UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

		WASHINGTON, D.C. 20460      

	

OFFICE OF CHEMICAL SAFETY 

AND POLLUTION PREVENTION

	                                                                   

				 

May 25, 2010

     

MEMORANDUM

SUBJECT:	Review of Product Characterization and Human Health Data for
Plant- Incorporated Protectant Bacillus thuringiensis (Bt) eCry3.1Ab
insect control protein and the genetic material necessary for its
production in Event 5307 maize (Zea mays) [EPA Reg. No. 67979-EUP-I] in
support for an Experimental Use Permit and Temporary Exemption from the
Requirement of a Tolerance [Petition No. 9F7561]; Decision Number:
409084, DP Barcode:  367108; submitted by Syngenta Seeds, Inc. – Field
Crops – NAFTA.

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)

TO:	Mike Mendelsohn, Senior Regulatory Action Leader

	Microbial Pesticides Branch, Biopesticides and

	Pollution Prevention Division (7511P)

ACTION REQUESTED: To review product characterization and human health
data submitted by Syngenta Seeds, Inc.-Field Crops – NAFTA, in support
for an Experimental Use Permit and a temporary tolerance exemption for
Bt eCry3.1Ab insect control protein expressed in Event 5307 maize.  

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 eCry3.1Ab protein and the genetic material
necessary for its production in Event 5307 maize. 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 for eCry3.1Ab protein are sufficient to support an
Experimental Use Permit and a temporary exemption from the requirement
of a tolerance.  

DATA REVIEW RECORD:

Active Ingredient:	Bacillus thuringiensis eCry3.1Ab insecticidal protein
and the genetic material necessary for its production in corn (via
plasmid vector PV-ZMIR245)

Product Name:	Event 5307 maize [EPA Reg. No. 67979-EUP-I]	

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

ID No: 	      	67979		

Decision Number:        409084

DP Barcode: 	367108

					

MRID No:	

		

Product Characterization Data 

477347-01	Characterization and Safety of eCry3.1Ab Protein and Corn
Plants Derived from Event 5307

477347-02 	Molecular characterization of Event 5307 maize

477347-03	Comparison of eCry3.1Ab protein produced in Event 5307-derived
maize plants and eCry3.1Ab protein produced in recombinant Escherichia
coli

477347-04  	Characterization of Test Substance ECRY3.1AB-0208 and
Certificate of Analysis

477347-05	Bioactivity of Test Substance ECRY3.1AB-0208 

477347-07	Evaluation of Transgenic Protein Levels in Multiple
Generations of Plants Derived from Transformation Event 5307 Maize

		

Human Health Data 

477539-01	ECRY3.1AB-0208: Single-dose Oral (Gavage) Toxicity Study in
Mice with a 14-Day Observation 

	Period

477347-09	eCry3.1Ab: Assessment of amino acid sequence homology with
known toxins

477347-10	eCry3.1Ab: Assessment of amino acid sequence homology with
known or putative allergens

477347-11	In vitro digestibility of eCry3.1Ab protein as contained in
test substance ECRY3.1AB-0208 and event 5307 maize under simulated
mammalian gastric conditions  

477347-12	Phosphomannose isomerase (Entrez Database Accession No
AAA24109): Assessment of amino acid sequence homology with known toxins

477347-13	Phosphomannose isomerase (Entrez Database Accession No
AAA24109): Assessment of amino acid 

	sequence homology with known or putative allergens

480384-01	Supplemental Information for Study SSB-024-08: In Vitro
Digestibility of eCry3.1Ab Protein as Contained in Test Substance
ECRY3.1AB-0208 and in 5307 Maize Under Simulated Mammalian Gastric
Conditions [EPA MRID 47734711]

BACKGROUND:

Based on amino acid sequence similarity and crystal structures, known
Cry proteins have a similar three-dimensional structure comprised of
three domains, Domain I, II and III (Nakamura et al., 1990; Li et al.,
1991; Ge et al., 1991; Honee et al., 1991). The toxin portions of Cry
proteins are characterized by having five conserved blocks (CB) across
their amino acid sequence. These are numbered CB1 to CB5 from the N-
terminus to the C-terminus (Hofte and Whiteley, 1989). The sequences
preceding and following these conserved blocks are highly variable and
are designated as variable regions V1 to V6. 

Syngenta Seeds, Inc. – Field Crops - NAFTA developed Event 5307 maize
(Zea mays) through Agrobacterium-mediated transformation (via plasmid
vector PV-ZMIR245) to express eCry3.1Ab protein for use as a
plant-incorporated protectant (PIP).  This proposed PIP is a chimeric
Bacillus thuringiensis protein, composed of portions of Cry1Ab and
modified Cry3A proteins.  The eCry3.1Ab protein was genetically
engineered via exchanging the variable regions (V1 to V6) between the
mCry3A and the Cry1Ab proteins for enhanced toxicity against western
corn rootworm (WCR, Diabrotica virgifera).  The eCry3.1Ab protein
consists of a fusion between the N-terminus (Domain I, Domain II, and a
portion of Domain III) of mCry3 A and the C-terminus (a portion of
Domain III and variable region 6) of Cry1Ab. The eCry3.1Ab protein is
654 amino acid residues in size and is approximately 73.7 kDa.

Event 5307 corn also contains the pmi gene, which was introduced along
with the eCry3.1Ab protein via the same PV-ZMIR245 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 testing on Event 5307 corn, hybrids thereof, and non-PIP
corn.   The proposed planting under the EUP will take place in 28 states
and Puerto Rico for a total of 14,619 acres over two years (7311 acres
in 2010 and 7308 acres in 2011).  The proposed planting also includes
field testing Event 5307 maize, as a combination PIP product, with one
or more of the following previously registered PIP corn events:  Bt11,
DAS-59122-7, MIR162, MIR604, and TC1507.  

Event Bt11 produces Bt Cry1Ab protein that protects against European
corn borer (Ostrinia nubilalis) and is currently registered for use as a
PIP (US EPA, 2001).

DAS-59122-7 produces Cry34Ab1 and Cry35Ab1 proteins derived from the Bt
strain PS149B1, to protect against coleopteran pests (corn rootworm) and
is currently registered for use as a PIP (US EPA, 2005b).

MIR162 produces Bt Vip3Aa20 insecticidal protein and is currently
registered for use as a PIP that protects against feeding damage caused
by many lepidopteran insect pests:  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)
(US EPA, 2009).

MIR604 produces a modified Bt mCry3A protein to protect against corn
rootworm (CRW) larval feeding and is currently registered for use as a
PIP (US EPA, 2007).  

TC1507 produces Bt Cry1F protein to selectively control larvae of the
European corn borer (O. nubilalis) and other lepidopteran insect pests
and is currently registered for use as a PIP (US EPA, 2005a)

To satisfy the data requirements specific for the EUP for eCry3.1Ab,
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 similarity assessment of PMI
to known toxins and allergens, which will supersede the previously
reviewed PMI data.  

RECOMMENDATION:  

The product characterization and human health data submitted for
eCry3.1Ab as expressed in Event 5307 maize [EPA Reg. No. 67979-EUP-I]
are sufficient to support an Experimental Use Permit and a temporary
exemption from the requirement of a tolerance.  

There are a few studies classified as “SUPPLEMENTAL” but sufficient
for the purposes of the EUP and satisfy the data needed to support the
preliminary safety assessment.  However, if the registrant pursues a
Sec. 3 Registration of eCry3.1Ab expressed in Event 5307 maize for use
as a PIP, additional data would be needed to complete the eCry3.1Ab
database.  Details of these studies and recommendations are provided
below:

MRID No. 477347-11 and 480384-01	

The provisional enzyme kinetic analysis of a previously conducted in
vitro digestibility study in SGF for the microbial-derived and the
plant-derived eCry3.1Ab test substances showed rapid pepsin degradation
rates  (DT50= <1 minute and DT50= <2 minutes, respectively). However,
the combination of the original in vitro digestibility study and
supplemental information are only sufficient to support the data needs
for the EUP request because these degradation rates are an estimated
range of values and based on a comparison of the degradation rate with
other non-allergenic proteins.  There is no exact quantified endpoint
for the DT50 endpoint to report.   

Therefore, if the registrant submits an application for a Sec. 3
registration for Event 5307 maize expressing eCry3.1Ab protein, the
Agency recommends conducting an appropriately designed in vitro
digestibility study of eCry3.1Ab test substances in SGF utilizing the
kinetic approach with the inclusion of additional data points in between
the first two minutes of the assay.  This study will serve as final
confirmation of the rapid digestibility of eCry3A.1Ab protein in pepsin
by providing earlier data points to plot the exact degradation rate of
the protein.  

 The Agency also recognizes that collecting enough data points early in
the decay curve is difficult (because the pepsinolysis reaction is
difficult to stop for rapidly degraded proteins), therefore, it is also
recommended the registrant consult with the Agency in a pre-registration
meeting to address the data needs for the Sec.3 registration.

The registrant should be prepared to provide data to verify  the buffer
ionic strength effect on other proteins to the Agency.

MRID No. 477347-07	

The study establishing field protein expression levels in 5307 corn
tissues and plants, the study is supplemental.  This study was a
preliminary greenhouse experiment and contained limited data points of
the protein expression and used a minimal sample size of eCry3.1Ab in
Event 5307 corn.  It does provide useful information for establishing
the consistency of expression of eCry3.1Ab and PMI protein
concentrations across four generations of Event 5307 maize (VT-R1
stages), but does not include quantification of eCry3.1Ab protein levels
expressed in various plant tissues and the whole corn plant, which is
necessary to determine exposure for non-target organisms, for IRM dose
levels, and dietary exposure estimates.  However, these data do not
affect the findings of the safety assessment for the proposed EUP.

This data requirement can be addressed in the Sec. 3 Registration of
eCry3.1Ab.  It is recommended that a field study be conducted to
determine the protein concentrations of eCry3.1Ab and PMI in various
plants tissues at multiple stages of plant growth development  (using
multiple geographic locations and a sufficient number of replicates for
the plant samples to ensure statistical validity). The study should also
include the following:

1) Protein concentration values should include:  the mean, range, and
standard deviations) and reported on a dry weight basis (µg protein/g
tissue).  An appendix should be submitted to contain the results of the
individual samples to serve

 as the basis of calculating the mean and range. 

2) The number of samples collected should be statistically valid to
represent a range of field conditions at various geographic regions
where the product is expected to be grown.

	3)  Standard curve data for the ELISA should be included. 

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

	5) Identification of the specific seed line and lot utilized as the
test material with number of plants used in analysis, field sites and
replicates.

Temporary Exemption from the Requirement of a Tolerance [Petition No.
9F7561]

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
eCry3.1Ab Bacillus thuringiensis insect control protein and the genetic
material necessary for its production in food and feed commodities of 
field corn, sweet corn, and popcorn.  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 (nucleic acids) that are part of any plant-incorporated
protectant are exempt from the requirement of a tolerance [40 CFR
174.507, effective Apr. 25, 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].  

Existing exemptions from the requirement of a tolerance has been
established for Cry1Ab protein in food and mCry3A protein in maize when
used as plant-incorporated protectants [see 40 CFR 174.511, effective
Apr. 25, 2007 and 40 CFR 174.506, effective Apr. 25, 2007,
respectively].  

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

An eCry3.1Ab Bacillus thuringiensis (Bt) insect control protein was
produced in Event 5307 maize (Zea mays) through Agrobacterium-mediated
transformation (via plasmid vector PV-ZMIR245) for use as a
plant-incorporated protectant (PIP).  This proposed PIP is a chimeric
Bacillus thuringiensis protein, composed of portions of Cry1Ab and
modified Cry3A proteins.  The eCry3.1Ab protein was genetically
engineered via exchanging the variable regions (V1 to V6) between the
mCry3A and the Cry1Ab proteins for enhanced toxicity against western
corn rootworm (WCR, Diabrotica virgifera).  The eCry3.1Ab protein
consists of a fusion between the N-terminus (Domain I, Domain II, and a
portion of Domain III) of mCry3 A and the C-terminus (a portion of
Domain III and variable region 6) of Cry1Ab. The eCry3.1Ab protein is
654 amino acid residues in size and is approximately 73.7 kDa.  The pmi
gene, which was introduced along with the eCry3.1Ab protein via the same
PV-ZMIR245 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 (Negrotto, et al. 2000).   

The ecry3.1Ab expression cassette consisted of the ecry3.lAb coding
region regulated by a cestrum yellow leaf curling virus promoter (CMP)
and a nopaline synthase (NOS) polyadenylation sequence. The pmi
expression cassette consisted of the pmi coding region regulated by a Z.
mays polyubiquitin promoter (ZmUbilnt) and the NOS polyadenylation
sequence. Southern blot analyses and DNA sequencing indicate that one
full length copy of each of the eCry3.1Ab and pmi genes were integrated
into the maize genome, without the backbone sequences from
transformation plasmid PV-ZMIR245 plasmid.   Therefore, the overall
integrity of the insert and the contiguousness of the functional
elements were confirmed.

Data have been submitted demonstrating the protein equivalency of
plant-expressed eCry3.1Ab protein and its respective protein test
substances, expressed in recombinant E. coli (ECRY3.1AB-0208) for use as
a surrogate in toxicity experiments based on  similar biochemical and
functional characteristics (see MRID No. 477347-03).  

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. 

1.   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 eCry3.1Ab
protein.  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 Sec. 158.740(b)(2)(i)). For microbial products, further toxicity
testing and residue data are triggered by significant adverse acute
effects in studies such as the mouse oral toxicity study, to verify and
quantify the observed adverse effects and clarify the source of these
effects (Tiers II & III). 

An acute oral toxicity study in mice (MRID No. 477539-01) indicated that
eCry3.1Ab is non-toxic. Two groups of ten male and ten female mice were
orally dosed (via gavage) with 2,000 milligrams/kilograms bodyweight
(eCry3.1Ab protein mg/kg bwt) of the ECRY3.1AB0208 test substance, the
microbial-produced eCry3.1Ab protein.  All treated animals gained weight
and had no test material-related clinical signs and no test
material-related findings at necropsy.  Since there were no significant
differences between the test and control groups related to the oral
administration of ECRY3.1AB-0208 test material; the eCry3.1Ab protein
does not appear to cause any significant adverse effects at an exposure
level of up to 2000 mg/kg bodyweight and supports the finding that the
eCry3.1Ab protein would be non-toxic to mammals.

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 eCry3.1Ab,
even at relatively high dose levels, the eCry3.1Ab protein is not
considered toxic.  Further, amino acid sequence comparisons showed no
similarities between the eCry3.1Ab protein and known toxic proteins in
protein databases that would raise a safety concern.     

Since eCry3.1Ab 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 (as recommended by CAC, 2003).  Current scientific
knowledge suggests that common food allergens tend to be resistant to
degradation by acid and proteases; may be glycosylated; and present at
high concentrations in the food. 

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
eCry3.1Ab with known allergens showed no significant overall sequence
similarity or identity at the level of eight contiguous amino acid
residues.

Prevalence in food.  Preliminary expression level analysis of eCry.1Ab
protein is present at relatively low levels.  Dietary exposure is
expected to be correspondingly low.  Expression in Event 5307 leaf is
<35 ppm; root is <6 ppm; pollen is < 0.15 ppm.  Thus, the expression has
been shown to be in the parts per million range.

Digestibility.  The eCry3.1Ab protein was rapidly digested in simulated
mammalian gastric fluid containing pepsin at a pH of 1.2 at 37°C.  The
enzyme kinetic analysis estimated the degradation rate as less than one
minute for eCry3.1Ab protein. 

Glycosylation.  The eCry3.1Ab protein expressed in corn was shown not to
be glycosylated.

Conclusion.  Considering all of the available information, EPA has
concluded that the potential for eCry3.1Ab to be a food allergen is
minimal.

2) Aggregate Exposures

In examining aggregate exposure, EPA considers 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 similarity
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 eCry3.1Ab 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 of 2 gm/kg showed no
adverse effects. Furthermore, the expected dietary exposure from corn
are several orders of magnitude lower than the amounts of eCry3.1Ab
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 eCry3.1Ab protein. 

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,
resulting from the plant-incorporated protectant, we conclude that there
are no cumulative effects for the eCry3.1Ab 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
eCry3.1Ab protein include the characterization of the expressed
eCry3.1Ab 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 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 eCry3.1Ab 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
eCry3.1Ab 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 eCry3.1Ab protein, even at relatively high
dose levels (2,000 mg eCry3.1Ab/kg bwt), the eCry3.1Ab 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, eCry3.1Ab showed no
sequence similarity to any known toxin.

Protein residue chemistry data for eCry3.1Ab 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 expression level analysis showed eCry3.1Ab protein
is present at relatively low levels.  Dietary exposure is expected to be
correspondingly low.  Expression in Event 5307 leaf, root, and pollen is
<35 ppm, <6 ppm, and < 0.15 ppm, respectively. 

Since eCry3.1Ab 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 eCry3.1Ab 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 eCry3.1Ab 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 eCry3.1Ab 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. 

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 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 eCry3.1Ab
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
eCry3.1Ab 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. 

5)  Other Considerations 

A. Endocrine Disruptors

As required under FFDCA section 408(p), EPA has developed the Endocrine
Disruptor Screening Program (EDSP) to determine whether certain
substances (including pesticide active and other ingredients) may have
an effect in humans or wildlife similar to an effect produced by a
“naturally occurring estrogen, or other such endocrine effects as the
Administrator may designate.”  The EDSP employs a two-tiered approach
to making the statutorily required determinations. Tier 1 consists of a
battery of 11 screening assays to identify the potential of a chemical
substance to interact with the estrogen, androgen, or thyroid (E, A, or
T) hormonal systems.  Chemicals that go through Tier 1 screening and are
found to have the potential to interact with E, A, or T hormonal systems
will proceed to the next stage of the EDSP where EPA will determine
which, if any, of the Tier 2 tests are necessary based on the available
data. Tier 2 testing is designed to identify any adverse endocrine
related effects caused by the substance, and establish a dose-response
relationship between the dose and the E, A, or T effect.

Between October 2009 and February 2010, EPA issued test orders/data
call-ins for the first group of 67 chemicals, which contains 58
pesticide active ingredients and 9 inert ingredients.  This list of
chemicals was selected based on the potential for human exposure through
pathways such as food and water, residential activity, and certain
post-application agricultural scenarios.  This list should not be
construed as a list of known or likely endocrine disruptors.

The proposed PIP, eCry3.1Ab insect control protein (as expressed in
Event 5307 maize), is not among the group of 58 pesticide active
ingredients on the initial list to be screened under the EDSP.  Under
FFDCA sec. 408(p) the Agency must screen all pesticide chemicals. 
Accordingly, EPA anticipates issuing future EDSP test orders/data
call-ins for all pesticide active ingredients. 

For further information on the status of the EDSP, the policies and
procedures, the list of 67 chemicals, the test guidelines and the Tier 1
screening battery, please visit our website:  http://www.epa.gov/endo/.

B. Analytical Method(s) 

A method for extraction and two test strip commercial kits to detect
eCry3.1Ab protein via ELISA analysis 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 eCry3.1Ab protein and the genetic
material necessary for its production in corn. 

SUMMARY OF DATA SUBMITTED: 

The submitted studies to support the registrant’s EUP request and
petition for a temporary exemption from the requirement of a tolerance
for eCry3A.1Ab protein as expressed in Event 5307 maize are listed below
with their respective MRID No. and classification (see Table 1).  The
Agency reviewed the submitted studies (including product
characterization, human toxicity, and allergenicity data) and they are
individually summarized in Data Evaluation Reports (DERs).  Each DER
contains the objective, test methods/materials, results and findings,
conclusion, and classification of each study.   This memorandum
represents the Agency’s overall recommendation for Syngenta’s EUP
request based on the summary of data reviewed in each DER (for detailed
information of each study, refer to the DER according to their
respective assigned MRID number).   

Table 1. Compilation of Submitted Data and Classification for Event 5307
expressing eCry3.1Ab protein [EPA Reg. No. 67676-EUP-I] in support of
Syngenta’s EUP request.

MRID NO.	STUDY TITLE	CLASSIFICATION

Product Characterization Data

477347-01	Characterization and Safety of eCry3.1Ab Protein and Corn
Plants Derived from Event 5307

	ACCEPTABLE

477347-02	Molecular characterization of Event 5307 maize

	ACCEPTABLE

477347-03	Comparison of eCry3.1Ab protein produced in Event 5307-derived
maize plants and eCry3.1Ab protein produced in recombinant Escherichia
coli

	ACCEPTABLE

477347-04	Characterization of Test Substance ECRY3.1AB-0208 and
Certificate of Analysis

	ACCEPTABLE

477347-05	Bioactivity of Test Substance ECRY3.1AB-0208

	ACCEPTABLE

477347-07	Evaluation of Transgenic Protein Levels in Multiple
Generations of Plants Derived from Transformation Event 5307 Maize

	SUPPLEMENTAL

Toxicity and Allergenicity Data

477539-01	ECRY3.1AB-0208: Single-dose Oral (Gavage) Toxicity Study in
Mice with a 14-Day Observation Period

	ACCEPTABLE

477347-09	eCry3.1Ab: Assessment of amino acid sequence homology with
known toxins

	ACCEPTABLE

477347-10	eCry3.1Ab: Assessment of amino acid sequence homology with
known or putative allergens

	ACCEPTABLE

477347-11	In vitro digestibility of eCry3.1Ab protein as contained in
test substance ECRY3.1AB-0208 and event 5307 maize under simulated
mammalian gastric conditions  	SUPPLEMENTAL

477347-12	Phosphomannose isomerase (Entrez Database Accession No
AAA24109): Assessment of amino acid sequence homology with known toxins

	ACCEPTABLE

477347-13	Phosphomannose isomerase (Entrez Database Accession No
AAA24109): Assessment of amino acid sequence homology with known or
putative allergens	ACCEPTABLE

480384-01	Supplemental Information for Study SSB-024-08: In Vitro
Digestibility of eCry3.1Ab Protein as Contained in Test Substance
ECRY3.1AB-0208 and in 5307 Maize Under Simulated Mammalian Gastric
Conditions [EPA MRID 47734711]	ACCEPTABLE- for the purposes of the EUP



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.

CAC (2003) Alinorm 03/34: Joint FAO/WHO Food Standard Programme. Codex
Alimentarius Commission, Twenty-Fifth Session, 30 July 2003. Rome,
Italy. Appendix III: Guideline for Conduct of Food Safety Assessments of
Foods Derived from Recombinant-DNA Plants; Appendix IV: Annex on
Assessment of Possible Allergenicity. CAC, 47–60.

Ge, A., D. Rivers, R. Milne, and D.H. Dean. (1991) Functional Domains of
Bacillus thuringiensis Insecticidal Crystal Proteins. Refinement of
Heliothis virescens and Trichoplusia ni Specificity Domains on Cry1A(c).
J. Biol. Chem. 266: 17954-17958.

Herman R.A., Storer N.A., Gao Y. (2006) Digestion assays in
allergenicity assessment of transgenic proteins.” Environmental Health
Perspectives. 114:1154–1157.

Hofte, H., and Whitley H.R. (1989) Insecticidal Crystal Proteins of
Bacillus thuringiensis. 

Microbiol. Rev. 53: 242-255.

Honee, G., D. Convents, J. Van Rie, S. Jansens, M. Peferoen, and B.
Visser. (1991) The C-Terminal Domain of the Toxic Fragment of a Bacillus
thuringiensis Crystal Protein Determines Receptor Binding. Mol.
Microbiol. 5: 2799-2806.

Li, J., J. Carroll, and D.J. Ellar. (1991) Crystal Structure of
Insecticidal δ-Endotoxin from 

	Bacillus thuringiensis at 2.5 A resolution. Nature 353: 815-821.

Nakamura, K., K. Oshie, M. Shimizu, Y. Takada, K. Oeda, and H. Ohkawa.
(1990) Construction of Chimeric Insecticidal Proteins Between the
130-kDa and 135-kDa Proteins of Bacillus thuringiensis subsp. aizawai
for Analysis of Structure-Function Relationship. Agric. Biol. Chem. 54:
715-724.

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.

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.

US EPA (2001)  Biopesticides Registration Action Document for Bacillus
thuringiensis (Bt) 

	Plant-Incorporated Protectants, dated October 2001.  US Environmental
Protection Agency (EPA), Washington, DC.

US EPA (2005a)  Biopesticides Registration Action Document for Bacillus
thuringiensis (Bt) 

	Cry1F Plant-Incorporated Protectant, dated August 2005.  US EPA,
Washington, DC.

US EPA (2005b)  Biopesticides Registration Action Document for Bacillus
thuringiensis (Bt) 

         Cry34Ab1 and Cry35Ab1 Plant-Incorporated Protectants, dated
October 2005. US EPA, Washington, D.C.

US EPA (2007) Biopesticides Registration Action Document for the
Bacillus thuringiensis (Bt) Modified Cry3A Plant-Incorporated
Protectant, dated March 2007. US EPA, Washington, D.C.

US EPA (2009) Biopesticides Registration Action Document for Bacillus
thuringiensis (Bt) 

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*THIS REPORT DOES NOT CONTAIN FIFRA CONFIDENTIAL BUSINESS INFORMATION*

