Document ID: EPA-HQ-OPP-2006-0783-0005
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2007-04-04T04:00Z

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY   

           		WASHINGTON, D.C. 20460

	

				

						FEB 08 2007		OFFICE OF PREVENTION,                                
              PESTICIDES AND TOXIC                                      
                                 SUBSTANCES       

MEMORANDUM

SUBJECT:	Review of Product Characterization and Human Health Data for
Plant- Incorporated Protectant Bacillus thuringiensis (Bt) Vip3Aa20
insect control protein (EPA Reg. No. 67979-EUP-A) and the genetic
material necessary for its production in Event MIR162 corn in support
for a temporary exemption from tolerances and Experimental Use Permit
(EUP), submitted by Syngenta Seeds, Inc. – Field Crops- NAFTA.

TO:	Mike Mendelsohn, Regulatory Action Leader

	Microbial Pesticides Branch, Biopesticides and

	Pollution Prevention Division (7511P)

FROM:	Annabel Waggoner, Environmental Protection Specialist	[signed]		

	Microbial Pesticides Branch, Biopesticides and		

	Pollution Prevention Division (7511P)

THROUGH:	John L. Kough, Ph.D., Biologist					[signed]

	Microbial Pesticides Branch, Biopesticides and			

	Pollution Prevention Division (7511P)

ACTION REQUESTED: To review product characterization, toxicological and
allergenicity data, human health data submitted by Syngenta Seeds,
Inc.-Field Crops – NAFTA, in support for an Experimental Use Permit
and a temporary exemption from tolerances for Bt Vip3Aa20 insect control
protein expressed in Event MIR162 corn.  

CONCLUSION:    The product characterization, toxicological and
allergenicity data support the finding that there is a reasonable
certainty that no harm will result from aggregate exposure to the U.S.
population, including infants and children, to the Vip3Aa20 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. Therefore, the product characterization and human
health data submitted are sufficient to support an EUP and a temporary
exemption from the requirement of a tolerance. There were several
studies classified as “SUPPLEMENTAL, but UPGRADEABLE”. These data
discrepancies do not ultimately affect the findings of the safety
assessment for the proposed EUP; however, these data should be submitted
with the Sec. 3 Registration in order to complete the Vip3Aa20 database
for Event MIR162.

*THIS REVIEW DOES NOT CONTAIN CONFIDENTIAL BUSINESS INFORMATION*

DATA REVIEW RECORD:

Active Ingredient:	Bacillus thuringiensis Vip3Aa20 insecticidal protein
and the genetic material necessary for their production (via pNOV1300)
in transgenic corn plants derived from Syngenta Seeds’ transformation
Event MIR162. 

Product Name:	Event MIR162 Corn Plants Expressing Vip3Aa20 Bacillus
thuringiensis Protein [EUP Reg. No. 67979-A]

Company Name:	Syngenta Seeds, Inc. – Field Corps- NAFTA

ID No:		67979

Chemical Number:	006599

Decision Number:	368367

DP Barcode:	332013						

MRID No:	

		

Product Characterization and Identity 

468648-01 	Molecular characterization of Event MIR162 maize

468648-02	Characterization of the Vip3A protein expressed in Event
MIR162-derived maize (corn) and comparison with microbially produced and
plant-derived Vip3A test substances

468648-03  	Characterization of Vip3A protein test substance
(VIP3A-0104) and certificate of analysis

468648-04  	Characterization of Vip3A protein test substance
(VIP3A-0204) and certificate of analysis

468648-05	Re-characterization of Vip3A protein test substance
(VIP3A-0204) and certificate of analysis

468648-06	Characterization of VIP3A protein produced in Pacha-derived
maize (corn) and comparison with VIP3A protein expressed in recombinant
E. coli

468648-07	Analytical method for the detection of Vip3Aa20 protein in
maize tissues from event MIR162

ive insecticidal protein Vip3A differs from that of Cry1Ab δ-endotoxin

		

Human Toxicity and Allergenicity

468648-08 	Vip3A as expressed in Event MIR162 maize: Assessment of amino
acid sequence homology with known toxins

468648-09	Vip3A as expressed in Event MIR162 maize: Assessment of amino
acid sequence homology with known allergens

468648-10	Phosphomannose isomerase protein: Assessment of amino acid
sequence homology with known toxins

468648-11	Phosphomannose isomerase: Assessment of amino acid sequence
homology with known allergens

BACKGROUND:

Vip3A is a vegetative insecticidal protein (Vip) from Bacillus
thuringiensis (Bt), a gram positive bacterium commonly found in soil. 
Vip proteins are produced during the vegetative stage of bacterial
growth and are active against the following lepidopteran pests of corn: 
Spodoptera frugiperda (fall armyworm), Pseudaletia unipunctata
(armyworm), Spodoptera exigua (beet armyworm), Helicoverpa zea (corn
earworm / cotton bollworm), Agrotis ipsilon (black cutworm), and
Striacosta albicosta (western bean cutworm).   

           Table 1.  Comparison of amino acid residues at positions 129
and 284 in                                                           
different variants of Vip3Aa protein

              Source of Vip3Aa Proteins	            Toxin Designation	  
          Amino Acids	Position  129 *            	          Position 284
*

              Bacillus thuringiensis strain AB88	Vip3Aa1	       789	M	K

              COT102 cotton	Vip3Aa19	       789	M	Q

              Pacha Corn	Vip3Aa19	      789	M	Q

              MIR162 corn	Vip3Aa20	      789	I	Q

 The native Vip protein, Vip3Aa1, was isolated from Bt strain AB88 and
characterized by Estruch et al. in 1996.  Syngenta has engineered a
variant of the native gene for incorporation into corn.  This engineered
gene has been designated, vip3Aa20, and it has been stably incorporated
(via pNOV1300 vector) into the genome of Event MIR162 corn by
Agrobacterium-mediated transformation.  The Vip3Aa20 protein encoded by
this gene is approximately 89 kDa molecular weight and 789 amino acids
in length differing by two amino acids from the native Vip3A.  The
sequence differences occur at positions 129 and 284 (M1291, K284Q). 
Another variant of Vip3Aa is also present as a PIP in Syngenta Event
COT102 cotton [EPA Reg. No. 67979-O] and Event Pacha corn; this variant
has been assigned the designation Vip3Aa19.  Vip3Aa19 differs from the
native Vip3Aa1 sequence by one amino acid at position 284, while
differing from the Vip3Aa20 by one amino acid at position 129 (Crickmore
et al. 2005). These substitutions are conservative and do not materially
impact insecticidal activity.  In fact, Vip3Aa20 shares >99.7% sequence
homology with the native protein (Vip3Aa1) and Vip3Aa19. Table 1
compares selected sequence information for different sources of Vip3Aa
discussed in this report.

* M = methionine, I = isoleucine, k = lysine, Q = glutamine

Event MIR162 corn also contains the pmi gene, which was introduced along
with the Vip3Aa20 protein via the same pNOV1300 transformation vector. 
The gene represents the manA gene from Escherichia coli and encodes the
enzyme phosphomannose isomerase (PMI), which was employed as a
selectable marker during the process of regenerating plant material
following transformation (Negrotto, et al., 2000).  

Syngenta Seeds, Inc. is applying for an experimental use permit (EUP) to
conduct field tests on Event MIR162 corn.  The EUP program for MIR162
corn will also involve the production and evaluation of combined trait
hybrids.  MIR162 will be crossed with corn Event Bt11 and with corn
Event MIR604 to produce combined trait hybrids that offer a broader
insect control spectrum than MIR162 alone.  Bt11 maize expressed the
insecticidal protein Cry1Ab for control of Ostrinia nubilalis (European
cornborer); MIR604 expresses the modified Cry3A (mCry3A) insecticidal
protein for control of certain species of Diabrotica (corn rootworms). 
Data have been previously submitted to the Agency demonstrating
mammalian safety of Cry1Ab produced in Bt11 maize (EPA, 2001) and mCry3A
produced in MIR604 maize (EPA, 2006).  

The proposed planting under the EUP will take place in 22 states and
Puerto Rico for a total of 3,099 total acres of MIR162, hybrids thereof,
and non-PIP corn. The objectives of the experimental field program are
to introgress Event MIR162 into elite inbred lines of corn, to evaluate
the insecticidal efficacy of the MIR162 corn hybrids, as well as
combined insecticidal trait hybrids (Bt11 x MIR162 and Bt11 x MIR162 x
MIR604) and to evaluate their agronomic performance, to conduct field
IRM studies, and to conduct field studies supporting regulatory
applications. The proposed experimental program and protocols were
reviewed and found acceptable by the Agency [see memorandum- from T.
Milofsky, M.S., through M. Hunter, to A. Reynolds, M.S., dated
12/06/2006].   

Studies have been conducted and previously submitted to EPA to evaluate
the safety of Vip3A proteins. Since the registrant is bridging this data
in support for an EUP for Vip3Aa20, a table summarizing the submitted
study titles, conclusions, and their MRID numbers (see Table 2) as well
as a table demonstrating the protein equivalence for all Vip3A variants
according to varying data parameters (see Table 3) are provided in this
report as well.  To satisfy the remaining data requirements specific for
the EUP for Vip3Aa20, Syngenta Seeds, Inc. has submitted product
characterization, toxicity, and allergenicity data, which are reviewed
in this report.   The registrant also submitted additional data for PMI
to be reviewed in this report- specifically an updated amino acid
homology assessment of PMI to known toxins and allergens, which will
supersede the previously reviewed PMI data.  

Temporary Exemption for the Requirement of a Tolerance 

In the Federal Register of  April 26, 2006 (71 FR 24582), the Agency
established a temporary exemption from the requirement of a tolerance
for Bacillus thuringiensis VIP3A insect control protein and the genetic
material necessary for their production in cotton [see 40 CFR 174.452],
which will expire May 1, 2007.  An exemption from the requirement of a
tolerance has been established for PMI in all crops when used as a
plant-incorporated protectant inert ingredient (see 40 CFR 180.1252,
effective May 14, 2004).  

In conjunction with the EUP, Syngenta Seeds, Inc. – Field Crops- NAFTA
has submitted a petition for a temporary exemption from the requirement
of a tolerance pursuant to section 408(d)(1) of the Federal Food, Drug,
and Cosmetic Act with respect to the plant-incorporated protectant
Vip3Aa20 Bacillus thuringiensis insect control protein and the genetic
material necessary for its production in all field corn, sweet corn, and
popcorn.  

 

Preliminary Safety Assessment

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. Section 408(b)(2)(C) of the FFDCA
requires 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. 

Product Characterization Profile

A Vip3Aa20 Bacillus thuringiensis (Bt) insect control protein is
produced in transgenic corn plants derived from transformation Event
MIR162.  A vip3Aa20 gene was synthetically created to optimize for
expression in corn with activity against several major lepidopeteran
corn pests.  Introduced via transformation vector pNOV1300, a vip3Aa20
specific probe, consisting of 2370 base pairs (bp), was incorporated
between a promoter region from the Zea mays polyubiquitin gene (ZmUbInt
(1993 bp)) and a terminator sequence from the 35S RNA from the
cauliflower mosaic virus genome.  An Escherichia coli manA gene encoding
a phosphomannose isomerase pmi gene (1176 bp) was incorporated between
the same promoter region from the Z. mays polyubiquitin gene (1993 bp)
and a terminator sequence from the nopaline synthase gene (NOS (253 bp))
 of Agrobacterium tumefaciens, which was used to provide a
polyadenylation site.  The pmi gene, which was introduced along with the
Vip3Aa20 protein via the same pNOV1300 transformation vector, encodes
the enzyme phosphomannose isomerase (PMI), which is employed as a
selectable marker during the process of regenerating plant material
following transformation.  The PMI protein is a common enzyme involved
in carbohydrate metabolism to allow for selection of transformants in
cell culture, by only allowing transformed corn cells to utilize mannose
as a sole carbon source, while corn cells lacking the pmi gene fail to
grow.   

Southern blot analyses and DNA sequencing indicate that one full length
copy of each of the vip3Aa20 and pmi genes were integrated into the
maize genome, without the backbone sequences from transformation plasmid
pNOV1300.  Therefore, the overall integrity of the insert and the
contiguousness of the functional elements were confirmed.

The native Vip protein, Vip3Aa1, was isolated from Bt strain AB88 and
characterized by Estruch et al. in 1996.  Syngenta has engineered a
variant of the native gene for incorporation into corn.  This engineered
gene has been designated, vip3Aa20, and it has been stably incorporated
(via pNOV1300 vector) into the genome of Event MIR162 corn by
Agrobacterium-mediated transformation.  The Vip3Aa20 protein encoded by
this gene is approximately 89 kDa molecular weight and 789 amino acids
in length differing by two amino acids from the native Vip3A.  The
sequence differences occur at positions 129 and 284 (M1291, K284Q). 
Another variant of Vip3Aa is also present as a PIP in Syngenta Event
COT102 cotton and Event Pacha corn; this variant has been assigned the
designation Vip3Aa19.  Vip3Aa19 differs from the native sequence by one
amino acid at position 284, while differing from the Vip3Aa20 by one
amino acid at position 129. These substitutions do not appear to
materially impact insecticidal activity.  Vip3Aa20 shares >99.7%
sequence homology with the native protein, Vip3Aa1 and Vip3Aa19.  

Data have been submitted demonstrating the protein equivalency among the
Vip3Aa1, Vip3Aa19, and Vip3Aa20 proteins and their respective protein
test substances, expressed in recombinant E. coli (VIP3A-0199,
VIP3A-0100, VIP3A-0104, and VIP3A-0204) or maize (LPPACHA-0199,
LPMIR162-0105, and IAPMIR162-0105) for use as a surrogate in toxicity
experiments (see MRID No. 458358-12 and 468648-03, -04, -05, and -06). 
Since equivalency has been established for the Vip3A protein variants,
all previous submitted data from Vip3Aa1 and Vip3Aa19 can be bridged to
Vip3Aa20.  

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. 

Data have been submitted demonstrating the lack of mammalian toxicity at
high levels of exposure to the pure Vip3A-0100 protein. These data
demonstrate the safety of the products at levels well above maximum
possible exposure levels that are reasonably anticipated in the crops.
This 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 158.740(b)(2)(i)). For microbial products, further toxicity
testing and residue data are triggered by significant acute effects in
studies such as the mouse oral toxicity study, to verify the observed
effects and clarify the source of these effects (Tiers II and III). 

An acute oral toxicity study was submitted for the Vip3Aa19 protein.
Male and female mice (16 of each) were dosed with 3,675
milligrams/kilograms bodyweight (mg/kg bwt) of Vip3Aa19 protein. All
mice survived the study, gained weight, had no test material-related
clinical signs, and had no test material-related findings at necropsy. 
The acute oral toxicity data submitted also supports the prediction that
the Vip3Aa20 protein would be non-toxic to humans. 

 

When proteins are toxic, they are known to act via acute mechanisms and
at very low dose levels (Sjoblad, Roy D., et al. 1992). Therefore, since
no effects were shown to be caused by the plant- incorporated
protectants, even at relatively high dose levels, the Vip3Aa20 protein
is not considered toxic. Amino acid sequence comparisons showed no
similarity between the Vip3Aa20 protein to known toxic proteins
available in public protein data bases.  According to the Codex
Alimintarius guidelines, the assessment of potential toxicity also
includes stability to heat (FAO/WH  O Standards Programme, 2001).  A
heat lability study demonstrated that Vip3Aa19 is inactivated against
FAW, when heated to 55 (C for 30 minutes.  

Since Vip3Aa20 is a protein, allergenic sensitivities were considered.
Currently, no definitive tests exist for determining the allergenic
potential of novel proteins.  Therefore, EPA uses a weight of the
evidence approach where the following factors are considered: source of
the trait; amino acid sequence similarity with known allergens;
prevalence in food; and biochemical properties of the protein, including
in vitro digestibility in simulated gastric fluid (SGF), and
glycosylation.  Current scientific knowledge suggests that common food
allergens tend to be resistant to degradation acid and proteases; may be
glycosylated; and present at high concentrations in the food. 

Data have been submitted that demonstrate that the Vip3A from
recombinant maize (LPPACHA-0199) and E. coli (VIP3A-0100) proteins are
rapidly degraded by gastric fluid in vitro. In a solution of simulated
gastric fluid (containing pepsin) and either 80 µL of LPPACHA-0199 or
320 µL of VIP3A-0100 test protein, both were shown to be susceptible to
pepsin degradation. These data support the conclusion that Vip3Aa
proteins expressed transgenic plants will be readily digested as
conventional dietary protein under typical mammalian gastric conditions.
Further data demonstrate that Vip3Aa20 is not glycoslylated and a
comparison of amino acid sequences of known allergens uncovered no
evidence of any homology with Vip3Aa20, even at the level of 8
contiguous amino acids residues. Preliminary data of the quantification
of Vip3Aa20 protein in various maize tissues were also submitted.  The
data demonstrated that mean Vip3Aa20 concentrations in kernels ranged
from ca. 24.6 - 40.3 µg Vip3Aa20/ g dry weight, representing ca. 0.003%
of the total protein in grain (assuming that corn grain contains 10%
total protein by weight).  Therefore, Vip3Aa20 is present in low levels
in corn tissue. 

Therefore, the potential for the Vip3Aa20 protein to be a food allergen
is minimal. As noted above, toxic proteins typically act as acute toxins
with low dose levels. Therefore, since no effects were shown to be
caused by the plant-incorporated protectant, even at relatively high
dose levels, the Vip3Aa20 protein is not considered toxic. 

Aggregate Exposures

In examining aggregate exposure, section 408 of the FFDCA directs EPA to
consider available information concerning 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 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. The amino acid homology assessment
included similarity to known aeroallergens.  It has been demonstrated
that there is no evidence of occupationally related respiratory
symptoms, based on a health survey on migrant workers after exposure to
Bt pesticides (Berstein et al. 1999).  Exposure via residential or lawn
use to infants and children is also not expected because the use sites
for the Vip3Aa20 protein are all agricultural for control of insects.
Oral exposure, at very low levels may occur from ingestion of processed
corn products and, potentially, drinking water. 

However, oral toxicity testing done at a dose in excess of 3 gm/kg
showed no adverse effects. Furthermore, the expected dietary exposure
from both cotton and corn are several orders of magnitude lower than the
amounts of Vip3Aa protein shown to have no toxicity. Therefore, even if
negligible aggregate exposure should occur, the Agency concludes that
such exposure would present no harm due to the lack of mammalian
toxicity and the rapid digestibility demonstrated for the Vip3Aa
proteins. 

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,
resulting from the plant-incorporated protectant, we conclude that there
are no cumulative effects for the Vip3Aa20 protein. 

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
Vip3Aa20 protein include the characterization of the expressed Vip3Aa20
protein in corn, as well as the acute oral toxicity, heat stability, and
in vitro digestibility of the proteins. The results of these studies
were determined applicable to evaluate human risk, and the validity,
completeness, and reliability of the available data from the studies
were considered. 

Adequate information was submitted to show that the Vip3Aa20 protein
test material derived from microbial cultures was biochemically and
functionally similar to the protein produced by the plant-incorporated
protectant ingredients in corn.  Microbially produced protein was chosen
in order to obtain sufficient material for testing.

The acute oral toxicity data submitted supports the prediction that the
Vip3Aa20 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. 1992). Since no effects
were shown to be caused by Vip3Aa20 protein, even at relatively high
dose levels (3,675 mg Vip3Aa19/kg bwt), the Vip3Aa20 protein is not
considered toxic. This 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 158.740(b)(2)(i)).   Moreover, Vip3Aa20 showed no
sequence similarity to any known toxin.

Protein residue chemistry data for Vip3Aa20 were not required for a
human health effects assessment of the subject plant-incorporated
protectant ingredients because of the lack of mammalian toxicity.
However, preliminary data (that was submitted with administrative
materials in the EUP application) demonstrated low levels of Vip3Aa20 in
corn tissues with less than 40 micrograms Vip3Aa20 protein/gram dry
weight in kernels and less than 75 micrograms Vip3Aa20 protein/gram dry
weight of whole corn plant. 

Since Vip3Aa20 is a protein, its potential allergenicity is also
considered as part of the toxicity assessment. Data considered as part
of the allergenicity assessment include that the Vip3Aa20 protein came
from Bacillus thuringiensis which is not a known allergenic source,
showed no sequence similarity to known allergens, was readily degraded
by pepsin, and was not glycosylated when expressed in the plant.
Therefore, there is a reasonable certainty that Vip3Aa20 protein will
not be an allergen. 

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 Vip3Aa20 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 in the crop. 

The genetic material necessary for the production of the
plant-incorporated protectant active ingredients are the nucleic acids
(DNA, RNA) which comprise genetic material encoding these proteins and
their regulatory regions. The genetic material (DNA, RNA) necessary for
the production of Vip3Aa20 protein has been exempted under the blanket
exemption for all nucleic acids (40 CFR 174.475). 

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(bfl2)(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 data base 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 Vip3Aa20
protein and the genetic material necessary for their production. 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 provisions 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
Vip3Aa20 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 previously discussed, no toxicity to mammals has
been observed, nor has there been any indication of allergenicity
potential for the plant-incorporated protectant. 

Other Considerations 

A. Endocrine Disruptors 

The pesticidal active ingredient is a protein, derived from sources that
are 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 method for extraction and ELISA analysis of Vip3Aa20 protein in corn
has been submitted and is under review by the Agency. 

C. Codex Maximum Residue Level 

No Codex maximum residue levels exist for the plant-incorporated
protectant Bacillus thuringiensis Vip3Aa20 protein and the genetic
material necessary for its production in corn. 

RECOMMENDATION:  As previously noted, there is a reasonable certainty
that no harm will result from aggregate exposure to the U.S. population,
including infants and children, to the Vip3Aa20 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. Therefore, the product characterization and human health
data submitted are sufficient to support an Experimental Use Permit and
a temporary exemption from the requirement of a tolerance.  However,
there were several studies classified as “SUPPLEMENTAL, but
UPGRADEABLE” with either clarifications of existing data or
“UNACCEPTABLE”, pending submission of new data. These include:

	MRID No. 468648-01   

The registrant should submit clarification of which specific vector DNA
sequences resulted in non-specific sequence binding in the Southern blot
analyses (Figure 6, lanes 9 and 11). In addition, visual verification is
also required by submitting a Southern blot containing genomic DNA from
MIR162, a negative control, the plasmid control pNOV1300 hybridized with
the pmi-specific probe, and use of a different molecular weight marker
(to avoid non-specific sequence binding). 

MRID No. 468648-06

In Figure 1, the smaller protein bands that the study author attributed
to VIP3A degradation products were not visible in determining the
molecular weight via SDS-PAGE.  The identity of the prominent band in
the E. coli- produced VIP3A sample (lane 7) was also not addressed. 
There was a similar data discrepancy in Figure 2, where smaller bands
were referred to in the text, but not present in the Western blot. 
Therefore, explanations for unidentified bands and better reproductions
of the gels should be provided by the registrant.

In the insect bioassay, the following data discrepancies should be
addressed by the registrant: why the % mortality was not evaluated for
all of the test substances and whether the experimental results were
corrected for control mortality.  

MRID No. 468648-07

A new residue analytical method should be submitted (concurrently with
the Sec. 3 registration of Event MIR162) and specifically conducted on
the MIR162 transgenic grain (single seed and composite) in order for the
ELISA to be verified as a suitable analytical method.   This experiment
should also be validated by an independent third party laboratory
according to OPPTS 860.1340(c)(6) and PR Notice 96-1 with GLP
compliance. The report should also include the following:  	

	1) Qualitative data to represent positive vs. negative transgenic
specific event results with percent accuracy; 

	2) Utilization of a negative control (non- transgenic convention corn
line) and positive control (confirmed transgenic corn line);

		3)  Testing of cross-reactivity against other transgenic events and
other proteins; and

		4)  The intra- and inter- assay coefficient of variation should be
reported.

Once the recommended report has been submitted and found acceptable,
EPA’s Analytical Method Laboratory located in Fort Meade (Maryland)
will have to independently validate Syngenta’s ELISA protocol for
accuracy, precision, and sensitivity.

In regards to establishing field protein expression levels in MIR162
corn tissues and plants, this study is supplemental because it provides
useful information for tissue expression levels to determine exposure
for non-target organisms, for IRM dose levels, and dietary exposure
estimates. However, it does not include quantification of Vip3Aa20
protein levels expressed in various plant tissues and the whole corn
plant. A full report determining the protein concentrations of Vip3Aa20
and PMI at different stages of plant development should be submitted
(including:  the mean, range, and standard deviations) and reported on a
dry weight basis (µg protein/g tissue) with GLP compliance.  This data
requirement can be addressed in the Sec. 3 Registration of Vip3Aa20. 
The study should also include the following:

		1) Standard curve data for the ELISA; 

	2) The calculation method for determining the dry weight conversion
factor from the fresh weight tissue samples; and

	3) Identification of the specific seed line and lot utilized as the
test material with number of field sites and replicates.

These data discrepancies do not ultimately affect the findings of the
safety assessment for the proposed EUP; however, these data should be
submitted with the Sec. 3 Registration in order to complete the Vip3Aa20
database for Event MIR162.

Summaries of each review supporting the safety findings in the areas of
product characterization, human toxicity, and allergenicity for this
product are provided below.

Table 2.  Summary of previously submitted product characterization,
toxicity, and allergenicity studies for Vip3Aa protein variants

MRID	Title	Summary

457665-01	Characteristics of Bacillus thuringiensis VIP3A Protein and
VIP3A Cotton Plants Derived from Event COT102	 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

458358-12	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 coli	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.  Amino acid sequences
corresponded identical to the predicted amino acid sequence of VIP3A and
no evidence of any post-translational modification of VIP3A was
observed. Peptides representing ca. 85% (673/789) of the complete VIP3A
amino acid sequence were identified by mass spectral analysis of cotton
produced VIP3A protein. Comparisons of bioactivity of E. coli-expressed
and cotton-expressed VIP3A protein in larvae of four lepidopteran
species demonstrated comparable activities.  These data indicate that
VIP3A proteins from recombinant E. coli, Pacha-derived maize and event
COT102-derived cotton are substantially equivalent.  

Classification:  Acceptable

457665-02	Summary of Mammalian Toxicology Data for  VIP3A Proteins
Produced by VIP3A Cotton Event COT102	Acute oral toxicity in male and
female mice was not observed 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.

Classification: Acceptable

457665-05	Acute Oral Toxicity Study with Test Substance VIP3A-0100
Protein in Mice	The test animals (male and female Crl-1® (ICR) BR mice,
16 each) 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.  All animals
sacrificed on day 15 had normal body weight gains.  Necropsy findings
showed no test material related macroscopic alterations.  In addition,
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 > 3675 mg VIP3A protein/kg body weight). 

Classification: Acceptable

458358-05	In vitro Digestibility of VIP3A Protein Under Simulated
Mammalian Gastric Conditions	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 via
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. Therefore, 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

458358-04	Summary of Mammalian Toxicology Data for the VIP3A and APH4
Proteins Produced by Transgenic VIP3A Cotton Event COT102; Supplement to
MRID No. 45766502	The study report is a summary of the results reported
in the various reports submitted for consideration of a Section 5 EUP
and a Section 3 registration.  This volume does not constitute a study
in the sense of data collection, but rather a compilation of data and
concepts related to the risk assessment for the VIP3A protein. The data
and information contained in this volume supplement information
previously submitted to the Agency in a summary volume titled “Summary
of Mammalian Toxicology Data for the VIP3A and APH4 Proteins Produced by
Transgenic VIP3A Cotton Event COT102” (MRID No. 45766502; Vlachos,
2002; submitted September 24, 2002). Briefly, the VIP3A protein, as
found in COT102, is non-toxic to mammals at the dose given (LD50 > 3675
mg VIP3A/kg body weight), susceptible to degradation in a simulated
gastric fluid assay, heat labile, and contains no homology to allergens
or toxins when compared to a database of known allergens and toxins. 

Classification: Not Applicable, summary of previously reviewed data.

SUMMARY OF DATA SUBMITTED: 

Summaries and discussion of each study on the product characterization,
human toxicity, and allergenicity of this product reviewed in this
memorandum are provided below.

MRID No. 468648-01   Molecular characterization of Event MIR162 maize

The purpose of this study was to present molecular characterization data
of the T-DNA insert and the genetic material required for its production
(via pNOV1300) in MIR162 plants.  Southern blot analysis and DNA
sequencing showed that Event MIR162 maize contains single copies of the
vip3A and phosphomannose isomerase (pmi) genes, but no backbone
sequences from the transformation plasmid pNOV1300.  Event MIR162 DNA
had two single nucleotide changes in the Vip3Aa coding sequence compared
to the Vip3Aa in pNOV1300, and was designated vip3Aa20.  The
substitution of thymine for guanine at base 387 changed the methionine
at position 129 to isoleucine (M129I), but the substitution of cytosine
for guanine at base 1683 was a silent mutation.  The pmi coding sequence
in Event MIR162 was identical to that in pNOV1300.  The stability of the
transgenic locus was shown by statistical analysis of the Event MIR162
segregation patterns over four generations, which confirmed the expected
Mendelian inheritance ratio for both the vip3Aa20 and pmi genes.

CLASSIFICATION:   SUPPLEMENTAL- but UPGRADEABLE- pending submission of
an additional Southern blot containing genomic DNA from MIR162, a
negative control, the plasmid control pNOV1300 hybridized with the
pmi-specific probe, and use a different molecular weight marker to avoid
non-specific sequence binding.

MRID No. 468648-02   Characterization of the Vip3A protein expressed in
Event MIR162-

derived maize (corn) and comparison with microbially produced and
plant-derived Vip3A test substances

The purpose of this study was to determine if Vip3Aa20 expressed in
maize plants derived from transformation Event MIR162 is substantially
equivalent to Vip3Aa19 or Vip3Aa1 present in various test substances
previously used in toxicity and/or test substance characterization
studies.  Vip3Aa produced in recombinant E. coli, MIR162 maize, and
Pacha maize were shown to be substantially equivalent based on the
finding that (1) Vip3Aa20 from MIR162 maize (test material LPMIR162-0105
and IAPMIR162-0105), Vip3Aa19 from Pacha maize (LPPACHA-0199), Vip3Aa19
from several of E. coli-derived samples (VIP3A-0204, VIP3A-0104,
VIP3A-0100), and Vip3Aa1 from E. coli-produced VIP3A-0199 each had the
expected predicted molecular weight of ~89 kDa, and were immunoreactive
with the same anti-Vip3Aa antibody on western blots, and (2) VIP3A-0204
and LPMIR162-0105 had comparable insecticidal activity against FAW (137
ng Vip3Aa19/cm2 vs. 154 ng Vip3Aa20 ng/cm2 diet surface).  Additionally,
there was no evidence of post-translational glycosylation of Vip3Aa from
LPMIR162-0105 or VIP3A-0204.  Therefore, the E. coli -produced Vip3A is
considered an appropriate substitute for Vip3Aa20 expressed in MIR162
maize in toxicity and/or protein characterization studies.  It was also
be noted that the VIP3A-0204 Vip3Aa19 protein N-terminal amino acid
sequence matched the predicted sequence however, the plant-expressed
Vip3A was not determined due to technical difficulties.    

CLASSIFICATION:   ACCEPTABLE

MRID No. 468648-03  	Characterization of Vip3A protein test substance
(VIP3A-0104) and 

		certificate of analysis

μg endotoxin /g VIP3A-0104 by lipopolysaccharide analysis.  Western
blots using goat anti-VIP3A polyclonal primary antibody and donkey
anti-goat alkaline phosphatase-linked secondary antibody revealed a
dominant immunoreactive band at the predicted molecular weight of
~89,800 Da.  VIP3A-0104 had insecticidal activity against first-instar
fall army worm (FAW) larvae in insect feeding assays, with an LC50 of
272 ng VIP3A/cm2 diet surface (95% confidence interval of 184 - 384 ng
VIP3A/cm2) after 168 hours.

CLASSIFICATION:   ACCEPTABLE

MRID No. 468648-04  	Characterization of Vip3A protein test substance
(VIP3A-0204) and 

		certificate of analysis

μg endotoxin/g VIP3A-0204 by lipopolysaccharide analysis.  Western
blots using goat anti-VIP3A polyclonal primary antibody and donkey
anti-goat alkaline phosphatase-linked secondary antibody revealed a
dominant immunoreactive band at the predicted molecular weight of
~89,800 Da.  VIP3A-0204 had insecticidal activity against first-instar
fall army worm (FAW) larvae in insect feeding assays, with an LC50 of
45.1 ng VIP3A/cm2 diet surface (95% confidence interval of 24.5 – 71.0
ng VIP3A/cm2 diet surface) after 120 hours.

CLASSIFICATION:   ACCEPTABLE

MRID No. 468648-05	Re-characterization of Vip3A protein test substance
(VIP3A-0204) and certificate of analysis

The purpose of this study was to re-characterize test substance
VIP3A-0204, containing the vegetative insecticidal protein VIP3A encoded
by the synthetic vip3A(a) gene.  VIP3A-0204 test material (Vip3Aa19
protein) produced from the synthetic vip3A(a) gene in an E. coli
over-expression system was previously purified and characterized (MRID
46864804).  It was shown to be ~89,800 Da and 89.7% pure (SDS-PAGE with
Coomassie blue staining and densitometric analysis), immunoreactive with
anti-VIP3A antibody (western blots), and to have insecticidal activity
against first-instar fall army worm (FAW) larvae (LC50 of 45.1 ng
VIP3A/cm2 diet surface after 120 hours).  In the present study, this
VIP3A-0204 sample was similarly re-characterized after seven months
storage lyophilized at -20°C, and shown to have retained its integrity
and bioactivity.  SDS-PAGE and western analysis determined a molecular
weight of ~89,800 Da and a purity of 91.8%, and insecticidal activity
assays with FAW larvae found an LC50 of 38.1 ng VIP3A/cm2 diet surface
after 120 hours.  Therefore, it can be concluded that the test substance
was stable when stored at -20°C, over ca. seven months.

CLASSIFICATION:   ACCEPTABLE

MRID No. 468648-06	Characterization of VIP3A protein produced in
Pacha-derived maize (corn) and comparison with VIP3A protein expressed
in recombinant E. coli

The purpose of this study was to demonstrate the equivalency of the
VIP3A protein as expressed in recombinant bacteria and transgenic maize
plants derived from the Pacha VIP3A Event.  Functional and biochemical
parameters were evaluated and compared in order to justify the use of
the microbially produced VIP3A test substance as a surrogate for
maize-expressed VIP3A protein in safety evaluations.  Comparisons
indicated that VIP3A protein produced by Pacha-derived maize
(LPPACHA-0199 sample; Vip3Aa19 protein) and by E. coli (VIP3A-0199
sample; Vip3Aa1 protein) was substantially equivalent.  SDS-PAGE and
western blot analysis showed that both proteins had a MW of ~89,000 and
were immunoreactive against the same anti-VIP3A antibody.  Edman
degradation was used to determine that the N-terminus of E. coli VIP3A
was MNKN, beginning with methionine-1, and of maize VIP3A was KNNXKL,
beginning with lysine-3 (X indicates that a definitive amino acid could
not be assigned).  The lack of two predicted amino acids at the
N-terminus of maize VIP3A was likely due to proteolytic degradation in
planta or in vitro.  The two VIP3A proteins had a similar insecticidal
activity profile against first-instar larvae, the E. coli-derived
protein being slightly more active.  Both were the most active against
black cutworm, with estimated 96-hour LC50 values of 70.4 and 88.5 ng
VIP3A/cm2 diet surface, respectively.  Both were slightly less active
against corn earworm and fall armyworm, and as expected, were inactive
against the European corn borer and diamondback moth.  Mass spectral
(MS) analysis of VIP3A proteolytic digests confirmed the predicted
complete sequence of ~95% of the E. coli and ~93% of the maize VIP3A
protein.  Neither MS nor an independent glycosylation analysis (DIG
Glycan) showed any evidence of post-translational glycosylation of
either the microbially-derived or maize-derived VIP3A.  

CLASSIFICATION:   SUPPLEMENTAL, but UPGRADEABLE- pending submission of
better reproductions of the SDS-PAGE and Western Blots of the E. coli-
and maize-derived VIP3A test proteins; as well as, clarifications to
data discrepancies noted in the insect bioassay.

MRID No. 468648-07	Analytical method for the detection of Vip3Aa20
protein in maize tissues from event MIR162

The purpose of this study was to determine the Vip3Aa20 extraction
efficiency from maize plant tissues in MIP162 maize, using an ELISA
assay (Tijssen, 1985) to measure Vip3Aa20 levels.  An ELISA procedure
was used to determine Vip3Aa20 levels in tissues of Event MIP162 maize
and the Vip3Aa20 protein tissue extraction efficiency.  The ELISA method
used 96-well plates, purified rabbit anti-Vip3A polyclonal primary
antibody, donkey anti-rabbit alkaline phosphatase conjugated secondary
antibody, and phosphatase substrate.  Each plate included the standard
test substance (MIR162-VIP3A-0106 or VIP3A-0104) that was used to
generate a standard curve, but this data was not shown.  The LOQ and LOD
for Vip3Aa20 ranged from, respectively, 0.04-0.25 and 0.003-0.032 µg
Vip3Aa20/gram fresh weight, and 0.21-0.35 µg and 0.029-0.045
Vip3Aa20/gram dry weight.  The average extraction efficiency of Vip3Aa20
was 82.7% in leaves, 81.0% in roots, 79.5% in pith, 88.3% in silk, 79.7%
in kernels, >98% in pollen, and 78.9% in whole plants at maturity.  

CLASSIFICATION:   This data packet is classified as UNACCEPTABLE for
residue analytical method. A new study should be submitted (concurrently
with the Sec. 3 registration of Event MIR162) and specifically conducted
on the MIR162 transgenic grain (single seed and composite) in order to
be verified as a suitable analytical method.   This experiment should
also be validated by an independent third party laboratory according to
OPPTS 860.1340(c)(6) and PR Notice 96-1 with GLP compliance. The report
should also include the following:  	

1) Qualitative data to represent positive vs. negative transgenic
specific event results with percent accuracy; 

2) Utilization of a negative control (non- transgenic convention corn
line) and positive control (confirmed transgenic corn line);

3)  Testing of cross-reactivity against other transgenic events and
other proteins; and

4)  The intra- and inter- assay coefficient of variation should be
reported.

Once the recommended report has been submitted and found acceptable,
EPA’s Analytical Method Laboratory located in Fort Meade (Maryland)
will have to independently validate Syngenta’s ELISA protocol for
accuracy, precision, and sensitivity.

In regards to establishing field protein expression levels in MIR162
corn tissues and plants, the study is supplemental.  It does provide
useful information for tissue expression levels to determine exposure
for non-target organisms, for IRM dose levels, and dietary exposure
estimates. However, it does not include quantification of Vip3Aa20
protein levels expressed in various plant tissues and the whole corn
plant. A full report determining the protein concentrations of Vip3Aa20
and PMI at different stages of plant development should be submitted
(including:  the mean, range, and standard deviations) and reported on a
dry weight basis (µg protein/g tissue) with GLP compliance.  This data
requirement can be addressed in the Sec. 3 Registration of Vip3Aa20. 
The study should also include the following:

1) Standard curve data for the ELISA; 

2) The calculation method for determining the dry weight conversion
factor from the fresh weight tissue samples; and

	3) Identification of the specific seed line and lot utilized as the
test material with number of field sites and replicates

f Cry1Ab δ-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

		

MRID No. 468648-08 	Vip3A as expressed in Event MIR162 maize: Assessment
of amino acid sequence homology with known toxins

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

MRID No. 468648-09	Vip3A as expressed in Event MIR162 maize: Assessment
of amino 

		acid sequence homology with known allergens

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

MRID No. 468648-10	Phosphomannose isomerase protein: Assessment of amino
acid 

		sequence homology with known toxins

The purpose of this study was to determine if phosphomannose isomerase
(PMI) derived from Escherichia coli had significant amino acid sequence
homology to known protein toxins.  The database identified 186 entries
with E values below 0.087.  All of these entries were known or putative
PMI enzymes (including mannose-6-phosphate isomerase (MPI) and ManA)
with no known toxic activity from 126 species, with E values of 0.0 –
0.067.  Therefore, no relevant similarities were found between the E.
coli PMI query sequence and known protein toxins.

CLASSIFICATION:   ACCEPTABLE

MRID No. 468648-11	Phosphomannose isomerase: Assessment of amino acid
sequence homology with known allergens

ent, that with the allergen α-parvalbumin from Rana species CH2001.
Hilger et al. (2002) identified α-parvalbumin as an allergen in an
individual who had severe anaphylaxis after eating frog legs of
Indonesian origin.  This patient’s serum was not cross-reactive to
related parvalbumins from the common edible frog (Rana esculenta). The
common amino acid sequence of DLSDKETT occurred at positions 327-334 in
PMI, and at positions 77-84 in α-parvalbumin. In order to determine if
the IgE antibodies present in this patient’s serum recognized PMI,
serum obtained from the one person with IgE-mediated allergy to
α-parvalbumin was not cross-reactive with PMI overexpressed in E. coli
(PMI-098; containing 61% w/w PMI protein and having PMI enzymatic
activity).  Therefore it is concluded that this 8-amino acid sequence
identity with α-parvalbumin was not biologically relevant, and that
there is no evidence that E. coli-derived PMI has significant amino acid
sequence homology to any known or putative allergenic proteins. EPA
previously reviewed this study and concurred with the study author’s
conclusion (see MRID No. 464252-01 and EPA 2005 a, b, and c).

CLASSIFICATION:   ACCEPTABLE

REFERENCES

Bernstein, I.L., Bernstein, J.A., Miller, M., Tierzieva, S., Bernstein,
D.I., Lummus, Z., Selgrade, M.K., Doerfler DL, Seligy VL. (1999)
“Immune responses in farm workers after exposure to Bacillus
thuringiensis pesticides.” Environ Health Perspect. 107(7):575-82

Crickmore, N., D.R. Zeigler, E. Schnepf, J. Van Rie, D. Lerecclus, J.
Baum, A. Bravo and D.H. Dean (2005).  Bacillus thuringiensis toxin
nomenclature.    HYPERLINK
"http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/" 
http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/  

EPA, 2001.  “Biopesticides Registration Action Document for the
Bacillus thuringiensis (Bt) Plant-Incorporated Protectants,” dated
October 15, 2001.

EPA, 2005a.  “Review of Product Characterization and Expression
Analysis for Modified Cry3A 

(mCry3A) Bacillus thuringiensis insect control protein and	maize (corn)
plants Derived from Event MIR604 in support for a temporary exemption
from tolerances and an Experimental Use Permit (EUP) Application.”
Memorandum- from A. Fellman, through J. Kough, Ph.D., to M. Mendelsohn,
dated February 11, 2005.

EPA, 2005b. “Addendum to Product Characterization and Expression
Analysis for Modified 

	Cry3A (mCry3A) B.t. insect control protein and maize (corn) plants
Derived from Event MIR604.”  Memorandum- from A. Fellman, through J.
Kough, Ph.D., to M. Mendelsohn, dated February 11, 2005.

EPA, 2005c. “Evaluation of Phosphomannose isomerase protein (PMI) as
expressed in 

Transgenic maize Event MIR604 with E. coli-derived PMI protein in
support for a temporary tolerance exemption for Modified Cry3A B.t.
insecticidal protein and the genetic material necessary for its
production in corn plants derived from Event MIR604.” Memorandum- from
A. Fellman, through J. Kough, Ph.D., to M. Mendelsohn, dated March 3,
2005.

EPA, 2006.  “Biopesticides Registration Action Document for the
Bacillus thuringiensis (Bt) Modified Cry3A (mCry3A) Plant-Incorporated
Protectant,” dated October 6, 2006.

Estruch, J. J., G. W. Warren, M. A. Mullins, G. J. Nye, J. A. Craig, and
M. G. Koziel (1996).  Vip3A, a novel Bacillus thuringiensis vegetative
insecticidal protein with a wide spectrum of activities against
lepidopteran insects.  Proc. Natl. Acad. Sci. 93:  5389 – 5394.

Food and Agriculture Organization of the United Nations and World Health
Organization. (2003).  Foods Derived from Biotechnology.  Codex
Alimentarius.  Sec. 4, No.38, pg. 16. 

Doolittle, R.F. (1990). Searching through Sequence Databases. Methods in
Enzymology, 183:  99-110.

Hilger, C., Grigioni, F., Thill, L., Mertens, L. and Hentges, F. (2002).
 Severe IgE-mediated anaphylaxis following consumption of fried frog
legs:  Definition of alpha-parvalbumin as the allergen in cause. 
Allergen  57 (11):  1053-1058.

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 hn6

vegetative insecticidal protein Vip3A from that of Cry1Ab
delta-endotoxin. Appl. Environ. Microbiol. 69(8):  4648-4657.

Myers, E.W. and W. Miller (1988). Optimal alignments in linear space. 
Comput. Appl. Biosci. 4:   

	11-17.

Negrotto, et al. (2000). The use of phosphomannose-isomerase as a
selectable marker to 

	recover transgenic maize plants (Zea mays L. via Agrobacterium
transformation). Plant Cell Reports, 19: 798-803.

Ogun, S.A. and A.A. Holder (1996). A high molecular mass Plasmodium
yoelii rhoptry protein binds to erythrocytes. Mol. Biochem. Parasitol.
76:  321-324.

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

Tijssen, P. (1985) Processing of data and reporting of results of enzyme
immuoassays.  In 

practice and theory of enzyme immunoassays. (Laboratory techniques in
Biochemistry and Molecular Biology, V. 15) Elsevier Science Publishers,
Amsterdam, The Netherlands, pp. 385-421.

Table 3.  Reference for establishing protein equivalence based on data
parameters for all Vip3A variants 

and corresponding test substances according to their respective MRID
Numbers.

Data Parameter	Vip3A Variants 

	Vip3Aa1	Vip3Aa19	Vip3Aa20

	Vip3A-0199	LPPacha-0199	Vip3A-0100	Vip3A-0104	Vip3A-0204	LPMIR162-0105
IAPMIR162-0105

Product Characterization

Southern blots

	458358-02

	468648-01	468648-01

DNA Sequencing

	458358-02

MW via SDS-PAGE	468648-06	458358-06 and  458358-12

468648-03	468648-04	468648-02	468648-02

MW via Western blot	468648-06	458358-06 and  458358-12

468648-03	468648-04	468648-02	468648-02

N-terminal AA sequencing

458358-06 and  458358-12

	468648-03

Glycosylation	468648-06	458358-12

	468648-02	468648-02

	Insect Bioactivity Assay	468648-06	458358-06 and  458358-12

468648-03	468648-02 and 468648-04	468648-02

	Mass Spectrometry 	468648-06	458358-06 and  458358-12

	Storage Stability

	468648-05

Toxicity and Allergenicity

Acute Oral toxicity test	457665-04	457665-06	457665-05

In vitro digestibility in SGF

458358-05	458358-05

AA sequence to known toxins

	458358-04

	468648-08

	AA sequence to known allergens

	458358-04

	468648-09

	Heat Stability

	458358-04



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