Document ID: EPA-HQ-OPP-2007-1204-0005
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2008-07-17T04:00Z

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

     WASHINGTON, D.C. 20460

 ADVANCE \d61 	

	OFFICE OF PESTICIDES AND TOXIC 

	SUBSTANCES        

MEMORANDUM			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. 

 

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:

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

PRODUCT CHARACTERIZATION

Event COT102 Cotton (OECD Unique Identifier:SYN-IR102-7) Expressing
Vip3Aa19 

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

Transformation System: 

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

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

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

Protein Characterization: 

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

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

Analytical Detection Methods:

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

Protein Expresson: 

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

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

Tissue Type	Vip3Aa19

(µg/g dry weight + standard deviation)	APH4

(µg/g dry weight + standard deviation)

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

Squares	116 + 22	2.2 + 0.4

Flowers	162	1.68

Pollen	3.47	64.3

Bolls	19 + 4	< 0.39

Whole Plants	25 + 4	< 0.37

Seed	7 + 2	1.4 + 0.3

Roots	16 + 2	0.53 + 0.11

*Ranges reflect means at different growth stages for leaves

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

Table 2. Product Characterization Data Submitted for Event COT102 Cotton

Study Type/Title	

Summary	

MRID #

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

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

Classification:  ACCEPTABLE	

45835801

Characterization of Inserted DNA/Molecular Characterization and Genetic
Stability of Event COT1022	

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

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

Superseded by MRID 47017603	

45835802

Characteristics of Bacillus thuringiensis VIP3A Protein and VIP3A Cotton
Plants Derived from Event COT1022	

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

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

45766501

Characterization of the active ingredient/Characterization of VIP3A
Protein Produced in COT102-Derived Cotton and Comparison with VIP3A
Protein Expressed in Both Maize (Corn) Derived from Event PACHA and
Recombinant Escherichia coli2	

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

Classification:  ACCEPTABLE	

45835812

Expression Level/ Analysis of Processed COT102 Cottonseed Products for
Yield and Presence of Gossypol and Vip3A Protein2	

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

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

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

45835803

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

Classification: ACCEPTABLE  

	46880801

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

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

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

Classification: ACCEPTABLE	

47017602

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

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

Classification: ACCEPTABLE	

47017603

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

Classification: ACCEPTABLE	

47017609

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

Event COT67B cotton, which was developed by Agrobacterium-mediated
transformation of cotton using elements of vectors pNOV4641 and
pNOV1914, expresses the insecticidal protein, modified Cry1Ab 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 + standard deviation)*

Leaves	65 + 9 – 158 + 40

Squares	93 + 13

Flowers	101

Pollen	12.1

Bolls	47 + 7

Whole Plants	26 + 2

Seed	29 + 5

Roots	17 + 1

*Range reflects means at different growth stages for leaves

Table 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 Type/Title	

Summary	

MRID #

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

Report No. SSB-125-06.	

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

Classification:  ACCEPTABLE	

46885901

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

Report No.SSB-022-064	

The purpose of this study was to quantify expression of Cry1Ab protein
in Event COT67B-derived cotton plants.  Quantifiable levels of Cry1Ab
protein in Event COT67B-derived cotton plants were determined by
enzyme-linked immunosorbent assay (ELISA) for various plant tissues and
whole plants at five developmental stages in four locations.
Corresponding, near-isogenic, non-transgenic control cotton plants were
analyzed in parallel. As expected, Cry1Ab protein was detected in all
COT67B plant tissues (i.e., young leaves, old leaves, roots, flowers,
pollen, bolls) except fiber 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 1st open boll), whole plants (collected at
pre-harvest), and seed (collected at pre-harvest) averaged 45.24, 42.87,
and 25.17 μg/g dw across locations, respectively. Cry1Ab concentrations
in flowers and pollen collected at the Winnsboro, LA site at peak-bloom
averaged 161.74 and 5.45 μg/g dw, respectively. Cry1Ab concentrations
in nectar taken from the same cotton plants was not detectable (limit of
detection = 0.0002 μg/mL). Cry1Ab concentrations in fiber samples
collected at this site at pre-harvest was <0.02 μg/g dw

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

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

Superseded by MRID 47017607.	

46885902

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

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

Classification: ACCEPTABLE	

47017604

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

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

Classification: ACCEPTABLE	

47017605

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

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

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

Classification: ACCEPTABLE	

47017608

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

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

Classification: ACCEPTABLE	

47017610

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

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

Classification: ACCEPTABLE	

47017611

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

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

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

Analytical Detection Methods:

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

Protein Expresson: 

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

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

Tissue Type	Vip3Aa19

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

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

(µg/g dry weight + standard deviation)*

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

Squares	132 + 18	2.1 + 0.5	94 + 10

Flowers	148	1.80	121

Pollen	3.06	74.7	10.7

Bolls	21 + 4	< 0.43	42 + 7

Whole Plants	25 + 7	< 0.40	29 + 7

Seed	7 + 1	1.6 + 4	27 + 4

Roots	11 + 3	0.46 + 0.05	20 + 4

*Ranges reflect means at different growth stages for leaves

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

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

Study Type/Title	

Summary	

MRID #

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

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

Classification:  ACCEPTABLE	

47017606

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

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

Classification: ACCEPTABLE	

47017607

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

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

Classification: ACCEPTABLE	

47074101

Human Health Assessment of Vip3Aa

 ADVANCE \d4 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 LD50 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:

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

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

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

Glycosylation.  Vip3Aa was shown not to be glycosylated. 

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 Type/Title	

Summary	

MRID #

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

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

Classification: SUPPLEMENTAL, 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.	

45766502

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

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

Classification: SUPPLEMENTAL. The VIP3A protein used in this study
differs from the VIP3A protein present in COT102 cotton by a 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.  	

45766503

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

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

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

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

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

Classification: Acceptable	

45766505

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

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

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

	45766506

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

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

Classification: ACCEPTABLE

	

45835805

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

Report No. SSB-122-064	

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

Classification:  Acceptable; Supersedes MRID 457665-02

	

46885903

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

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

Classification: Acceptable; Supersedes MRID 457665-02

	46885906

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

Classification: ACCEPTABLE

	46864808

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

Classification: ACCEPTABLE

	46864809

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

Classification: ACCEPTABLE 	47017613

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

Classification: ACCEPTABLE	47017617

Human Health Assessment of Modified Cry1Ab Containing 26 Additional
Amino Acids

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.    

 ADVANCE \d4 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:

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

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

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

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

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

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

Summary	

MRID #

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

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

Classification: ACCEPTABLE	

47017614

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

Classification: ACCEPTABLE	47017615

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

5˚C or 95˚C for 30 minutes substantially decreased or eliminated the
insecticidal activity of the protein.  No significant effect on the
protein’s insecticidal properties was found following incubation for
30 minutes at temperatures ≤37˚C.

Classification: ACCEPTABLE	

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ver successive 80-amino acid peptides and a search for eight or more
identical contiguous amino acids. 

Classification: ACCEPTABLE 	47017619

HUMAN HEALTH ASSESSMENT Hygromycin B Phosphtransferase (APH4)

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

Summary of new data submitted for APH4

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

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

Classification: ACCEPTABLE

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

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

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

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

	

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