post-dosing and at least   |      |
|               | daily thereafter for 14 days. All decedent   |      |
|               | or euthanized animals were necropsied. All   |      |
|               | mice survived the study, gained weight and   |      |
|               | appeared normal during the study. The oral   |      |
|               | LD~50~ for males, females, and combined was  |      |
|               | greater than 18 mg/kg VIP3A protein/kg body  |      |
|               | weight. The net concentration of VIP3A (18   |      |
|               | mg / kg body weight) is significantly lower  |      |
|               | than the prescribed 2000 to 5000 mg / kg     |      |
|               | body weight suggested in the guideline       |      |
|               | requirements. At this concentration and with |      |
|               | the mix of other proteins present in the     |      |
|               | leaf preparation, no toxicity was evident in |      |
|               | the test animals.                            |      |
|               |                                              |      |
|               | **Classification: SUPPLEMENTAL.**            |      |
|               | Information is supportive, but not part of   |      |
|               | guideline requirements; no further           |      |
|               | information required.                        |      |
|               |                                              |      |
|               | **Note: this study provides additional       |      |
|               | support for the conclusion that Vip3Aa       |      |
|               | proteins are non-toxic to mammals.**         |      |
+---------------+----------------------------------------------+------+
| *In Vitro*    | VIP3A from recombinant maize (field corn)    | 4583 |
| Digestibility | plants was prepared as sample LPPACHA-0199   | 5805 |
| of VIP3A      | by extracting the leaves of recombinant corn |      |
| Protein Under | plants and concentra­ting the VIP3A by        |      |
| Simulated     | ammonium sulfate precipitation, dialysis of  |      |
| Mammalian     | the resulting salt, and lyophilization of    |      |
| Gastric       | the collected protein. ELISA showed VIP3A    |      |
| Conditions    | constituted \~0.36 % by weight of the sample |      |
| ^2^           | and retained insecticidal activity against   |      |
|               | sensitive lepidopteran species. VIP3A from   |      |
|               | *E. coli* was prepared as sample VIP3A-0100  |      |
|               | in an *E. coli* strain BL21DE3pLysS          |      |
|               | over-expression system. The synthetic        |      |
|               | *vip3A(a)* gene was cloned into the          |      |
|               | inducible over-expression pET-3a® vector.    |      |
|               | Following collection, purifi­cation,          |      |
|               | dialysis, and lyophilization, the sample was |      |
|               | estimated by ELISA to contain \~73.5% VIP3A  |      |
|               | by weight and it retained its insecticidal   |      |
|               | activity against sensitive lepidopteran      |      |
|               | species. The reactions were initiated by the |      |
|               | addition of 80 µL of LPPACHA-0199 or         |      |
|               | VIP3A-0100 to 320 µL of simulated gastric    |      |
|               | fluid containing pepsin incubated at 37°C.   |      |
|               | Immediately after sample addition, an        |      |
|               | aliquot was removed and quenched with an     |      |
|               | equal volume of Laemmli buffer (pH not       |      |
|               | reported) and inactivated at \>75°C for 10   |      |
|               | minutes. Additional aliquots were removed    |      |
|               | and treated as above following 2, 5, 10, 20, |      |
|               | 30, and 60 minutes of incubation. Digestion  |      |
|               | of the protein samples was evaluated using   |      |
|               | SDS-PAGE and Western blotting. The digestion |      |
|               | of VIP3A protein in a simulated gastric      |      |
|               | environment proceeds at a rapid rate and     |      |
|               | demonstrates the lability of this protein to |      |
|               | conditions typical of a monogastric          |      |
|               | mammalian stomach. The presence of a small   |      |
|               | amount of immunoreactive protein             |      |
|               | (approximately 6 to 9 kD) indicates that a   |      |
|               | portion or domain of the protein is less     |      |
|               | readily digested in this environment,        |      |
|               | although these bands do degrade beyond the   |      |
|               | point of immunorecognition with time.        |      |
|               | Results of this study indicate VIP3A         |      |
|               | protein, whether isolated from recombinant   |      |
|               | corn plants or from genetically modified *E. |      |
|               | coli,* will be rapidly digested in a         |      |
|               | simulated gastric environment.               |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Amino acid    | A.  The purpose of this study was to         | 4688 |
| sequence      |     determine if Vip3Aa19 had any            | 5903 |
| comparison/   |     significant amino acid sequence homology |      |
| Vip3Aa19:     |     to known protein toxins. No relevant     |      |
| Assessment of |     similarities between the Vip3Aa19 query  |      |
| amino acid    |     sequence and known protein toxins were   |      |
| sequence      |     found other than with other              |      |
| homology with |     insect-specific vegetative insecticidal  |      |
| known toxins. |     proteins of *B. thuringiensis*.          |      |
|               |                                              |      |
| Report No.    | B.  **Classification: Acceptable; Supersedes |      |
| SSB-122-06^4^ |     MRID 457665-02**                         |      |
+---------------+----------------------------------------------+------+
| Amino acid    | C.  The purpose of this study was to         | 4688 |
| sequence      |     determine if Vip3Aa19 had any            | 5906 |
| comparison/   |     significant amino acid sequence homology |      |
| Vip3Aa19:     |     to known protein allergens. Vip3Aa19 had |      |
| Assessment of |     no significant amino acid sequence       |      |
| amino acid    |     homology to known or putative allergenic |      |
| sequence      |     proteins.                                |      |
| homology with |                                              |      |
| known         | D.  **Classification: Acceptable; Supersedes |      |
| allergens.    |     MRID 457665-02**                         |      |
|               |                                              |      |
| Report No.    |                                              |      |
| SSB-130-06^4^ |                                              |      |
+---------------+----------------------------------------------+------+
| Amino acid    | The purpose of the study was to determine if | 4686 |
| sequence      | Event MIR162 Vip3A protein had any           | 4808 |
| comparison/   | significant amino acid sequence homology to  |      |
| Vip3A as      | known or putative protein toxins. The        |      |
| expressed in  | database identified 32 entries with E values |      |
| Event MIR162  | below 6 x 10^-6^, of which 30 were           |      |
| maize:        | vegetative insecticidal proteins of *B.      |      |
| Assessment of | thuringiensis* and had E values of 0.0 to 1  |      |
| amino acid    | x 10^-10^. Two proteins were identified as   |      |
| sequence      | rhoptry proteins from *Plasmodium yoelii*, a |      |
| homology with | pathogen that causes malaria in rodents      |      |
| known         | *via* erythrocyte binding and invasion (Ogun |      |
| toxins^3^     | and Holder, 1996). Despite the pathogenic    |      |
|               | nature of *P. yoelii*, the low overall       |      |
|               | sequence similarity between MIR162 Vip3A and |      |
|               | the rhoptry proteins (3.9 or 11.4% overall   |      |
|               | amino acid sequence identity) suggests that  |      |
|               | the E values are of no biological            |      |
|               | significance (Doolittle, 1990). Furthermore, |      |
|               | a global protein alignment (Myers and        |      |
|               | Miller, 1988) demonstrates that there are no |      |
|               | more than three contiguous identical amino   |      |
|               | acids between Vip3A and the rhoptry          |      |
|               | proteins. Therefore, no relevant             |      |
|               | similarities between the Event MIR162 Vip3A  |      |
|               | query sequence and known protein toxins were |      |
|               | found.                                       |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
|               |                                              |      |
|               | E.                                           |      |
+---------------+----------------------------------------------+------+
| Amino acid    | The purpose of this study was to determine   | 4686 |
| sequence      | if Event MIR162 Vip3Aa20 had any significant | 4809 |
| comparison/   | amino acid sequence homology to known or     |      |
| Vip3A as      | putative protein allergens. No significant   |      |
| expressed in  | sequence homology was found between any      |      |
| Event MIR162  | sequential MIR162 Vip3A 80-amino acid        |      |
| maize:        | peptides and any entry in the SBI Allergen   |      |
| Assessment of | Database. No alignments of eight or more     |      |
| amino acid    | contiguous identical amino acids were        |      |
| sequence      | identified between MIR162 Vip3A and proteins |      |
| homology with | in the SBI Allergen Database. Therefore, no  |      |
| known         | significant amino acid sequence homology was |      |
| allergens^3^  | found between the MIR162 Vip3A and any known |      |
|               | or putative protein allergens.               |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Analysis of   | The purpose of this study was to determine   | 4701 |
| Vip3A or      | whether Vip3A or Vip3A-like proteins are     | 7613 |
| Vip3A-Like    | detectable and quantifiable in commercial    |      |
| Proteins in   | formulations of *Bacillus thuringiensis*     |      |
| Six Different | (Bt)-based microbial insecticide products.   |      |
| Commercial    | ELISA (enzyme-linked immunosorbent assay)    |      |
| Microbial     | and Western blot analyses were used to       |      |
| *Bacillus     | detect and analyze Vip3A or Vip3A-like       |      |
| t             | proteins in the formulations. Vip3A or       |      |
| huringiensis* | Vip3A-like proteins were detected in all six |      |
| Products      | commercial products, with concentrations     |      |
|               | ranging from a low of *ca*. 2.0 µg/g product |      |
|               | to a high of *ca*. 209 µg/g. Those products  |      |
|               | showing the highest protein concentrations   |      |
|               | were all derived from the *kurstaki*         |      |
|               | subspecies of *B. thuringiensis*.            |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+
| Amino acid    | Two amino acid sequences comparisons of      | 4701 |
| sequence      | Vip3Aa19 with known allergens were conducted | 7617 |
| comparison/   | using the Syngenta Biotechnology, Inc (SBI)  |      |
| Vip3Aa19:     | Allergen Database. The results indicate that |      |
| Assessment of | Vip3Aa19 has no significant amino acid       |      |
| amino acid    | sequence homology to known or putative       |      |
| sequence      | allergenic proteins based on a search for    |      |
| homology with | greater than 35% sequence identity over      |      |
| known         | successive 80-amino acid peptides and a      |      |
| allergens     | search for eight or more contiguous          |      |
|               | identical amino acids.                       |      |
|               |                                              |      |
|               | **Classification: ACCEPTABLE**               |      |
+---------------+----------------------------------------------+------+

**[Human Health Assessment of Modified Cry1Ab Containing 26 Additional
Amino Acids]{.underline}**

**Background**

EPA has previously established an exemption from the requirement of a
tolerance for *Bacillus thuringiensis* Cry1Ab protein in all plants when
used as a plant-incorporated protectant in all food commodities (40 CFR
174.511). Event COT67B cotton produces 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. The additional amino acids have no
impact on insecticidal activity, as they are in the protoxin portion of
the protein, which is cleaved off during toxin activation. However,
because Cry1Ab proteins in nature do not contain these additional amino
acids, EPA has determined that the tolerance exemption at 40 CFR 174.511
does not cover this modified protein and is therefore establishing a new
tolerance exemption for the modified Cry1Ab protein.

Section 408(c)(2)(A)(i) of the FFDCA allows EPA to establish an
exemption from the requirement for a tolerance (the legal limit for a
pesticide chemical residue in or on a food) only if EPA determines that
the exemption is "safe." Section 408(c)(2)(A)(ii) of the FFDCA defines
"safe" to mean that "there is a reasonable certainty that no harm will
result from aggregate exposure to the pesticide chemical residue,
including all anticipated dietary exposures and all other exposures for
which there is reliable information." This includes exposure through
drinking water and in residential settings, but does not include
occupational exposure. Pursuant to section 408(c)(2)(B), in establishing
or maintaining in effect an exemption from the requirement of a
tolerance, EPA must take into account the factors set forth in section
408(b)(2)(C), which require EPA to give special consideration to
exposure of infants and children to the pesticide chemical residue in
establishing a tolerance and to "ensure that there is a reasonable
certainty that no harm will result to infants and children from
aggregate exposure to the pesticide chemical residue\...."

Additionally, section 408(b)(2)(D) of the FFDCA requires that the Agency
consider "available information concerning the cumulative effects of a
particular pesticide's residues" and "other substances that have a
common mechanism of toxicity." EPA performs a number of analyses to
determine the risks from aggregate exposure to pesticide residues.
First, EPA determines the toxicity of pesticides. Second, EPA examines
exposure to the pesticide through food, drinking water, and through
other exposures that occur as a result of pesticide use in residential
settings.

**Toxicological Profile**

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

**Mammalian Toxicity and Allergenicity Assessment**

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'.
These data demonstrate the safety of the product at a level well above
maximum possible exposure levels that are reasonably anticipated in the
crop. Basing this conclusion on acute oral toxicity data without
requiring further toxicity testing and residue data is similar to the
Agency position regarding toxicity testing and the requirement of
residue data for the microbial *Bacillus thuringiensis* products from
which this plant-incorporated protectant was derived (See 40 CFR
158.2140). 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, Roy D., et al., "Toxicological
Considerations for Protein Components of Biological Pesticide Products,"
Regulatory Toxicology and Pharmacology 15, 3-9 (1992)). Therefore, since
no acute effects were shown to be caused by modified Cry1Ab, even at
relatively high dose levels, the modified Cry1Ab protein is not
considered toxic.

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

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

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

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

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

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

Although modified Cry1Ab was only shown not to be glycosylated in
cotton, it is unlikely to be glycosylated in any other crops because in
order for a protein to be glycoslyated, it needs to contain specific
recognition sites for the enzymes involved in glycosylation, and the
mechanisms of protein glycosylation are similar in different plants
(Lerouge, P. Cabanes-Macheteau, M., Rayon, C., Fichette-Lainé, A-C.,
Gomord, V., and Faye, L., "N-Glycoprotein biosynthesis in plants: recent
developments and future trends," *Plant Molecular Biology* **38**:
31-48, 1998).

**2. 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. Oral exposure, at very low levels, may occur
from ingestion of processed corn products and, theoretically, drinking
water. However, oral toxicity testing showed no adverse effects.

**3. 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.

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

**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, Roy D., et al.,
"Toxicological Considerations for Protein Components of Biological
Pesticide Products," Regulatory Toxicology and Pharmacology 15, 3-9
(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.

**b) 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.

**c) 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.

**5. Other Considerations**

**a) 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.

**b) Analytical Method(s)**

A validated lateral flow enzyme-linked immubsorbent assay (ELISA)
protocol has been provided to the Agency for detecting modified Cry1Ab
in cotton.

**c) Codex Maximum Residue Level**

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

**References**

Sjoblad, R. D., McClintock, J. T., and Engler, R., "Toxicological
Considerations for Protein Components of Biological Pesticide Products,"
*Reg. Toxicol. Pharmacol*. 15(1), **1992**, 3-9.

Table 8. Summary of Modified Cry1Ab Human Health Data

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

**[HUMAN HEALTH ASSESSMENT Hygromycin B Phosphtransferase
(APH4)]{.underline}**

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**

[47017618---APH4 (Entrez Database accession No. CAA85741): Assessment of
Amino Acid Sequence Homology with Known Allergens:]{.underline}

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

**Classification: ACCEPTABLE**

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

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

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

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