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**UNITED STATES ENVIRONMENTAL PROTECTION AGENCY**

WASHINGTON, D.C. 20460

> OFFICE OF PESTICIDES AND TOXIC
>
> SUBSTANCES
>
> JUN 14 2006
>
> **[MEMORANDUM]{.underline}**
>
> **SUBJECT:** Review of Human Health and Product Characterization Data
> for a Temporary Tolerance Exemption for Bt Cry 1A.105 and Cry2Ab2 in
> corn and an EUP Request for Insect-Protected Corn MON 89034, MON
> 89017, and MON 89034 x MON 89017
>
> **TO:** Mike Mendelsohn
>
> Regulatory Action Leader
>
> Microbial Pesticides Branch, Biopesticides and
>
> Pollution Prevention Division (7511P)
>
> **FROM:** Rebecca L. Edelstein, Ph.D., Chemist /s/
>
> Ibrahim Barsoum, Ph.D., Biologist /s/
>
> Microbial Pesticides Branch, Biopesticides and
>
> Pollution Prevention Division (7511P)
>
> **THROUGH:** John L. Kough, Ph.D., Biologist /s/
>
> Microbial Pesticides Branch, Biopesticides and
>
> Pollution Prevention Division (7511P)
>
> **ACTION REQUESTED:** To review the human health and product
> characterization data for a temporary tolerance exemption petitions
> for Bt Cry1A.105 and EUP request for MON 89034, MON 89017, and MON
> 89034 x MON 89017 corn
>
> **CONCLUSION:**

Monsanto has submitted an EUP request for events MON 89034, MON 88017,
and MON 89034 x MON 88017, as well as petitions for temporary tolerance
exemptions for Cry1A.105 and Cry2Ab2 in corn. The product
characterization and human health data submitted on event MON 89034 and
the data previously submitted on event MON 88017 are sufficient to
support the EUP request. In addition, the product characterization and
human health data submitted on Cry1A.105 and cited for Cry2Ab2 are
sufficient to support the temporary tolerance exemption requests. The
Cry3Bb1 and CP4 EPSPS proteins are already covered by tolerance
exemptions (40 CFR 180.1214: Cry3Bb1 protein and the genetic material
necessary for its production in corn when used as plant-incorporated
protectants in field corn, sweet corn, and popcorn; 40 CFR 180.1174: CP4
EPSPS and the genetic material necessary for its production in all
plants).

MON 89034, which was developed by *Agrobacterium*-mediated
transformation of corn using the 2T-DNA plasmid vector PV-ZMIR245,
produces two *Bacillus thuringiensis* proteins, Cry1A.105 and Cry2Ab2.
These proteins are intended to provide protection from feeding damage
caused by a number of lepidopteran pests. Cry1A.105 is a chimeric
protein composed of portions of Cry1Ab, Cry1Ac, and Cry1F proteins. MON
88017, developed by *Agrobacterium*-mediated transformation of corn with
plasmid vector PV-ZMIR39, produces Cry3Bb1 and CP4 EPSPS proteins. The
Cry3Bb1 protein is intended to provide protection from feeding damage by
corn rootworm larvae, and the CP4 EPSPS protein confers tolerance to the
Roundup® family of herbicides.

Characterization of the DNA isolated from event MON 89034 corn using
restriction enzyme digests and Southern blot analysis indicated that the
expected DNA inserted in the corn genome at a single locus and contains
one copy each of the *cry1A.105* and *cry2Ab2* expression cassettes.
There were no other elements detectable other than those associated with
the respective cassettes. No backbone sequences from plasmid PV-ZMIR245
or *nptII* coding sequences were detected in the corn genome.

Protein characterization data demonstrate that the plant-produced
Cry1A.105 has characteristics and activities that are similar to those
of the *E. coli*-produced Cry1A.105 used in several safety studies. The
following techniques were used to characterize and compare the
plant-produced and the *E. coli*-produced proteins: sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot
analysis, densitometry, matrix assisted laser desorption time of flight
(MALDI-TOF) mass spectrometry, glycosylation analysis, and a Cry1A.105
insecticidal activity assay. Glycoslyation analysis indicated that the
proteins are not glycoslyated. These analyses demonstrated the
structural and functional similarity between the plant-produced and the
*E. coli*-produced Cry1A.105 proteins and justified the use of *E.
coli*-produced protein in toxicity studies. Monsanto also provided
information showing the similarity between Cry1A.105, Cry1Ab, and
Cry1Ac.

In support of the safety of the Cry1A.105 protein, Monsanto submitted an
acute oral toxicity study, which demonstrated the lack of mammalian
toxicity at high levels of exposure to the pure Cry1A.105 protein. An
*in vitro* digestibility study demonstrated that the protein digested
rapidly (within \~ 30 seconds in simulated gastric fluid). In addition,
amino acid sequence comparisons showed no similarities between Cry1A.105
and known allergens or mammalian toxins. A heat stability study showed
that Cry1A.105 is no longer detectable by Western blot after heating at
204 ºC for 20 minutes.

In support of the safety of the Cry2Ab2 protein, Monsanto cited
previously submitted data demonstrating the rapid digestion of Cry2Ab2
in simulated digestive fluid, lack of mammalian toxicity, and lack of
amino acid similarities between Cry2Ab2 and known allergens or mammalian
toxins. Monsanto did not submit protein characterization data on the
plant-produced Cry2Ab2 from event MON 89034 to demonstrate equivalence
with the *B. thuringiensis*-produced protein used in several of the
safety studies. However, Monsanto previously submitted data
demonstrating similarity of the protein produced in event MON 840 and
the *B. thuringiensis*-produced protein, and Monsanto provided
information indicating that the Cry2Ab2 coding sequence (including the
transit peptide sequence *SSU-CTP* targeting sequence) inserted in event
MON 89034 is identical to the coding sequence inserted in event MON 840
corn: In an attachment to an email sent by Natalia Bogdanova of Monsanto
on June 1, 2006, Monsanto states "The *cry2Ab2* expression cassette in
vector PV-ZMIR245 that was used to produce MON 89034 contains the same
elements as the *cry2Ab2* cassette in vector ZMBK28 with one exception.
The e35S promoter that was used to regulate expression of the *cry2Ab2*
in MON 84006 was replaced by the FMV promoter in PV-ZMIR245." In the EUP
data package reviewed here, Monsanto also submitted data demonstrating
that the Cry2Ab2 protein is no longer detectable by western blot after
heating at 204 ºC for 20 minutes.

Expression level data were provided for Cry1A.105, Cry2Ab2, and Cry3Bb1
in the individual events and in the conventional cross. All three
proteins are expressed at relatively low levels. The data were produced
using ELISA methods specific for each protein.

**DATA REVIEW RECORD**

Active Ingredient: *Bacillus thuringiensis* Cry1A.105, Cry2Ab2, and
Cry3Bb1 insecticidal proteins and the genetic material necessary for
their production in corn

Product Name: MON 89034, MON 88017, MON 89034 x MON 88017

Company Name: Monsanto Company

ID No:

Chemical Number: 006498, 006514, 006515, 006502

Decision Number: 353287

DP Barcode: 324861

MRID/Study Titles: 46471601---Data in Support of an Application for an
Experimental Use Permit to Test ZMIR245xMON 88017 Combined Corn Trait
along with ZMIR245 and MON 88017

46694501---Summary of Southern Blot Analyses of MON 89034 and MON 89697
Corn

46694502---Assessment of the Cry1A.105, Cry2Ab2, and Cry3Bb1 Protein
Levels in Tissues of Insect-Protected Corn MON 89034, MON 89034 x MON
88017, MON 89597, and MON 89597 x MON 88017

46694503---Qualitative Detection Method for the Cry2Ab2 Protein in Corn
Leaf and Seed of MON 89034 and MON 89597

46694601---Structural and Functional Similarity of the Cry1A.105 Protein
to Cry1A Class of *Bacillus thuringiensis* Proteins: Final Report

46694603---Acute Oral Toxicity Study in Mice with Cry1A.105 Protein

46694604---Characterization of the Cry1A.105 Protein Purified from the
Corn Grain of MON 89034 and Comparison of the Physiochemical and
Functional Properties of the Plant-Produced and *E. coli*-Produced
Cry1A.105 Proteins

46694605---Bioinformatics Analysis of the Cry1A.105 Protein Utilizing
the AD6, Toxin5, and Allpeptides Databases

46694606---Assessment of the In Vitro Digestibility of the Cry1A.105
Protein in Simulated Gastric Fluid

46694607---Immunodetection of Cry2Ab2 and Cry1A.105 Proteins in Corn
Grain from MON 89034 Following Heat Treatment

45086305---Assessment of the Equivalence of Proteins Expressed in Corn
Event MON 840

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

Monsanto has submitted an EUP request for events MON 89034, MON 88017,
and MON 89034 x MON 88017. MON 89034, which was developed by
*Agrobacterium*-mediated transformation of corn using the 2T-DNA plasmid
vector PV-ZMIR245, produces two *Bacillus thuringiensis* proteins,
Cry1A.105 and Cry2Ab2. These proteins are intended to provide protection
from feeding damage caused by a number of lepidopteran pests. Cry1A.105
is a chimeric protein composed of portions of Cry1Ab, Cry1Ac, and Cry1F
proteins. EPA granted a temporary tolerance exemption for Cry2Ab2 in
corn and cotton in 2001, and removed the time limitation for cotton in
2004; however, the exemption has expired for Cry2Ab2 in corn. Monsanto
has submitted petitions to establish temporary tolerance exemptions for
both Cry1A.105 and Cry2Ab2 in corn. Monsanto is relying on previously
submitted data to support the tolerance exemption for Cry2Ab2 but has
submitted new data, reviewed in this memorandum, to support the
tolerance exemption for Cry1A.105. Safety data on Cry2Ab2 were reviewed
previously in a memorandum from M. Watson to M. Mendelsohn dated Feb.
12, 2001 (MRIDs 44966602 Acute Oral Toxicity Study; 44966603 *In vitro*
Digestibility; 44966604 Bioinformatics Analysis---Allergen Database;
44966605 Bioinformatics Analysis---Toxin and Public Domain Genetic
Databases). Monsanto is also relying on previously submitted data to
demonstrate equivalence of the plant-produced Cry2Ab2 protein and the
microbially-produced protein, which was used in some of the safety
studies. Monsanto had previously submitted the protein characterization
data to support an EUP request for MON 840; however, the data were never
reviewed since the EUP request was withdrawn. The data are therefore
reviewed in this memorandum.

MON 88017, developed by *Agrobacterium*-mediated transformation of corn
with plasmid vector PV-ZMIR39, produces Cry3Bb1 and CP4 EPSPS proteins.
The Cry3Bb1 protein is intended to provide protection from feeding
damage by corn rootworm larvae, and the CP4 EPSPS protein confers
tolerance to the Roundup® family of herbicides. On March 31, 2004, EPA
established a tolerance exemption for Bt Cry3Bb1 protein and the genetic
material necessary for its production in corn when used as
plant-incorporated protectants in field corn, sweet corn, and popcorn
(40 CFR 180.1214). In addition, EPA has established a tolerance
exemption for CP4 EPSPS and the genetic material necessary for its
production in all plants (40 CFR 180.1174). Bt Cry3Bb1 protein and the
genetic material necessary for its production in event MON 88017 corn is
currently registered (EPA Reg. No. 524-LLR). The human health and
product characterization data submitted for registration of the product
(reviewed in a memorandum dated July 25, 2005 from A. Fellman to M.
Mendelsohn) supports this EUP, and Bt Cry3Bb1 produced in event MON
88017 and MON 88017 x MON 89034 is covered by the tolerance exemption
for Bt Cry3Bb1 (40 CFR 180.1214).

This memorandum reviews the product characterization and human health
data submitted in support of Monsanto's EUP request and petitions for
establishing tolerance exemptions for Cry1A.105 and Cry2Ab2.

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

The submitted human health data are sufficient to support an EUP and
temporary exemptions from the requirement of a food tolerance. However,
the following are needed for section 3 registration:

-   Validated detection method for Cry1A.105 and Cry2Ab2 in corn

-   Southern blot analysis (or other appropriate molecular
    characterization) of the event(s) and conventional crosses (stacks)
    being registered

-   Protein characterization of plant-expressed Cry2Ab2 from event MON
    89034 to show equivalence with the microbial protein used in safety
    testing

-   Expression levels for EPSPS in the stacked product

-   Plant-produced Cry1A.105 shows both a higher-molecular weight
    immunoreactive band and lower molecular weight immunoreactive bands
    in the western blot analysis in addition to the full-length protein.
    These additional bands appear to be absent in the *E. coli*-produced
    protein preparation. Monsanto should address whether these
    differences are a result of protein purification or whether these
    different molecular weight proteins are likely present in the plant.
    Monsanto should also address whether use of the *E. coli* protein as
    a surrogate for safety testing is appropriate, given the differences
    in the protein preparations.

-   Provide cited protocols for Southern blot analysis

-   Discuss determination of equivalence between microbially-produced
    and plant-produced proteins using bioactivity assays. Are statistics
    used? Why was the acceptance criteria determined to be \<4 fold
    difference between the mean EC~50~ values in the corn earworm
    diet-incorporation insect biassay.

-   Provide the following SOPs: SOP BR-ME-0044-03, SOP BR-ME-0044-02,
    and BR-ME-0956-02.

**[HUMAN HEALTH ASSESSMENT Cry1A.105]{.underline}**

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

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

**Toxicological Profile**

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

**1. Mammalian Toxicity and Allergenicity Assessment**

Monsanto has submitted acute oral toxicity data demonstrating the lack
of mammalian toxicity at high levels of exposure to the pure Cry1A.105
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 the
observed adverse effects and clarify the source of these effects (Tiers
II & III).

An acute oral toxicity study in mice (MRID 46694603) indicated that
Cry1A.105 is non-toxic to humans. Cry1A.105 produced from microbial
culture was dosed by gavage as two doses separated by 4 hours (±20
minutes) to 10 females and 10 males (2072 mg/kg body weight). Two
control groups were also included in the study: a bovine serum albumin
protein control, and a vehicle control. One male in the test protein
group was moribund and sacrificed on day 1 due to a mechanical dosing
error; this death was not attributed to the test material. All other
mice survived the study. There were no significant differences in body
weight or body weight change among the three groups during the study,
and no treatment-related gross pathological findings were observed. The
oral LD~50~ for males, females, and combined mice was greater than 2072
mg/kg.

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 Cry1A.105, even at
relatively high dose levels, the Cry1A.105 protein is not considered
toxic. Further, amino acid sequence comparisons showed no similarities
between the Cry1A.105 and known toxic proteins in protein databases that
would raise a safety concern.

Since Cry1A.105 is a protein, allergenic potential was also considered.
Currently, no definitive tests for determining the allergenic potential
of novel proteins exist. Therefore, EPA uses a weight-of- evidence
approach where the following factors are considered: source of the
trait; amino acid sequence 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 by heat, acid, and proteases, may be
glycosylated, and can be present at high concentrations in the food.

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

2.  Amino acid sequence. A comparison of the amino acid sequence of
    Cry1A.105 with known allergens showed no overall sequence similarity
    or identity at the level of eight contiguous amino acid residues.

3.  Prevalence in food. Expression level analysis indicated that the
    protein is present at relatively low levels in corn: approximately 3
    µg/g in grain on a dry weight basis. Thus, the expression has been
    shown to be in the parts per million range.

4.  Digestibility. The Cry1A.105 protein was digested within 30 seconds
    in simulated gastric fluid containing pepsin.

5.  Glycosylation. Cry1A.105 expressed in corn was shown to have not to
    be glycosylated []{.mark}

6.  Conclusion. Considering all of the available information, EPA has
    concluded that the potential for Cry1A.105 to be a food allergen is
    minimal.

**2. Aggregate Exposures**

Pursuant to FFDCA section 408(b)(2)(D)(vi), EPA considers available
information concerning aggregate exposures from the pesticide residue in
food and all other non-occupational exposures, including drinking water
from ground water or surface water and exposure through pesticide use in
gardens, lawns, or buildings (residential and other indoor uses).

The Agency has considered available information on the aggregate
exposure levels of consumers (and major identifiable subgroups of
consumers) to the pesticide chemical residue and to other related
substances. These considerations include dietary exposure under the
tolerance exemption and all other tolerances or exemptions in effect for
the plant-incorporated protectants chemical residue, and exposure from
non-occupational sources. Exposure via the skin or inhalation is not
likely since the plant- incorporated protectant is contained within
plant cells, which essentially eliminates these exposure routes or
reduces these exposure routes to negligible. In addition, even if
exposure can occur through inhalation, the potential for Cry1A.105 to be
an allergen is low, as discussed above. Although the allergenicity
assessment focuses on potential to be a food allergen, the data also
indicate a low potential for Cry1A.105 to be an inhalation allergen.
Exposure via residential or lawn use to infants and children is also not
expected because the use sites for the Cry1A.105 protein is
agricultural. Oral exposure, at very low levels, may occur from
ingestion of processed corn products and, theoretically, drinking water.
However oral toxicity testing showed no adverse effects. Furthermore,
the expression of the Cry1A.105 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 amount of Cry1A.105
shown to have no toxicity. Therefore, even if negligible aggregate
exposure should occur, the Agency concludes that such exposure would
result in no harm due to the lack of mammalian toxicity and low
potential for allergenicity demonstrated for the Cry1A.105 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 from the
plant-incorporated protectant, we conclude that there are no cumulative
effects for the Cry1A.105 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
Cry1A.105 protein includes the characterization of the expressed
Cry1A.105 protein in corn, as well as the acute oral toxicity study,
amino acid sequence comparisons to known allergens and toxins, and in
vitro digestibility of the protein. The results of these studies were
used to evaluate human risk, and the validity, completeness, and
reliability of the available data from the studies were also considered.

Adequate information was submitted to show that the Cry1A.105 test
material derived from microbial culture was biochemically and
functionally equivalent to the protein produced by the
plant-incorporated protectant ingredient in corn. Microbially produced
protein was used in the safety studies so that sufficient material for
testing was available.

The acute oral toxicity data submitted support the prediction that the
Cry1A.105 protein would be non-toxic to humans. As mentioned above, when
proteins are toxic, they are known to act via acute mechanisms and at
very low dose levels (Sjoblad, Roy D., et al., "Toxicological
Considerations for Protein Components of Biological Pesticide Products,"
Regulatory Toxicology and Pharmacology 15, 3-9 (1992)). Since no
treatment-related adverse effects were shown to be caused by the
Cry1A.105 protein, even at relatively high dose levels, the Cry1A.105
protein is not considered toxic. Basing this conclusion on acute oral
toxicity data without requiring further toxicity testing and residue
data is similar to the Agency position regarding toxicity and the
requirement of residue data for the microbial *Bacillus thuringiensis*
products from which this plant-incorporated protectant was derived (See
40 CFR 158.740(b)(2)(i)). For microbial products, further toxicity
testing and residue data are triggered when significant adverse effects
are seen in studies such as the mouse oral toxicity study. Further
studies verify the observed adverse effects and clarify the source of
these effects (Tiers II and III).

Residue chemistry data were not required for a human health effects
assessment of the subject plant-incorporated protectant ingredients
because of the lack of mammalian. However, data submitted demonstrated
low levels of the Cry1A.105 in corn tissues.

Since Cry1A.105 is a protein, potential allergenicity is also considered
as part of the toxicity assessment. Considering all of the available
information (1) Cry1A.105 originates from a non-allergenic source; (2)
Cry1A.105 has no sequence similarities with known allergens; (3)
Cry1A.105 is not glycosylated; (4) Cry1A.105 will only be present at low
levels in food; and (5) Cry1A.105 is rapidly digested in simulated
gastric fluid; EPA has concluded that the potential for Cry1A.105 to be
a food allergen is minimal.

Neither available information concerning the dietary consumption
patterns of consumers (and major identifiable subgroups of consumers
including infants and children) nor safety factors that are generally
recognized as appropriate for the use of animal experimentation data
were evaluated. The lack of mammalian toxicity at high levels of
exposure to the Cry1A.105 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 ingredient include the nucleic
acids (DNA, RNA) that encode these proteins and regulatory regions. The
genetic material (DNA, RNA), necessary for the production of the
Cry1A.105 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(b)(2)(C) also provides that EPA shall apply
an additional tenfold margin of safety for infants and children in the
case of threshold effects to account for prenatal and postnatal toxicity
and the completeness of the database unless EPA determines that a
different margin of safety will be safe for infants and children.

In this instance, based on all the available information, the Agency
concludes that there is a finding of no toxicity for the Cry1A.105
protein and the genetic material necessary for its 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 considerations of consumption patterns, special
susceptibility, and cumulative effects do not apply.

**c) Overall Safety Conclusion**

There is a reasonable certainty that no harm will result from aggregate
exposure to the U.S. population, including infants and children, to the
Cry1A.105 protein and the genetic material necessary for its production.
This includes all anticipated dietary exposures and all other exposures
for which there is reliable information. The Agency has arrived at this
conclusion because, as discussed above, no toxicity to mammals has been
observed, nor any indication of allergenicity potential for the
plant-incorporated protectant.

**5. Other Considerations**

**a) Endocrine Disruptors**

The pesticidal active ingredient is a protein, derived from a source
that is not known to exert an influence on the endocrine system.
Therefore, the Agency is not requiring information on the endocrine
effects of the plant-incorporated protectant at this time.

**b) Analytical Method(s)**

A short description of an enzyme-linked immunosorbent assay for the
detection and quantification of Cry1A.105 in corn tissue has been
submitted.

**c) Codex Maximum Residue Level**

No Codex maximum residue level exists for the plant-incorporated
protectant *Bacillus thuringiensis* Cry1A.105 protein and the genetic
material necessary for its production in corn.

**[HUMAN HEALTH ASSESSMENT Cry2Ab2]{.underline}**

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

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

**Toxicological Profile**

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

**1. Mammalian Toxicity and Allergenicity Assessment**

Monsanto has submitted acute oral toxicity data demonstrating the lack
of mammalian toxicity at high levels of exposure to the pure Cry2Ab2
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 the observed adverse effects and clarify the
source of these effects (Tiers II & III).

An acute oral toxicity study in mice (MRID 44966602) indicated that
Cry2Ab2 is non-toxic to humans. Three groups of ten male and ten female
mice were dosed by oral gavage with 30, 300, or 1000 mg/kg bodyweight of
microbially-produced Cry2Ab2 protein. Two negative control groups were
also included in the study: bovine serum albumin protein control, and a
vehicle control (purified water). Two deaths occurred in control group
animals; both deaths were attributed to gavage injury. All other mice
survived the study. Several animals in both the control and test groups
lost weight during the study, and several abnormalities were observed by
gross necropsy in several animals in both the test and control groups.
There were no significant differences between the test and control
groups; therefore, the Cry2Ab2 protein does not appear to cause any
significant adverse effects at an exposure level of up to 1000 mg/kg
bodyweight.

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 Cry2Ab2, even at relatively
high dose levels, the Cry2Ab2 protein is not considered toxic. Further,
amino acid sequence comparisons showed no similarities between the
Cry2Ab2 protein and known toxic proteins in protein databases that would
raise a safety concern.

Since Cry2Ab2 is a protein, allergenic potential was also considered.
Currently, no definitive tests for determining the allergenic potential
of novel proteins exist. Therefore, EPA uses a weight-of- evidence
approach where the following factors are considered: source of the
trait; amino acid sequence 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 by heat, acid, and proteases, may be
glycosylated, and can be present at high concentrations in the food.

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

2.  Amino acid sequence. A comparison of the amino acid sequence of
    Cry2Ab2 with known allergens showed no significant overall sequence
    similarity or identity at the level of eight contiguous amino acid
    residues.

3.  Prevalence in food. Expression level analysis indicated that the
    protein is present at relatively low levels in corn: approximately
    2.3 µg/g in grain on a dry weight basis. Thus, the expression has
    been shown to be in the parts per million range.

4.  Digestibility. The Cry2Ab2 protein was digested within 15 seconds in
    simulated gastric fluid containing pepsin.

5.  Glycosylation. Cry2Ab2 expressed in corn was shown not to be
    glycosylated.

6.  Conclusion. Considering all of the available information, EPA has
    concluded that the potential for Cry2Ab2 to be a food allergen is
    minimal.

**2. Aggregate Exposures**

Pursuant to FFDCA section 408(b)(2)(D)(vi), EPA considers available
information concerning aggregate exposures from the pesticide residue in
food and all other non-occupational exposures, including drinking water
from ground water or surface water and exposure through pesticide use in
gardens, lawns, or buildings (residential and other indoor uses).

The Agency has considered available information on the aggregate
exposure levels of consumers (and major identifiable subgroups of
consumers) to the pesticide chemical residue and to other related
substances. These considerations include dietary exposure under the
tolerance exemption and all other tolerances or exemptions in effect for
the plant-incorporated protectants chemical residue, and exposure from
non-occupational sources. Exposure via the skin or inhalation is not
likely since the plant- incorporated protectant is contained within
plant cells, which essentially eliminates these exposure routes or
reduces these exposure routes to negligible. In addition, even if
exposure can occur through inhalation, the potential for Cry2Ab2 to be
an allergen is low, as discussed above. Although the allergenicity
assessment focuses on potential to be a food allergen, the data also
indicate a low potential for Cry2Ab2 to be an inhalation allergen.
Exposure via residential or lawn use to infants and children is also not
expected because the use sites for the Cry2Ab2 protein is agricultural.
Oral exposure, at very low levels, may occur from ingestion of processed
corn products and, theoretically, drinking water. However, oral toxicity
testing showed no adverse effects. Furthermore, the expression of the
Cry2Ab2 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 amount of Cry2Ab2 shown to have no toxicity.
Therefore, even if negligible aggregate exposure should occur, the
Agency concludes that such exposure would result in no harm due to the
lack of mammalian toxicity and low potential for allergenicity
demonstrated for the Cry2Ab2 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 from the
plant-incorporated protectant, we conclude that there are no cumulative
effects for the Cry2Ab2 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
Cry2Ab2 protein includes the characterization of the expressed Cry2Ab2
protein in corn, as well as the acute oral toxicity study, amino acid
sequence comparisons to known allergens and toxins, and in vitro
digestibility of the protein. The results of these studies were used to
evaluate human risk, and the validity, completeness, and reliability of
the available data from the studies were also considered.

Adequate information was submitted to show that the Cry2Ab2 test
material derived from microbial culture was biochemically and
functionally equivalent to the protein produced by the
plant-incorporated protectant ingredient in corn. Microbially produced
protein was used in the safety studies so that sufficient material for
testing was available.

The acute oral toxicity data submitted support the prediction that the
Cry2Ab2 protein would be non-toxic to humans. As mentioned above, when
proteins are toxic, they are known to act via acute mechanisms and at
very low dose levels (Sjoblad, Roy D., et al., "Toxicological
Considerations for Protein Components of Biological Pesticide Products,"
Regulatory Toxicology and Pharmacology 15, 3-9 (1992)). Since no
treatment-related adverse effects were shown to be caused by the Cry2Ab2
protein, even at relatively high dose levels, the Cry2Ab2 protein is not
considered toxic. Basing this conclusion on acute oral toxicity data
without requiring further toxicity testing and residue data is similar
to the Agency position regarding toxicity and the requirement of residue
data for the microbial *Bacillus thuringiensis* products from which this
plant-incorporated protectant was derived (See 40 CFR 158.740(b)(2)(i)).
For microbial products, further toxicity testing and residue data are
triggered when significant adverse effects are seen in studies such as
the mouse oral toxicity study. Further studies verify the observed
adverse effects and clarify the source of these effects (Tiers II and
III).

Residue chemistry data were not required for a human health effects
assessment of the subject plant-incorporated protectant ingredients
because of the lack of mammalian toxicity. However, data submitted
demonstrated low levels of the Cry1A.105 in corn tissues.

Since Cry2Ab2 is a protein, potential allergenicity is also considered
as part of the toxicity assessment. Considering all of the available
information (1) Cry2Ab2 originates from a non-allergenic source; (2)
Cry2Ab2 has no sequence similarities with known allergens; (3) Cry2Ab2
is not glycosylated; (4) Cry2Ab2 will only be present at low levels in
food; and (5) Cry2Ab2 is rapidly digested in simulated gastric fluid;
EPA has concluded that the potential for Cry2Ab2 to be a food allergen
is minimal.

Neither available information concerning the dietary consumption
patterns of consumers (and major identifiable subgroups of consumers
including infants and children) nor safety factors that are generally
recognized as appropriate for the use of animal experimentation data
were evaluated. The lack of mammalian toxicity at high levels of
exposure to the Cry2Ab2 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 ingredient include the nucleic
acids (DNA, RNA) that encode these proteins and regulatory regions. The
genetic material (DNA, RNA), necessary for the production of the Cry2Ab2
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(b)(2)(C) also provides that EPA shall apply
an additional tenfold margin of safety for infants and children in the
case of threshold effects to account for prenatal and postnatal toxicity
and the completeness of the database unless EPA determines that a
different margin of safety will be safe for infants and children.

In this instance, based on all the available information, the Agency
concludes that there is a finding of no toxicity for the Cry2Ab2 protein
and the genetic material necessary for its 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
considerations of consumption patterns, special susceptibility, and
cumulative effects do not apply.

**c) Overall Safety Conclusion**

There is a reasonable certainty that no harm will result from aggregate
exposure to the U.S. population, including infants and children, to the
Cry2Ab2 protein and the genetic material necessary for its production.
This includes all anticipated dietary exposures and all other exposures
for which there is reliable information. The Agency has arrived at this
conclusion because, as discussed above, no toxicity to mammals has been
observed, nor any indication of allergenicity potential for the
plant-incorporated protectant.

**5. Other Considerations**

**a) Endocrine Disruptors**

The pesticidal active ingredient is a protein, derived from a source
that is not known to exert an influence on the endocrine system.
Therefore, the Agency is not requiring information on the endocrine
effects of this plant-incorporated protectant at this time.

**b) Analytical Method(s)**

A short description of an enzyme-linked immunosorbent assay for the
detection and quantification of Cry2Ab2 in corn tissue has been
submitted, and a commercially available qualitative
immunochromatographic test strip was shown to detect the Cry2Ab2 protein
in corn tissues.

**c) Codex Maximum Residue Level**

No Codex maximum residue level exists for the plant-incorporated
protectant *Bacillus thuringiensis* Cry2Ab2 protein and the genetic
material necessary for its production in corn.

**References**

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

**[SUMMARY OF DATA SUBMITTED:]{.underline}**

**MRID 46471601---Data in Support of an Application for an Experimental
Use Permit to Test ZMIR245xMON 88017 Combined Corn Trait along with
ZMIR245 and MON 88017---**This MRID presents summary information for
events MON 89034, 89597 and 89771. Only brief summary information,
figures and tables are presented for the genetic constructs, molecular
characterization and protein (Cry1A.105, Cry2Ab2, Cry3Bb1) expression
levels for each hybrid. No detailed information is presented for
materials, methods or results. It is noted that detailed data for MON
89034 and 89597 were submitted to EPA by Monsanto in November 2005
(MRIDs 46694501 - 04 and MIRDs 46494601 - 07) which appears to replace
this earlier submission. The detailed data in the November 2005 MRIDs
for MON 89034 and 89597 provides a full suite of data in support of the
EUP application and are reviewed in this memorandum.

**MRID 46694501---Summary of Southern Blot Analyses of MON 89034 and MON
89697 Corn---**Southern blot analyses indicate that MON 89034 and MON
89597 have the introduced DNA inserted in the corn genome at a single
locus and contain one copy each of the *cry1A.105* and *cry2Ab2*
expression cassettes. All expression elements are shown to be present in
each of the inserts, and there are no other elements detectable other
than those associated with the respective cassettes. No backbone
sequences from plasmid PV-ZMIR245 or *nptII* coding sequences were
detected in the corn genome.

Classification: ACCEPTABLE---however, need Southern blot data for
MON89034 x MON88017 (or whatever event(s) and cross(es) are being
registered) for section 3 registration

**MRID 46694502---Assessment of the Cry1A.105, Cry2Ab2, and Cry3Bb1
Protein Levels in Tissues of Insect-Protected Corn MON 89034, MON 89034
x MON 88017, MON 89597, and MON 89597 x MON 88017---**The levels of
Cry1A.105, Cry2Ab2 and Cry3Bb1 proteins in corn leaf (OSL1 and OSL4),
grain, forage and pollen tissues collected from MON 89034, MON 89034 x
MON 88017, MON 89597, MON 89597 x MON 88017 were determined using ELISA
methods. Corn tissue samples were collected from plants grown in a
greenhouse except for MON 89034 forage samples which were collected from
the field. The mean Cry1A.105 protein levels in MON 89034 OSL1, OSL4,
grain, forage and pollen tissues were 72, 37, 3.2, 30, and 15 µg/g dwt,
respectively. The mean Cry2Ab2 protein levels in MON 89034 OSL1, OSL4,
grain and forage tissues were 49, 84, 1.9,and 29 µg/g dwt, respectively.
The mean Cry1A.105 protein levels in MON 89034 x MON 88017 OSL1, OSL4,
grain, forage and pollen tissues were 71, 60, 3.1, 83, and 7.6 µg/g dwt,
respectively. The mean Cry2Ab2 protein levels in MON 89034 x MON 88017
in OSL1, OSL4, grain and forage tissues were 50, 74, 2.5, and 53 µg/g
dwt, respectively.

The mean Cry1A.105 protein levels in MON 89597 OSL1, OSL4, grain,
forage, and pollen tissues were 78, 29, 2.3, 36, and 9.4 µg/g dwt,
respectively. The mean Cry2Ab2 protein levels in MON 89597 OSL1, OSL4,
grain and forage tissues were 33, 41, 2.1, and 29 µg/g dwt,
respectively. The mean Cry1A.105 protein levels in MON 89597 x MON 88017
OSL1, OSL4, grain, forage, and pollen tissues were 81, 14, 1.6, 17, and
4.3 µg/g dwt, respectively. The mean Cry2Ab2 protein levels in MON 89597
x MON 88017 in OSL1, OSL4, grain and forage tissues were 32, 36, 1.9,
and 26 µg/g dwt, respectively. Cry2Ab2 protein levels were less than the
assay LOQ (0.625 µg/g) for pollen from all lines analyzed.

Classification: ACCEPTABLE.

**MRID 46694503---Qualitative Detection Method for the Cry2Ab2 Protein
in Corn Leaf and Seed of MON 89034 and MON 89597---**A commercially
available qualitative immunochromatographic test strip (QuickStix™ kit
AS 005 LS) was obtained from EnviroLogix Inc. to determine if the strips
can detect the Cry2Ab2 protein produced in MON 89034 and MON 89597. The
QuickStix™ kit AS 005 LS detected the presence of Cry2Ab2 in MON 89034
and 89597. It was demonstrated that extracts of leaves or seed from MON
89034 or MON 89597 (both expressing Cry2Ab2) can be distinguished from
corn plants that do not express the Cry2Ab2 protein.

**MRID 46694601---Structural and Functional Similarity of the Cry1A.105
Protein to Cry1A Class of *Bacillus thuringiensis* Proteins: Final
Report**--- A summary of current information about the structural and
functional similarities of the Cry1A.105 protein to other *Bt* Cry1
proteins is presented in this submission. The Cry1A.105 protein is a
chimeric protein with overall amino acid sequence identity to the
Cry1Ac, Cry1Ab and Cry1F proteins of 93.6, 90.0 and 76.7%, respectively.
A structure model of the Cry1A.105 protein was developed using the X-ray
crystal structure of the Cry1Aa protein. The model demonstrated high
overall main chain structural similarity with Cry1Aa. Models of Cry1Ab
and Cry1Ac were also prepared using the Cry1A.105 model. Comparison of
the aligned folds of all three proteins showed that Cry1Ab and Cry1A.105
have essentially the same main chain structure and that Cry1Ac differs
slightly in its main chain structure from the other two in domain III.
Thus, comparison of the modeled crystal structures of the Cry1A.105,
Cry1Ab, and Cry1Ac with the experimental Cry1Aa X-ray crystal structure
demonstrated high structure similarity between the four proteins.

Monsanto also summarized results from bioactivity assays using
Cry1A.105, Cry1Ab, and Cry1Ac in this submission and indicates that
complete study reports will be submitted for registration. Monsanto
states that purified *E.coli*-produced Cry1A.105 protein had significant
activity against each representative lepidopteran insect larvae in
laboratory diet bioassays. Tests species included; black cutworm
(*Agrotis ipsilon*), corn earworm (*Helicoverpa zea*), fall armyworm
(*Spodoptera frugiperda*) and European corn borer (*Ostrinia
nubilalis*). Cry1A.105 insecticidal activity was similar to other Cry1
proteins (i.e., Cry1Ac, Cry1F, Cry1Ab). Coleopteran and heteropteran
larvae showed no indication of sensitivity to the Cry1A.105 protein.
Results from testing purified Cry1A.105 protein against non-target
invertebrates from different orders such as honey bee, minute pirate
bug, earthworms, parasitic hymenoptera and ladybird beetle demonstrated
no meaningful activity. Corn tissues from MON 89034 were tested in a
bioassay to assess potential activity of the Cry1A.105 and Cry2Ab2
proteins against Collembola (*Folsomia candida*), *Daphnia magna* and
bobwhite quail with results indicating no effect on the tested
non-target organisms.

**MRID 46694603---Acute Oral Toxicity Study in Mice with Cry1A.105
Protein---** The Cry1A.105 test protein (2072 mg/kg body weight) was
dosed by gavage as two doses separated by 4 hours (±20 minutes). The BSA
protein control (1998 mg/kg body weight) was dosed using the same
procedure as for the test protein group. The vehicle control group was
dosed with carbonate-bicarbonate with reduced glutathione. Body weight
was recorded prior to fasting, prior to dosing, and on days 7 and 14.
The test animals were observed for clinical signs of toxicity two times
post-dosing and for 14 days. A general health/mortality check was done
twice daily. All animals were necropsied. One male in the test protein
group was moribund and sacrificed on day 1 due to a mechanical dosing
error, which resulted in a perforated esophagus. All other mice survived
the study. There were no significant differences in body weight or body
weight change among the three groups during the study. The oral LD~50~
for males, females, and combined mice was greater than 2072 mg/kg. This
places Cry1A.105 Protein in TOXICITY CATEGORY III due to dose amounts
only; no signs of toxicity were observed.

Classification: ACCEPTABLE.

**MRID 46694604---Characterization of the Cry1A.105 Protein Purified
from the Corn Grain of MON 89034 and Comparison of the Physiochemical
and Functional Properties of the Plant-Produced and *E. coli*-Produced
Cry1A.105 Proteins---** The physicochemical properties and functional
properties of the plant-produced Cry1A.105 were analyzed and compared
with the properties of the *E. coli* produced Cry1A.105 using sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western
blot analysis, densitometry, matrix assisted laser desorption time of
flight (MALDI-TOF) mass spectrometry, glycosylation analysis, and a
Cry1A.105 insecticidal activity assay. Similar immunoreactive bands
migrating between approximately 85 and 130 kDa were observed in the
plant-produced Cry1A.105 and *E. coli*-produced reference samples, and
the full-length Cry1A.105 protein (\~130 kDa) was observed in both the
plant-produced and *E. coli*-produced protein samples. MALDI-TOF mass
spectrometry analysis of the \~130 kDa band after trypsin digestion
yielded peptide masses consistent with peptide masses of the predicted
sequence of the Cry1A.105 protein. The identified peptide masses yielded
43.8% overall coverage of the expected peptide sequence (516 of the 1177
amino acids). Immunoreactivity with the N-terminal peptide antibody
demonstrated that the N-terminus in the plant-produced full-length
Cry1A.105 protein was intact. Glycosylation analysis demonstrated that
neither the plant-produced nor the *E. coli*-produced Cry1A.105 protein
is glycosylated. The plant-produced and *E. coli*-produced proteins gave
similar results in the corn earworm diet-incorporation bioactivity
assay: the mean EC~50~ values for the plant-produced Cry1A.105 protein
and *E. coli*-produced reference standard were determined to be 0.0074
and 0.012 µg Cry1A.105 per mL diet, respectively. The results of this
study demonstrate the structural and functional similarity between the
plant-produced and the *E. coli*-produced Cry1A.105 proteins.

Classification: ACCEPTABLE.

**MRID 46694605---Bioinformatics Analysis of the Cry1A.105 Protein
Utilizing the AD6, Toxin5, and Allpeptides Databases---**Bioinformatic
analyses were used to search for sequence similarities between the
Cry1A.105 protein and toxins and allergens. The FASTA alignment tool and
the allergen (AD5), toxin (TOXIN5), and public domain (ALLPEPTIDES)
database sequences were used to assess structural similarity. No
significant similarities were found, other than with the Cry1Ac protein;
this alignment is not surprising, since the Cry1A.105 protein contains a
significant portion of the Cry1Ac protein. The Cry1A.105 protein
sequence was also screened against the AD5 sequence database using a
pair-wise comparison algorithm. No matches of 8 amino acids or more were
found for the Cry1A.105 protein in the AD5 database. No similarities
between Cry1A.105 protein and known allergens, human or animal toxins,
or pharmacologically active proteins were found in the study.

Classification: ACCEPTABLE.

**MRID 46694606---Assessment of the In Vitro Digestibility of the
Cry1A.105 Protein in Simulated Gastric Fluid---**No bands representative
of intact Cry1A.105 protein were identified by SDS-PAGE after ≥30
seconds incubation with simulated gastric fluid containing pepsin. A
very faint band of 4.5 kDaltons was observed between the 30 second and
20 minute digestions but was not observed after 20 minutes. The limit of
detection for the SDS-PAGE method was determined to be 5 ng for the
full-length Cry1A.105 protein. Both the pepsin stability and test
material stability controls gave appropriate responses. In the Western
Blot assay, Cry1A.105 was not immunologically identifiable within 30
seconds of incubation. The limit of detection for the method was
determined to be 1 ng for the full-length Cry1A.105 protein. The pepsin
stability and test material stability controls gave appropriate
responses.

Classification: ACCEPTABLE.

**MRID 46694607---Immunodetection of Cry2Ab2 and Cry1A.105 Proteins in
Corn Grain from MON 89034 Following Heat Treatment---**The
immunodetectability of the Cry1A.105 and Cry2Ab2 proteins in corn grain
from MON 89034 following heat treatment was assessed. MON 89034 and
conventional grain were ground, mixed with water, and then heated in an
oven at 204 ºC for 20 minutes to simulate the heating process used
commercially to process grain. Heated and unheated grain was extracted
with two buffers: 50 mM CAPS and 50 mM NLS (CAPS containing 2% N-Lauroyl
sarcosine). The extracts were analyzed using western blot to detect the
presence of the Cry1A.105 and Cry2Ab2 proteins. The amount of
immunodetectable Cry2Ab2 protein in either CAPS or NLS buffer extracts
of MON 89034 after heating was below the lower LOD. Based on the LOD and
the estimated amount of protein in the unheated extract, it was
determined that the Cry2Ab2 protein decreased at least 77% and 70%,
respectively following heat treatment, relative to their original
values. Likewise, the amount of immunodetectable Cry1A.105 protein in
either CAPS or NLS buffer extracts of MON 89034 after heating was below
the limit of detection (LOD) and had decreased by at least 94% and 78%,
respectively, relative to their original values. This loss is likely due
to protein degradation or aggregation into an insoluble complex as a
result of heat treatment.

Classification: ACCEPTABLE.

**MRID 45086305---Assessment of the Equivalence of Proteins Expressed in
Corn Event MON 840---**The physicochemical properties and functional
properties of the plant-produced Cry2Ab2 were analyzed and compared with
the properties of the *B. thuringiensis-* produced Cry2Ab2 using sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western
blot analysis, matrix assisted laser desorption time of flight
(MALDI-TOF) mass spectrometry (conducted prior to this study; results
summarized in appendix), glycosylation analysis, and a Cry2Ab2
insecticidal activity assay. In the SDS-PAGE analysis, the
plant-produced and *B. thuringiensis*-produced proteins both migrated at
an approximate molecular weight of 64 kDa. In the western blot analysis,
Monsanto states that both proteins comigrated and had similar
immunoreactivity However, the bands for the plant-produced protein
appear to be fainter compared to the *B.t*.-produced protein, although
the same amounts of protein were reportedly loaded on the gel. MALDI-TOF
mass spectrometry analysis of the plant-produced protein allowed \>58%
of the Cry2Ab2 protein to be identified using observed masses that
correspond to the expected masses. Glycosylation analysis demonstrated
that neither the plant-produced nor the *B. t.*-produced Cry2Ab2 protein
is glycosylated. The plant-produced and *B. t.*-produced proteins gave
similar results in the corn earworm bioactivity assay: the
plant-produced protein had 2-fold more activity than the
microbially-produced protein, which falls within the range of assay
variability. The results of these analyses demonstrate the structural
and functional similarity between the plant-produced and the *B.
thuringiensis*-produced Cry2Ab2 proteins. The plant-produced NPT II
protein was also analyzed and compared with the bacterially-produced
protein to demonstrate equivalence.

Classification: ACCEPTABLE. For section 3 registration, Cry2Ab2 produced
in the event being registered should be characterized.

**Comparison of the genetic elements that regulate expression of the
*cry2Ab2* genes as produced in MON 840 and MON 89034---**This submission
provides summary information on the expression cassettes used to produce
MON 840 and MON 89034 corn. Monsanto previously submitted product
characterization data for MON 840 and compares MON 840 with MON 89034 to
support bridging protein characterization data from MON 840 to support
the EUP for MON 89034 and a temporary tolerance exemption for Cry2Ab2.
Monsanto states that vector PV-ZMIR245 used to produce MON 89034
contains the same *cry2Ab2* cassette as that in vector ZMBK28, used to
produce MON 840, except that the e35S promoter that regulates *cry2Ab2*
expression in MON 840 was replaced by the FMV promoter in PV-ZMIR245.
Since the *cry2Ab2* expression cassettes are so similar, data on the
plant-expressed protein from event MON 840 showing equivalence with the
microbially-expressed Cry2Ab2 protein can be used in support of the EUP
for MON 89034 and MON 89045 x MON 88017 and the temporary tolerance
exemption.