![](media/image1.jpeg){width="0.875in" height="0.8548611111111111in"}

> **UNITED STATES ENVIRONMENTAL PROTECTION AGENCY**
>
> WASHINGTON, D.C. 20460

**FEB 15 2006**

> OFFICE OF PREVENTION, PESTICIDES AND TOXIC SUBSTANCES

**[MEMORANDUM]{.underline}**

**SUBJECT:** Review of Product Characterization and Human Health Data
for Modified Cry3A (mCry3A) *Bacillus thuringiensis* insect control
protein and maize (Corn) Plants Derived from Event MIR604 (EPA Reg. No.
67979-L) in support for a Permanent Exemption from Tolerances and
Section 3 Registration, submitted by Syngenta Seeds, Inc. -- Field
Crops- NAFTA.

**TO:** Mike Mendelsohn, Regulatory Action Leader

Microbial Pesticides Branch, Biopesticides and

Pollution Prevention Division (7511C)

**FROM:** Annabel Fellman, Environmental Protection Specialist
\[signed\]

Microbial Pesticides Branch, Biopesticides and

Pollution Prevention Division (7511C)

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

Microbial Pesticides Branch, Biopesticides and

Pollution Prevention Division (7511C)

**ACTION REQUESTED:** To review additional product characterization,
human health and analytical methods data submitted by Syngenta Seeds,
Inc.-Field Crops - NAFTA in support for section 3 registration and a
permanent exemption from tolerances for Modified Cry3A insect control
protein and maize (Corn) Plants Derived from Event MIR604.

**CONCLUSION:** The product characterization, protein expression,
toxicological and allergenicity data support the finding that there is a
reasonable certainty of no harm to humans from the aggregate exposure to
the residues of the mCry3A protein, including all anticipated dietary
exposures and all other exposures for which there is reliable
information.

**\*REVIEW DOES NOT CONTAIN CONFIDENTIAL BUSINESS INFORMATION\***

**[DATA REVIEW RECORD:]{.underline}**

**Active Ingredient:** Modified Cry3A (mCry3A) *Bacillus thuringiensis*
insecticidal protein and the genetic material necessary for their
production (via pZM26) in transgenic maize (corn) plants derived from
Syngenta Seeds' transformation Event MIR604.

**Product Name:** Event MIR604 Maize Plants Expressing Modified Cry3A
*Bacillus thuringiensis* Protein

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

**ID No:** 67979

**Chemical Number:** 006509

**Decision Number:** 343070

**DP Barcode:** 319746

**MRID No:**

**[465974-01]{.underline}** **Independent Laboratory Validation of ELISA
method for the Detection of mCry3A in Event MIR604 corn grain**

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

Syngenta synthetically modified a *cry3A* gene from *Bacillus
thuringiensis* subsp. *tenebrionis* to optimize the gene for expression
in maize (corn) and to enhance its activity against the western corn
rootworm (WCRM; *Diabrotica virgifera vigifera*) and northern corn
rootworm (NCRM; *D. longicornis barberi*) (Sekar *et al.,* 1987).
Syngenta's maize Event MIR604 corn plants were a result of a corn plant
transformation with the synthetic modified *cry3A* gene, which provides
resistance to these pests (Chen, E. and Stacy, C., 2003). Transformation
was conducted using immature maize embryos derived from a proprietary
*Zea mays* line, *via* *Agrobacterium*-mediated transformation. By this
method, genetic elements within the left and right border regions of the
transformation vector are efficiently transferred and integrated into
the genome of the plant cell.

Event MIR604 maize also contains the *pmi* gene, which was introduced
along with the mCry3A protein via the same pZM26 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). 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. An existing permanent 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). This regulation eliminates the
need to establish a maximum permissible level for residues in or on all
plant commodities of phosphomannose isomerase and the genetic material
necessary for its production in all plants when part of a
plant-incorporated protectant.

In the Federal Register of April 6, 2005 (70 FR 17323), the Agency
established a temporary exemption from the requirement of a tolerance
for Modified Cry3A and the genetic material necessary for their
production in corn which will expire October 15, 2006. In addition, EPA
issued an experimental permit for the use *Bt* mCry3A protein and the
genetic material necessary for their production (via plasmid pZM26) in
Event MIR604 corn and associated activities (such as collection of field
data; harvesting & processing of seed after last planting) on 575 acres
of field corn. To support their requests for EUP and a temporary
tolerance, Syngenta Seeds, Inc. submitted product characterization and
protein expression analyses, toxicological and allergenicity data \[see
memoranda from A. Fellman through J. Kough to M. Mendelsohn, dated
02/11/2005, 02/23/2005, 03/03/2005, and 05/31/2005\]. The submitted
study titles, conclusions, and their MRID numbers are provided (see
Table 1) in this report. The final study to complete data requirements
for Section 3 registration for Modified Cry3A protein and the genetic
material necessary for their production (via plasmid pZM26) in Event
MIR604 corn is reviewed in this report as well.

**[Permanent Exemption for the Requirement of a Tolerance
(4F6838)]{.underline}**

EPA has established an exemption from tolerance requirements pursuant to
FFDCA section 408(j)(3) for *Bacillus thuringiensis* Cry3A
delta-endotoxin and the genetic material necessary for its production in
potatoes, and this tolerance exemption has been reassessed and meets the
408(c)(2) standard (see 40 CFR 180.1147). 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 the Federal Register of April 6, 2005 (70 FR 17323), the Agency
established a temporary exemption from the requirement of a tolerance
for Modified Cry3A and the genetic material necessary for their
production in corn which will expire October 15, 2006. In addition, EPA
issued an experimental permit for the use *Bt* mCry3A protein and the
genetic material necessary for their production (via plasmid pZM26) in
Event MIR604 corn and associated activities (such as collection of field
data; harvesting & processing of seed after last planting) on 575 acres
of field corn.

Syngenta Seeds, Inc. -- Field Crops- NAFTA has submitted a petition for
a permanent 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 modified Cry3A *Bacillus
thuringiensis* insect control protein and the genetic material necessary
for its production in all corn.

Previously submitted studies demonstrated the lack of toxicity of the
mCry3A protein following acute oral high-dose exposure to mice, rapid
degradation of mCry3A upon exposure to simulated mammalian gastric fluid
and the lack of significant amino acid sequence homology of the mCry3A
protein to proteins known to be mammalian toxins or human allergens.
Therefore, dietary exposure to mCry3A in corn is not anticipated to pose
any harm for the U.S. population as stated in the memorandum from A.
Fellman, through J. Kough, Ph.D., to M. Mendelsohn, dated May 31, 2005.

The only outstanding data gap identified in the May 31, 2005 memorandum
was the submission of a validated analytical method for detection of
mCry3A in corn. A new submission for the analytical method employing a
monoclonal antibody is reviewed (MRID No. 465974-01) in this report.

## 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 Asafe.@ Section 408(c)(2)(A)(ii) of the FFDCA defines
Asafe@ to mean that Athere 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 Aensure
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 Aavailable 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 mCry3A *Bacillus thuringiensis* (*Bt*) insect control protein is
produced in transgenic corn plants derived from transformation Event
MIR604. A *cry3A* gene from *Bt* subsp. *tenebrionis* was recreated
synthetically to optimize for expression in corn. Additional changes in
this corn-optimized gene were made, such that the encoded mCry3A protein
has enhanced activity against larvae of the western corn rootworm (WCRM;
*Diabrotica virgifera vigifera*) and northern corn rootworm (NCRM; *D.
longicornis barberi*). Introduced *via* transformation vector pZM26, a
*mcry3A* specific probe, consisting of 1797 base pairs (bp), was
incorporated between an MTL promoter (2556 bp) from the *Zea mays*
metallothionein-like gene and a terminator sequence from the nopaline
synthase (NOS) gene of *Agrobacterium tumefaciens* used to provide a
polyadenylation site. An *Escherichia coli manA* gene encoding a
phosphomannose isomerase *pmi* gene (1176 bp) was incorporated between a
promoter region from the *Zea mays* polyubiquitin gene (ZmUbInt (1993
bp)) and the same NOS terminator sequence described above. This *pmi*
gene, which was introduced along with the mCry3A protein via the same
pZM26 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.

Hybridization patterns indicate that one full length copy of each of the
*mcry3A* and *pmi* genes were integrated into the maize genome.
Moreover, DNA sequencing revealed that there was a 44 and 43 bp
truncation at the right and left break points of the T-DNA insert,
respectively, during the transformation process that resulted in MIR604.
Therefore, the overall integrity of the insert and the contiguousness of
the functional elements were confirmed.

The native Cry3A protein of *Bt* subsp. *tenebrionis* is a *ca.* 73 kDa
polypeptide of 644 amino acids. By comparison, the mCry3A protein
expressed in Event MIR64 corn is a *ca.* 67 kDa polypeptide of 598 amino
acids. The amino acid sequence of the mCry3A protein corresponds to that
of the native Cry3A protein, except: 1) Its N-terminus corresponds to
methionine-48 of the native protein; and 2) A cathepsin-G protease
recognition site has been introduced, beginning at amino acid residue
155 of the native protein. This cathepsin-G recognition site has the
sequence alanine-alanine-proline-phenylalanie, and has replaced the
amino acids valine-155, serine- 156, and serine-157 in the native
protein.

**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 mCry3A 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 mCry3A protein. The
acute oral toxicity data submitted support the prediction that the
mCry3A protein would be non-toxic to humans. Male and female mice (5 of
each) were dosed with 2,377 milligrams/kilograms bodyweight (mg/kg bwt)
of mCry3A protein. With the exception of one female in the test group
that was euthanized on day 2 (due to adverse clinical signs consistent
with a dosing injury), all other mice survived the study, gained weight,
had no test material-related clinical signs, and had no test
material-related findings at necropsy.

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 mCry3A protein is
not considered toxic. Amino acid sequence comparisons showed no
similarity between the mCry3A 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/WHO Standards Programme, 2001). Further data demonstrate
that mCry3A is inactivated against WCRM, when heated to 95 °C for 30
minutes.

Since mCry3A is a protein, allergenic sensitivities were considered.
Currently, no definitive tests for determining the allergenic potential
of novel proteins exist. 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 mCry3A protein is
rapidly degraded by gastric fluid *in vitro.* In a solution of simulated
gastric fluid 1 mg/mE mCry3A test protein mixed with simulated gastric
fluid (pH 1.2, containing 2 mg/mL NaCl, 14 *pL* 6 N HC1, and 2.7 mg/mL
pepsin) resulting in 10 pepsin activity units/ jag protein (complies
with 2000 US Pharmacopoeia recommendations), complete degradation of
detectable mCry3A protein occurred within 2 minutes. A comparison of
amino acid sequences of known allergens uncovered no evidence of any
homology with mCry3A, even at the level of 8 contiguous amino acids
residues. Further data demonstrate that mCry3A is not glycoslylated, is
inactivated when heated to 95 °C for 30 minutes, and is present in low
levels in corn tissue.

Therefore, the potential for the mCry3A 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 mCry3A 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. Exposure via residential or lawn
use to infants and children is also not expected because the use sites
for the mCry3A 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 2 gm/kg
showed no adverse effects. Furthermore, the expression of the modified
Cry3A protein in corn kernels has been shown to be in the parts per
million range,which makes the expected dietary exposure several orders
of magnitude lower than the amounts of mCry3A 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 mCry3A protein.

**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 mCry3A 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
mCry3A protein include the characterization of the expressed mCry3A
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 mCry3A 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
mCry3A 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 mCry3A protein, even at relatively high dose
levels (2,377 mg mCry3A/kg bwt), the mCry3A 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, mCry3A showed no sequence similarity
to any known toxinand was inactivated by heat against WCRM.

Protein residue chemistry data for mCry3A were not required for a human
health effects assessment of the subject plant-incorporated protectant
ingredients because of the lack of mammalian toxicity. However, data
submitted demonstrated low levels of mCry3A in corn tissues with less
than 2 micrograms mCry3A protein/gram dry weight in kernels and less
than 30 micrograms mCry3A protein/gram dry weight of whole corn plant.

Since modified Cry3A 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 modified Cry3A 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 modified Cry3A
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 mCry3A 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 mCry3A 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 mCry3A 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
mCry3A 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 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 mCry3A protein in corn
has been submitted and found acceptable by the Agency.

*C. Codex Maximum Residue Level\
*No Codex maximum residue levels exist for the plant-incorporated
protectant *Bacillus thuringiensis* mCry3A protein and the genetic
material necessary for its production in corn.

**[RECOMMENDATION:]{.underline}** 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 mCry3A
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 Section 3 registration and a permanent exemption from the
requirement of a tolerance.

The data submitted for the ELISA method for determining mCry3A protein
in Event MIR604 grain are classified as **ACCEPTABLE** and satisfies the
EPA Residue Chemistry Guidelines OPPTS 860.1340(c)(6) Residue Analytical
Methods and PR Notice 96-1. However, the EPA's Analytical Method
Laboratory located in Fort Meade (Maryland) will have to independently
validate Syngenta's ELISA protocol for accuracy, precision, and
sensitivity.

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

+--------+-------------------+-----------------------------------------+
| **Ta   |                   |                                         |
| ble 1. |                   |                                         |
| Prev   |                   |                                         |
| iously |                   |                                         |
| sub    |                   |                                         |
| mitted |                   |                                         |
| p      |                   |                                         |
| roduct |                   |                                         |
| chara  |                   |                                         |
| cteriz |                   |                                         |
| ation, |                   |                                         |
| tox    |                   |                                         |
| icity, |                   |                                         |
| and    |                   |                                         |
| a      |                   |                                         |
| llerge |                   |                                         |
| nicity |                   |                                         |
| stu    |                   |                                         |
| dies** |                   |                                         |
+--------+-------------------+-----------------------------------------+
| **     | **Title**         | **Summary**                             |
| MRID** |                   |                                         |
+--------+-------------------+-----------------------------------------+
| 461    | Review of         | The mCry3A protein contains 598 amino   |
| 556-01 | Characterization  | acids (*ca*. 67 kDa), whereas the       |
|        | and Safety of     | native Cry3A protein is *ca*. 73 kDa    |
|        | Modified Cry3A    | polypeptide of 644 amino acids. The     |
|        | protein and maize | amino acid sequence of the mCry3A       |
|        | (corn) plants     | protein corresponds to that of the      |
|        | derived from      | native Cry3A protein, except: 1) Its    |
|        | Event MIR604 with | N-terminus corresponds to methionine-48 |
|        | comparison to     | of the native protein; and 2) A         |
|        | native Cry3A      | cathepsin-G protease recognition site   |
|        | protein           | has been introduced, beginning at amino |
|        |                   | acid residue 155 of the native protein. |
|        |                   | The susceptibility of insect pest       |
|        |                   | species and insecticidal properties,    |
|        |                   | such as solubilization, proteolytic     |
|        |                   | processing, receptor binding, and       |
|        |                   | membrane pore forming properties, were  |
|        |                   | also determined for mCry3A protein and  |
|        |                   | differentiated with native Cry3A        |
|        |                   | protein. The mCry3A protein has a       |
|        |                   | similar spectrum of activity to the     |
|        |                   | native Cry3A, but with enhanced         |
|        |                   | toxicity to NCRW and WCRW.              |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 461    | Molecular         | Corn Event MIR604 does not contain any  |
| 556-02 | characterization  | of the backbone sequences from the      |
|        | of event MIR604   | transforming plasmid pZM26. Three       |
|        | maize (corn)      | nucleotide changes were identified, one |
|        | expressing a      | in a regulatory region of the *mCry3A*  |
|        | modified Cry3A    | gene and two in the *pmi* coding        |
|        | bacillus          | sequence. The *mCry3A* and *pmi* genes  |
|        | thuringiensis     | are closely linked.                     |
|        | protein           |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 461    | Characterization  | This study evaluated the similarity of  |
| 556-03 | of modified Cry3A | modified Cry3A (mCry3A) insecticidal    |
|        | protein produced  | protein expressed in corn event MIR604  |
|        | in event          | and mCry3A protein expressed in a       |
|        | MIR604-derived    | recombinant *E. coli* test system. The  |
|        | maize (corn) and  | mCry3A protein derived from corn event  |
|        | comparison with   | MIR604 and recombinant *E. coli* had    |
|        | modified Cry3A    | the same approximate molecular weight   |
|        | protein expressed | (ca. 67,700 Da.) based on mass spectral |
|        | in recombinant    | analysis (from MRID 461556-06). The     |
|        | Escherichia coli  | mCry3A protein from both sources was    |
|        |                   | immunologically cross-reactive with the |
|        |                   | same anti-mCry3A antibody. Both         |
|        |                   | proteins produced comparable toxicities |
|        |                   | toward Western Corn Rootworm larvae,    |
|        |                   | based on LC~50~ values. There was no    |
|        |                   | evidence of post-translational          |
|        |                   | glycosylation of mCry3A protein from    |
|        |                   | either source. It was concluded that    |
|        |                   | the mCry3A proteins from corn event     |
|        |                   | MIR604 and from recombinant *E. coli*   |
|        |                   | were substantially the same.            |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 461    | Quantification of | The plant extracts (including leaves,   |
| 556-04 | modified Cry3A    | roots, kernels, silk, pollen, silage,   |
|        | and pmi proteins  | and whole plants) from inbred and       |
|        | in transgenic     | hybrid corn lines derived from MIR604   |
|        | maize (corn)      | field plants were quantitatively        |
|        | tissues, whole    | analyzed for mCry3A by ELISA. The       |
|        | plants, and       | magnitude of expression for tissue      |
|        | silage derived    | types was as follows in descending      |
|        | from              | order: leaves, roots, silage, and       |
|        | transformation    | kernels. All control tissues were       |
|        | event MIR604      | negative for the expression of mCry3A.  |
|        |                   | Number range CHAPTERThe mean extraction |
|        |                   | efficiency for mCry3A over all tissues  |
|        |                   | (except for pollen or silk tissue,      |
|        |                   | where levels were too low to be         |
|        |                   | determined) was 76.6%. Low, but         |
|        |                   | quantifiable, levels of PMI protein     |
|        |                   | were found in most of the Event         |
|        |                   | MIR604-derived plant tissues analyzed   |
|        |                   | including pollen. Number range          |
|        |                   | CHAPTERThe mCry3A and PMI proteins were |
|        |                   | stably expressed in four backcross      |
|        |                   | generations in leaf tissue analyzed at  |
|        |                   | anthesis stage. Mean levels across all  |
|        |                   | backcross generations were *ca.* 11.8 - |
|        |                   | 15.5 μg/g dry weight and 1.1 - 1.3 μg/g |
|        |                   | dry weight for mCry3A and PMI proteins, |
|        |                   | respectively.                           |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 461    | Characterization  | This study characterized test material  |
| 556-05 | of modified Cry3A | MCRY3A-0102, a microbially produced     |
|        | test substance    | protein preparation containing a        |
|        | mCry3A-0102 and   | modified Cry3A (mCry3A) protein. The    |
|        | certificate of    | purity of mCry3A in the test material   |
|        | analysis          | was shown to be *ca.* 90.3% by weight   |
|        |                   | using SDS-PAGE analysis. Western blot   |
|        |                   | analysis of the test material showed a  |
|        |                   | single immunoreactive band              |
|        |                   | corresponding to the predicted          |
|        |                   | molecular weight of ca. 67,700 Da. The  |
|        |                   | test material was insecticidally active |
|        |                   | and had a 144-hour LC~50~ of 1.4 µg/mL  |
|        |                   | diet (95% confidence interval: 0.7 -    |
|        |                   | 2.2 µg/mL) against Western corn         |
|        |                   | rootworm (WCRW) larvae. Two forms of    |
|        |                   | mCry3A were found in the test material, |
|        |                   | designated mCry3A-SF and mCry3A-LF,     |
|        |                   | respectively, and were both             |
|        |                   | insecticidally active against WCRW. On  |
|        |                   | this basis, and taking into account the |
|        |                   | high degree of structural homology      |
|        |                   | (97.4% amino acid identity), the two    |
|        |                   | forms of mCry3A in test material        |
|        |                   | MCRY3A-0102 were considered to be       |
|        |                   | equivalent. The test material was       |
|        |                   | re-analyzed ca. 9 months after its      |
|        |                   | initial characterization and found to   |
|        |                   | be substantially stable when stored at  |
|        |                   | -20 °C.                                 |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 461    | Further           | Number range CHAPTERThe test material,  |
| 556-06 | Characterization  | MCRY3A-0102, containing mCry3A protein  |
|        | of Modified Cry3A | was shown, by SDS-PAGE and MALDI TOF    |
|        | Test Substance    | mass spectrometry, to contain two       |
|        | MCRY3A-0102       | closely related components in a ratio   |
|        |                   | of *ca.* 2:3. The lesser of the two     |
|        |                   | components, with the lower molecular    |
|        |                   | weight, corresponded to the intended    |
|        |                   | mCry3A protein with 598 amino acids.    |
|        |                   | The other component contained the same  |
|        |                   | 598 amino acids as the first component  |
|        |                   | but also contained an additional 16     |
|        |                   | amino acids at the N--terminal end of   |
|        |                   | the protein. The identity of the two    |
|        |                   | components was conclusively determined  |
|        |                   | by peptide mapping using tandem (MS/MS) |
|        |                   | mass spectrometry and by MALDI TOF mass |
|        |                   | spectrometry of the intact proteins.    |
|        |                   | The molecular weight data showed the    |
|        |                   | two proteins to have masses of 67,519   |
|        |                   | and 69,138 Da.                          |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 461    | In vitro          | The susceptibility of mCry3A protein to |
| 556-07 | digestibility of  | proteolytic degradation was evaluated   |
|        | modified Cry3A    | in simulated gastric fluid (SGF)        |
|        | protein           | containing pepsin. Modified Cry3A       |
|        | (mCry3A-0102 and  | protein from transgenic corn and        |
|        | IAPMIR604-0103)   | recombinant E. coli (test material      |
|        | under simulated   | MCRY3A-0102) was readily degraded in    |
|        | mammalian gastric | SGF. The data support a conclusion that |
|        | conditions        | mCry3A protein expressed in transgenic  |
|        |                   | plants will be readily digested as      |
|        |                   | conventional dietary protein under      |
|        |                   | typical mammalian gastric conditions.   |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 461    | Effect of         | At 95°C mCry3A protein was completely   |
| 556-08 | temperature on    | inactivated against WCRM. At 4°C, 25°C, |
|        | the stability of  | and 37° C there was little or no effect |
|        | modified Cry3A    | on mCry3A bioactivity, while at 65°C    |
|        | protein           | there was some reduction in the         |
|        | (mCry3A-0102)     | bioactivity.                            |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 461    | Analysis for the  | Among the wet-milled fractions, the     |
| 556-09 | presence of       | medium fiber (0.46 µg mCry3A/g), fine   |
|        | modified Cry3A    | fiber (0.26 µg mCry3A/g), and gluten    |
|        | protein in wet    | meal (0.24 µg mCry3A/g) fractions       |
|        | and dry milled    | yielded quantifiable amounts of mCry3A. |
|        | fractions, corn   | Among the dry-milled fractions the      |
|        | oil and corn      | highest concentrations were found in    |
|        | chips from corn   | the flaking grits (2.12 µg mCry3A/g),   |
|        | (maize) event     | the corn hulls (1.42 µg mCry3A/g), and  |
|        | MIR604            | the coarse grit (0.92 µg mCry3A/g)      |
|        |                   | fractions. Levels of mCry3A found in    |
|        |                   | the other dry-milled fractions,         |
|        |                   | including fine grits, corn meal, corn   |
|        |                   | cone and corn flour, were between 0.32  |
|        |                   | and 0.69 µg mCry3A/g. Although the      |
|        |                   | concentration of mCry3A protein         |
|        |                   | measured in the flour used to prepare   |
|        |                   | the corn chips was 0.32 µg mCry3A /g,   |
|        |                   | no mCry3A protein was detected in the   |
|        |                   | corn chips. Similarly, mCry3A protein   |
|        |                   | was not detectable in oil, whereas the  |
|        |                   | starting material, flaking grits,       |
|        |                   | contained 2.12µg mCry3A/g.              |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 461    | Acute oral        | MCRY3A-0102 was not acutely toxic to    |
| 556-10 | toxicity study of | mice. There was no evidence of toxicity |
|        | modified Cry3A    | at 2,632 mg MCry3A-0102/kg body weight, |
|        | protein           | representing ca. 2,377 mg mCry3A        |
|        | (mCry3A-0102) in  | protein/kg body weight. The estimated   |
|        | the mouse         | LD~50~ value for pure mCry3A protein in |
|        |                   | male and female mice was \> 2,377 mg/kg |
|        |                   | body weight, the single dose used.      |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 461    | Modified Cry3A    | The National Center for Biotechnology   |
| 556-11 | protein as        | Information (NCBI) GenBank Database     |
|        | expressed in      | (NCBI, 2003) containing all publicly    |
|        | transgenic maize  | available protein sequences was queried |
|        | event MIR604:     | for proteins with amino acid sequences  |
|        | assessment of     | having significant homology to mCry3A   |
|        | amino acid        | protein and that were toxins. The query |
|        | homology with     | found no significant amino acid         |
|        | known toxins      | homology between any protein toxin and  |
|        |                   | the mCry3A protein.                     |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 461    | Modified Cry3A    | No significant similarity was found     |
| 556-12 | protein as        | between any of the mCry3A 80-amino acid |
|        | expressed in      | peptides and any entries in the SBI     |
|        | transgenic maize  | Allergen Database. Also, there were no  |
|        | event MIR604:     | alignments of eight or more contiguous  |
|        | assessment of     | amino acids between the mCry3A protein  |
|        | amino acid        | and any of the proteins in the allergen |
|        | homology with     | database. Overall, the mCry3A protein   |
|        | known allergens   | showed no significant amino acid        |
|        |                   | homology to any known or putative       |
|        |                   | allergenic protein.                     |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 465    | Number range      | Number range CHAPTERAn ELISA method to  |
| 974-01 | CHAPTERAnalytical | quantify modified Cry3A (mCry3A)        |
|        | Method for the    | protein in corn tissues was validated   |
|        | Detection of the  | by an independent third-party           |
|        | P                 | laboratory (EnvironLogix, Inc.) using   |
|        | lant-Incorporated | grain from Event MIR604, which contains |
|        | Protectant        | mCry3A, and 25 other                    |
|        | Modified Cry3A    | commercially-available transgenic and   |
|        | Protein in Event  | non-transgenic corns which do not       |
|        | MIR604 Corn Grain | contain mCry3A. The average mCry3A LOD  |
|        | and Independent,  | for three different lots of reagents    |
|        | Third-Party       | tested was 5.5 ± 4.6 ppb, which         |
|        | Validation of     | established the sensitivity of the      |
|        | Said Method       | ELISA assay at approximately 25%. In a  |
|        |                   | ground grain assay, the mean mCry3A     |
|        |                   | protein detected was 159.1 ± 70.2 ng/g  |
|        |                   | fresh weight with a coefficient of      |
|        |                   | variation (%CV) of 44.1%. However, the  |
|        |                   | data submitted showed: less than        |
|        |                   | optimal LOD; low sensitivity; high      |
|        |                   | standard deviations; the %CV exceeding  |
|        |                   | the recommended value (20%); anomalous  |
|        |                   | frequency of false positives in the     |
|        |                   | non-MIR604 corn (both transgenic and    |
|        |                   | conventional) as well as false          |
|        |                   | negatives in the MIR604 corn; and       |
|        |                   | unexpected cross reactivity with other  |
|        |                   | Cry3Bb1 samples.                        |
|        |                   |                                         |
|        |                   | **Classification: Supplemental**        |
+--------+-------------------+-----------------------------------------+
| 461    | Phosphomannose    | Two nucleotide changes were discovered  |
| 556-13 | Isomerase as      | in the *pmi* gene sequence inserted in  |
|        | expressed in      | corn Event MIR604. This resulted in two |
|        | transgenic maize  | changes in the PMI protein; valine-61   |
|        | event MIR604:     | was replaced by alanine, and            |
|        | assessment of     | glutamine-210 was replaced by           |
|        | amino acid        | histidine. These substitutions have not |
|        | homology with     | resulted in any apparent functional     |
|        | known toxins      | change in the PMI protein. The NCBI     |
|        |                   | GenBank Database containing all         |
|        |                   | publicly available protein sequences    |
|        |                   | was queried for proteins with amino     |
|        |                   | acid sequences having significant       |
|        |                   | homology to this modified PMI protein   |
|        |                   | that were toxins. The query found no    |
|        |                   | significant amino acid homology between |
|        |                   | any protein toxin and the PMI protein   |
|        |                   | expressed in corn Event MIR604.         |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+
| 464    | Phosphomannose    | No significant similarity was found     |
| 252-01 | Isomerase as      | between any of the PMI 80-amino acid    |
|        | expressed in      | peptides and any entries in the SBI     |
|        | transgenic maize  | Allergen Database. However, in the      |
|        | event MIR604:     | eight or more contiguous amino acids    |
|        | assessment of     | homology search, there was an alignment |
|        | amino acid        | between the PMI protein and a recently  |
|        | homology with     | identified allergen, α-parvalbumin from |
|        | known allergens   | *Rana species* CH2001 (a frog of        |
|        |                   | Indonesian origin). However, a serum    |
|        |                   | screening concluded that there is no    |
|        |                   | cross-reactivity between PMI and serum  |
|        |                   | IgE (obtained from an allergic          |
|        |                   | individual who displayed food-induce    |
|        |                   | anaphylaxis from α-parvalbumin). Bovine |
|        |                   | serum albumin was also tested as an     |
|        |                   | internal check. The observed low degree |
|        |                   | of sequence identity between MIR604 PMI |
|        |                   | and α-parvalbumin is not biologically   |
|        |                   | relevant.                               |
|        |                   |                                         |
|        |                   | **Classification: Acceptable**          |
+--------+-------------------+-----------------------------------------+

**[465974-01]{.underline} Independent Laboratory Validation of
Monoclonal-based ELISA method for the Detection of mCry3A in Event
MIR604 corn grain**

A monoclonal based enzyme-linked immunosorbent assay (ELISA) method was

developed by Syngenta to detect modified Cry3A (mCry3A) protein
expressed in MIR604 seed and leaf tissues. An independent, third party
laboratory (EnvironLogix, Inc.) utilized the assay methodology, Event
MIR604 seed, negative isoline seed, and three lots of mCry3A monoclonal
antibody for validation of assay protocol, sensitivity and
cross-reactivity, according to the USDA GIPSA directive. The assay
sensitivity was estimated at 1 positive Event MIR604 kernel in 999
non-MIR604 kernels (0.1%), which was based on a minimum of 120 ground
seed samples (with a LOD of 0.33 ppb for seed material). The average
quantification was 2.07 ppb (ng/g fresh weight corn). No cross
reactivity with other commercial, conventional and transgenic corn was
detected with the exception of products expressing the Cry3Bb1 protein.
Moreover, extraction efficiency was determined at 59% for ground seed
and 75% for leaf tissue.

**[CLASSIFICATION:]{.underline} ACCEPTABLE-** Number range CHAPTERThe
monoclonal assay antibody-based commercial ELISA detection assay (tested
by EnviroLogix, Inc.) satisfies the EPA Residue Chemistry Guidelines
OPPTS 860.1340(c)(6) Residue Analytical Methods and PR Notice 96-1.
However, the EPA's Analytical Method Laboratory located in Fort Meade
(Maryland) will have to independently validate Syngenta's ELISA protocol
for accuracy, precision, and sensitivity.

**REFERENCES**

Chen, E. & Stacy, C. (2003) Modified Cry3A toxins and nucleic acid
sequences coding

therefore. WO Patent No. 03/018810.

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.

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.

Sekar et al. (1987) Molecular cloning and characterization of the
insecticidal crystal

protein gene of *Bacillus thuringiensis* var. *tenebrionis.* **Proc.
Natl. Acad. Sci.**

**USA** 8: 7036-7040.

Sjoblad, R. D., McClintock, J. T., and Engler, R. (1992) AToxicological
Considerations

for Protein Components of Biological Pesticide Products,@ **Reg.
Toxicol.**

**Pharmacol**. 15(1): 3-9.