Biopesticide Registration Action Document Bacillus Thuringiensis Cry1a.105 and Cry2ab2 Insecticidal Proteins and the Genetic Material Necessary for Their Production in Corn [pc Codes 006515 (cry2ab2) 006514 (cry1a.105)]


BIOPESTICIDE REGISTRATION ACTION DOCUMENT

Bacillus thuringiensis Cry 1 A. 105 and Cry2Ab2 Insecticidal Proteins and the Genetic Material

Necessary for Their Production in Corn
[PC Codes 006515 (Cry2Ab2), 006514 (Cry 1 A. 105)]

U.S. Environmental Protection Agency
Office of Pesticide Programs
Biopesticides and Pollution Prevention Division

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TABLE of CONTENTS

I.	OVERVIEW	4

A.	BACKGROUND	4

B.	EXECUTIVE SUMMARY	6

C.	USE PROFILE	10

D.	REGULATORY HISTORY	10

II.	SCIENCE ASSESSMENT	12

A.	PRODUCT CHARACTERIZATION	12

B.	HUMAN HEALTH ASSESSMENT OF Cry 1 A. 105	19

C.	HUMAN HEALTH ASSESSMENT Cry2Ab2	27

D.	ENVIRONMENTAL ASSESSMENT for MON 89034	34

E.	INSECT RESISTANCE MANAGEMENT (IRM)	65

F.	BENEFITS AND PUBLIC INTEREST FINDING	112

III.	REGULATORY POSITION FOR CRY1A.105, AND CRY2AB2	137

IV.	BIBLIOGRAPHY:	143

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BIOPESTICIDE REGISTRATION ACTION TEAM
Office of Pesticide Programs:

Biopesticides and Pollution Prevention Division

Product Characterization and Human Health

John Kough, Ph.D.

Rebecca Edelstein, Ph.D.

Environmental Fate and Effects

Zigfridas Vaituzis, Ph.D.

Tessa Milofsky, M.S.

Mika Hunter

Insect Resistance Management

Sharlene Matten, Ph.D.

Alan Reynolds, M.S.

Jeannette Martinez

Benefits Assessment

Jeannine Kausch

Registration Support

Mike Mendelsohn
Susanne Cerrelli
Jeannine Kausch
Matthew Thompson

Office of General Council

Chris Kaczmarek, Esq.

Keith Matthews, Esq.

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I. OVERVIEW

A. BACKGROUND

On June 10, 2008, EPA conditionally registered a plant-incorporated protectant product, event
MON 89034 corn, containing two new active ingredients, Bacillus thuringiensis Cry 1 A. 105 and
Cry2Ab2 insecticidal proteins, and the genetic material necessary for their production. EPA also
conditionally registered another product, MON 89034 x MON 88017 corn, which contains a
previously registered Bacillus thuringiensis Cry3Bbl protein in addition to the two new active
ingredients. The MON 89034 corn registration will expire on midnight September 30, 2022 and
the. MON 89034 x MON 88017 corn registration will expire on midnight September 30, 2015.
The Agency determined that the use of these pesticide products is in the public interest and that
their use will not cause any unreasonable adverse effects on the environment during the time the
products are registered. The registrant for both products is Monsanto Company ("Monsanto").

Event MON 89034 corn produces its own insecticide derived from Bacillus thuringiensis (Bt), a
naturally occurring soil bacterium. The Bt proteins produced in this product, called Cry 1 A. 105,
and Cry2Ab2, have been shown to effectively control highly destructive lepidopteran corn pests,
including European corn borer (ECB), corn earworm (CEW), southwestern corn borer (SWCB),
fall armyworm (FAW), and sugarcane borer (SCB), in field trials conducted during the 2003-
2004 growing seasons in Puerto Rico and the United States. These pests feed on the base of
seedlings and on the stalk, leaf, and ear tissue of corn plants, thereby destroying the entire plant,
weakening the stalk, and/or damaging the ear. In areas where one or more of these pests is
prevalent (e.g., the corn belt), significant financial losses are realized from decreased corn yields
and increased expenditures on chemical pest control agents, including organophosphate,
carbamate and pyrethroid insecticides.

On June 10, 2008, when the conditional, time-limited registrations of MON 89034, and MON
89034 x MON 88017 were issued, the non-Bt corn borer refuge was required to be at least 20%
for the corn belt. On December 15, 2008, EPA amended these product registrations to allow a
reduction in the structured corn borer refuge requirement (5%) in the non-cotton-growing regions
of the corn belt.

The data required to satisfy the conditions of these registrations are listed in Section III,
"Regulatory Position for Cry 1 A. 105, and Cry2Ab2."

On October 1, 2009, EPA announced a policy to provide a more meaningful opportunity for the
public to participate on major registration decisions before they occur. According to this policy,
EPA intends to provide a public comment period prior to making a registration decision for, at
minimum, the following types of applications: new active ingredients; first food uses; first
outdoor uses; first residential uses; and other actions for which the Agency anticipates that there
will be significant public interest.

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Consistent with the policy of making registration actions more transparent, the amendments to
the expiring MON 89034 corn products were subject to a 30-day comment period because the
Agency believed, given past experiences with PIPs in general, these actions would be of
significant interest to the public. During this comment period, several comments were received
from the following stakeholders: Mycogen Seeds c/o Dow AgroSciences LLC; Pioneer Hi-Bred
International, Incorporated; Monsanto Company; National Corn Growers Association;
Agricultural Biotechnology Stewardship Technical Committee; Center for Science in the Public
Interest; and Association of American Seed Control Officials. After reviewing and considering
all of the public comments received, the Agency still maintains that, based on all data submitted
in support of the MON 89034 corn registrations (both for initial registrations and as responses to
conditions of registration), it is in the best interest of the public and the environment to amend
the currently existing MON 89034 registrations by extending their expiration dates (September
30, 2022 for MON 89034 corn; September 30, 2015 for MON 89034 x MON 88017 corn). The
basis for this decision can be found in both the risk assessment for the MON89034 corn
products, which is characterized throughout this Biopesticides Registration Action Document
(BRAD), and the Agency's response to comments document.

All data and findings for the MON 89034 corn products are presented within the standard BRAD
configuration for PIPs (i.e., information is placed into separate and distinct chapters according to
scientific discipline or regulatory focus); this should be the most familiar format to outside
stakeholders interested in reading further about these actions. In addition to the MON 89034 corn
products, there are other Bt corn PIPs, expressing different proteins effective in controlling
various lepidopteran pests or corn rootworm, that were due to expire in 2010, and for which the
associated registrants formally requested an extension to expiration dates. Therefore, within the
same docket (EPA-HQ-OPP-2010-0607) as this document, the following information21 is also
available for public examination:

•	CrylF and CrylAb BRAD (Draft - August 2010; Final - Sept. 2010)

•	Cry3Bbl BRAD (Draft - July 2010; Final - Sept. 2010)

•	mCry3A BRAD (Draft - July 2010; Final - Sept. 2010)

•	Cry 1 A. 105 and Cry2Ab2 BRAD (Draft - August 2010; Final - September 2010)

•	Optimum® AcreMax™ B.t. Seed Blends BRAD (Draft - August 2010; Final - Sept. 2010)

•	Current Registration Terms and Conditions for Bt Corn Registrations Set to Expire in 2010

•	Proposed Registration Terms and Conditions for Bt Corn Registrations Set to Expire in 2010

•	Registration Terms and Conditions Established with the Finalized Amendments

•	BPPD mCry3A, Cry3Bbl, and Cry34/35Abl Rootworm Monitoring Reviews (June 2010)

•	Public Comments on EPA Docket Number EPA-HQ-OPP-2010-0607

•	EPA's Response to Comments

a Each of the Biopesticides Registration Action Documents in this action are modified from previous versions to
account for data/information submitted to fulfill terms and conditions of registration (see draft and final versions)
and to respond, in part, to comments received on the information presented in Docket Number EPA-HQ-OPP-2010-
0607 (see final versions only). All documents presented in the list can be retrieved from the following website:

httv: //www .regulations, gov.

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EPA made the decision to amend the registrations of eighteen (18) expiring Bt corn PIP
registrations to extend the expiration dates. We conducted comprehensive assessments of each of
these registrations, considering all toxicity and environmental effects data, data from insect
resistance monitoring, and insect resistance refuge compliance reports, received and obtained
since the last comprehensive evaluation of these products in 2001. Based upon our
comprehensive assessment, we reached significant conclusions regarding the positive
environmental impact of Bt corn PIPs, and we took several actions to strengthen the insect
resistance management requirements to ensure continued success in the prevention of the
evolution of resistance in target pests.

Since the commercialization of Bt crops, there have been a significant number of published field
studies that, combined with the post-registration field studies required to be submitted to the
Agency, have demonstrated that non-target invertebrates are generally more abundant in Bt
cotton and Bt corn fields than in non-transgenic fields managed with chemical insecticides. Thus,
these published and registrant-produced studies demonstrate that, not only are the Bt crops not
causing any unreasonable adverse effects in the environment, but, arthropod prevalence and
diversity is greater in Bt crop fields.

To strengthen insect resistance management of these corn PIPs and to address reports that
compliance with the mandated refuge requirements has been decreasing, EPA is requiring
enhanced compliance assurance programs (CAPs), and a phased requirement for seed bag
labeling that clearly shows the refuge requirements. Also, given the increasing variety of PIP
products and combinations, and the differing risk of resistance evolution that the various
products represent, we are granting registrations for the corn PIP products for different
timeframes, based on assessments of their likelihood of forestalling the evolution of insect
resistance. We are registering differing categories of products for differing time periods to reflect
the assessed level of risk of resistance posed by the various corn PIP products. The scheme that
we are following includes registration periods generally of five, eight, and twelve years; with the
possibility of a fifteen-year registration period for products that are demonstrated to meet
specified criteria. We retain, however, the discretion to register products for time periods
differing from these defaults where circumstances warrant.

B. EXECUTIVE SUMMARY

Product Characterization

MON 89034 was developed by Agrobacterium-mQdi&tQd transformation of corn using the 2T-
DNA plasmid vector PV-ZMIR245. The transformation produces two Bacillus thuringiensis
proteins, Cry 1 A. 105 and Cry2Ab2. Cry 1 A. 105 is a chimeric protein composed of portions of
CrylAb, CrylAc, and CrylF proteins.

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Mammalian Toxicity and Allergenicity Assessment

The acute oral toxicity data submitted by Monsanto demonstrated the lack of mammalian
toxicity in rats and mice exposed to pure Cry 1 A. 105 and Cry2Ab2 protein at doses well above
the maximum levels anticipated in treated crops, based upon the demonstrated expression values
of these two proteins.

Data demonstrating no mammalian toxicity at high levels of exposure confirm the safety of the
product at levels well above any possible maximum exposure levels anticipated for a plant-
incorporated protectant. This is similar to the Agency position regarding toxicity and the
requirement of residue data for the microbial Bacillus thuringiensis products from which these
plant-incorporated protectants were derived. [See 40 CFR Sec. 158.2130 and 158.2140.] For
microbial products, further toxicity testing to verify the observed effects and clarify the source of
the effects (Tiers II & III) and residue data are triggered by significant acute effects in studies
such as the mouse oral toxicity study.

Since no acute effects were observed in the submitted studies, even at relatively high dose levels,
the Cry 1 A. 105 and Cry2Ab2 proteins are not considered to be toxic. This conclusion was
supported by amino acid sequence comparisons of the Cry 1 A. 105 and Cry2Ab2 proteins with
databases of known toxic proteins, which showed no similarities that would raise a safety
concern. In addition, the data submitted by Monsanto demonstrated that the Cry 1 A. 105 and
Cry2Ab2 proteins were substantially degraded by heat when examined by immunoassay. This
instability to heat would decrease the potential for dietary exposure to intact Cry 1 A. 105 and
Cry2Ab2 proteins in cooked or processed foods. These biochemical features, along with the lack
of adverse results in the acute oral toxicity tests, support the Agency's conclusion that there is a
reasonable certainty of no harm from dietary exposure to Cry 1 A. 105 and Cry2Ab2 containing
crops.

Since Cry 1 A. 105 and Cry2Ab2 are proteins, their potential for food allergenicity was also
considered. Currently, no definitive tests for determining the allergenic potential of novel
proteins exist. Therefore, EPA uses a "weight-of-evidence" approach when considering the
allergenic potential for a PIP protein, and bases its conclusions upon the following factors: the
source of the trait, the amino acid sequence compared with known allergens, and the biochemical
properties of the protein, including in vitro digestibility in simulated gastric fluid (SGF) and
glycosylation. This is consistent with the approach outlined in the Annex to the Codex
Alimentarius "Guideline for the Conduct of Food Safety Assessment of Foods Derived from
Recombinant-DNA Plants." The Agency's allergenicity assessment for the Cry 1 A. 105 and
Cry2Ab2 proteins follows:

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

2. Amino acid sequence. A comparison of the amino acid sequences of Cry 1 A. 105 and
Cry2Ab2 with known allergens showed no sequence similarity or identity at the level of

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eight contiguous amino acid residues, which is considered to be the smallest amino acid
sequence necessary to induce an immune response.

3. Digestibility. The CrylA.105 and Cry2Ab2 proteins were digested rapidly in simulated
gastric fluid containing pepsin, the enzyme produced by the stomach that digests proteins
so they can be absorbed as nutrients into the body via the small intestine. The rapid
degradation of Cry 1 A. 105 and Cry2Ab2 in the simulated gastric environment indicated
that the intact protein will not pass from the stomach into the intestinal lumen, where
sensitization of the immune system to food allergens occurs.

4. Glycosylation. Cry 1 A. 105 and Cry2Ab2 proteins expressed in corn are not
glycosylated.1

5. Conclusion. EPA concluded that the potential for Cry 1 A. 105 and Cry2Ab2 to be a food
allergen is minimal.

The information on the safety of pure Cry 1 A. 105 and Cry2Ab2 proteins provides adequate
justification to address possible exposures in all corn crops.

Environmental Hazard Assessment

Maximum hazard dose toxicity testing on representative beneficial organisms from several taxa
was performed in support of the registrations of Cry 1 A. 105 and Cry2Ab2 proteins expressed in
corn. The toxicity of the Cry 1 A. 105 and Cry2Ab2 proteins was evaluated on several species of
invertebrates, including the lady beetle, minute pirate bug, parasitic hymenoptera, Collembola,
Daphnia, honey bee, and earthworm. Developmental observations were also made in the lady
beetle, minute pirate bug, and honeybee studies. Observations of possible reproductive effects
were also made in the Collembolan studies. In addition, earthworm studies were voluntarily
submitted to the Agency to ascertain the potential effects of the Cry 1 A. 105 and Cry2Ab2
proteins on beneficial decomposer species. Avian dietary studies and soil fate data were also
submitted.

The test substances used for the studies submitted in support of the MON 89034 registrations
included bacterially produced, purified Cry 1 A. 105 and Cry2Ab2 proteins, and MON 89034 corn
leaf tissue, pollen, and grain. The October 2000 FIFRA Science Advisory Panel (SAP)
recommended that while actual plant material is the preferred test material, bacterially derived
protein is also a valid test substance, particularly in scenarios where test animals do not normally
consume corn plant tissue and where large amounts of Cry protein (Cry protein concentrations
that exceed levels present in plant tissue) are needed for maximum hazard dose testing. An
insect feeding study, which compared the relative potency of plant produced Cry 1 A. 105 and
Cry2Ab2 proteins to the microbe produced proteins, indicated that plant produced protein was
similar in toxicity to the bacterially produced protein (Edelstein Memo, November 7, 2007).

1 Although this was only demonstrated in corn, these expressed proteins are unlikely to be glycosylated if produced in
any other crops since the mechanisms of protein glycosylation are similar in different plants (Lerouge, P. Cabanes-Macheteau,
M., Rayon, C., Fichette-Laine, A-C., Gomord, V., and Faye, L., "N-Glycoprotein biosynthesis in plants: recent developments and
future trends," Plant Molecular Biology 38: 31-48, 1998

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The potential interactions between the Cry 1 A. 105 and Cry2Ab2 proteins was addressed in a
memorandum for the MON 89034 Experimental Use Permit accompanying the Agency's
review, "Evaluation of the Potential for Interactions Between the Bacillus thuringiensis Proteins
Cry 1 A. 105 and Cry2Ab2," (Hunter, M., July 6, 2006). The study provided evidence that the
proteins do not interact in either an antagonistic or synergistic manner, and that there will not be
any unexpected interactions with regard to target and non-target insects. New data on the
potential interaction between the combined CrylA.105, Cry2Ab2 and Cry3Bbl proteins were
submitted. The results from that study demonstrated that the combined Cry 1 A. 105 and
Cry2Ab2 activity was not affected by the Cry3Bbl protein, and that the Cry3Bbl activity was
unaffected by combined Cry 1 A. 105 and Cry2Ab2 activity (MRIDs 469513-05 & 469513-06).

Insect Resistance Management

Monsanto has demonstrated that the Cry 1 A. 105 and Cry2Ab2 toxins have different modes of
action, and consequently, a low likelihood of cross-resistance. Therefore, Cry 1 A. 105 and
Cry2Ab2 are suitable partners in a pyramided product. Monsanto has also shown that there is a
low likelihood of cross-resistance between Cry 1 A. 105 and CrylAb. Monsanto has previously
demonstrated that there is a low likelihood of cross-resistance between Cry2Ab2 and Cryl Ac.
Both CrylAb and Cry 1 Ac are expressed in other registered Bt corn and Bt cotton PIPs.

Monsanto did not, however, address the likelihood of cross-resistance between Cryl A. 105 and
Cryl Ac, and CrylFa (Bt proteins already in existing Bt corn and Bt cotton products), and what
impact such cross-resistance would have on the durability of MON 89034. As a result,
Monsanto was required to provide additional information on cross-resistance of Cryl A. 105 and
CrylFa and Cryl Ac (including binding site models and use of resistant colonies) for the target
pests and determine how such cross-resistance could impact the durability of MON 89034.

Monsanto originally proposed that a 5% structured refuge, rather than the 20% structured refuge
required for other Bt corn registrations, be applied to field corn uses of MON 89034 in the U.S.
Corn Belt. But, the data and simulation modeling in Monsanto's initial application did not
support the 5% proposed refuge for MON 89034 in the Corn Belt. There were uncertainties
regarding the dose determination for susceptible and heterozygote (i.e., partially resistant) insects
(ECB, SWCB, CEW, and FAW), the cross-resistance potential of Cryl A. 105, Cryl Ac and
CrylFa and any impacts on the durability of MON 89034, and limitations in the simulation
modeling. Therefore, the field corn uses of MON 89034 in the Corn Belt were registered with a
20% refuge requirement until such time as Monsanto could address the uncertainties. EPA
determined, however, that the data did support reduction of the refuge from 50% to 20% in
cotton-growing regions in the southeastern U.S., where a 50% non-Bt corn refuge has been
required for other Bt corn registrations.

Subsequent to the registrations of the event MON 89034 corn and MON 89034 x MON 88017
corn products, Monsanto submitted additional data and an analysis of potential resistance risks to
support an amendment to reduce the required non -Bt corn refuge for MON 89034 corn from 20%

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to 5% in the U.S. Corn Belt. After reviewing these data, EPA determined that a 5% refuge in the
U.S. Corn Belt should not significantly increase the risk of resistance for ECB, CEW, and
SWCB. Monsanto sufficiently addressed the requirement to analyze potential cross resistance in
existing Bt corn and Bt cotton products for Cry 1 A. 105 and Cry lFa, but additional analysis and
information is still needed to fully assess the cross resistance potential for Cryl Ac and
CrylA.105.

C. USE PROFILE

Active Ingredient Name: Bacillus thuringiensis Cryl A. 105, and Cry2Ab2 insecticidal

protein and the genetic material necessary for their
production in corn

Trade and Other Name(s): MON 89034

OPP Chemical Codes: 006515 (Cry2Ab2) and 006514 (CrylA.105)

Basic Manufacturer: Monsanto Company

800 North Lindbergh Blvd.

St. Louis, MO 63167

Type of Pesticide: Plant-incorporated Protectant

Uses: Field Corn and Sweet Corn

Target Pests for Active Ingredient: European corn borer (Ostrinia nubilalis),
Southwestern corn borer (Diatraea grandiose lid), Southern cornstalk borer (Diatraea
crambidoides), Corn earworm (Helicoverpa zea), Fall army worm (Spodoptera
frugiperda), Corn stalk borer (.Papaipema nebris), and Sugarcane borer (Diatreae
saccharalis)

D. REGULATORY HISTORY

Monsanto previously submitted an Experimental Use Permit (EUP) application for events MON
89034, MON 88017, and MON 89034 x MON 88017. MON 89034 was developed by
Agrobacterium-mQdi&tQd transformation of corn using the 2T-DNA plasmid vector PV-
ZMIR245, and produces two Bacillus thuringiensis (Bt) proteins, CrylA.105 and Cry2Ab2.
These proteins are intended to provide protection from feeding damage caused by a number of
lepidopteran pests. CrylA.105 is a chimeric protein composed of portions of CrylAb, Cryl Ac,
and Cry IF proteins. On July 17, 2006, EPA established temporary exemptions from the
requirement of a tolerance for both CrylA.105 (71 FR 40427 and 72 FR 20434; 40 CFR

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174.502) and Cry2Ab2 (71 FR 40431 and 72 FR 20434; 40 CFR 174.503) in the food and feed
commodities of corn; these exemptions were set to expire on June 30, 2009.

On November 15, 2006, Monsanto submitted petitions to EPA under the Federal Food, Drug,
and Cosmetic Act (FFDCA), as amended by the Food Quality Protection Act of 1996 (FQPA),
requesting amendment of the existing temporary tolerances in 40 CFR 174.503 for the Bacillus
thuringiensis Cry2Ab2 insecticidal protein to establish a permanent exemption from the
requirement of a tolerance for the Bacillus thuringiensis Cry2Ab2 insecticidal protein and in 40
CFR 174.502 for the Bacillus thuringiensis Cry 1 A. 105 insecticidal protein to establish a
permanent exemption from the requirement of a tolerance for the Bacillus thuringiensis Cry
1A.105 insecticidal protein in field corn, sweet corn, and popcorn.

On September 29, 2006, Monsanto submitted an application to register MON 89034 and MON
89034 x MON 88017 under Section 3 of the Federal Insecticide, Fungicide and Rodenticide Act
(FIFRA).

On March 9, 2007, Monsanto resubmitted petitions to EPA under the Federal Food, Drug, and
Cosmetic Act (FFDCA), requesting amendment of the existing temporary tolerances in 40 CFR
174.503 for the Bt Cry2Ab2 insecticidal protein to establish a permanent exemption from the
requirement of a tolerance for the Bt Cry2Ab2 insecticidal protein, and in 40 CFR 174.502 for
the Bt Cry 1 A. 105 insecticidal protein to establish a permanent exemption from the requirement
of a tolerance for the Bt Cry 1 A. 105 insecticidal protein in all crops and agricultural commodities

On June 10, 2008, conditional registrations were issued for MON 89034 and MON 89034 x
MON 88017 products.

On July 2, 2008 (73 FRNo. 128), the existing permanent exemption from the requirement of a
tolerance for residues of the Bacillus thuringiensis Cry2Ab2 protein under 174.519 was amended
to include corn or cotton when used as a plant-incorporated protectant in the food and feed
commodities: field corn, sweet corn, popcorn, cotton seed, cotton oil, cotton meal, cotton hay,
cotton hulls, cotton forage, and cotton gin byproducts in accordance with good agricultural
practices.

On July 16, 2008 (73 FRNo. 137), the Agency established permanent exemptions from the
requirement of a tolerance for residues of the Bacillus thuringiensis Cry 1 A. 105 protein in or on
the food and feed commodities: field corn, sweet corn, and popcorn when used as plant
incorporated protectant in all food commodities in accordance with good agricultural practices.

On December 15, 2008, the conditional registrations were amended for MON 89034 and MON
89034 x MON 88017, to allow for a 5% structured refuge in the corn belt (in non-cotton growing
regions) for corn borers.

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II. SCIENCE ASSESSMENT

A. PRODUCT CHARACTERIZATION

MON 89034 was developed by Agrobac/erium-med\ated transformation of corn using the 2T-
DNA plasmid vector PV-ZMIR245 and produces two Bacillus thuringiensis proteins, Cry 1 A. 105
and Cry2Ab2. These proteins are intended to provide protection from feeding damage caused by
a number of lepidopteran pests. Cryl A. 105 is a chimeric protein composed of portions of
CrylAb, CrylAc, and CrylF proteins.

Transformation System:

PV-ZMIR245 is a binary vector containing two separate transfer DNAs (2T-DNA). The first T-
DNA contains the cryl A. 105 and the cry2Ab2 expression cassettes. The second T-DNA contains
the nptll (neomycin phosphotransferase II) expression cassette. The cryl A. 105 expression
cassette contains the cryl A. 105 coding sequence under the regulation of the e35S promoter,

Ractl intron, and the Hspll 3' end sequence. The cry2Ab2 expression cassette contains the
cry2Ab2 coding sequence under the regulation of the FMV promoter, the Hsp70 intron, a
chloroplast transit peptide (TS-XS71-C'l'P), and the nos 3' end sequence. The nptll expression
cassette contains the nptll coding sequence under the regulation of the CaMV 35S promoter and
the nos 3' end sequence. During transformation, both T-DNAs were inserted into the genome.
The nptll selectable marker gene was used to select for transformed cells. Traditional breeding
was then used to isolate plants that only contain the cryl A. 105 and cry2Ab2 expression cassettes
and not the nptll expression cassette.

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

Characterization of the DNA isolated from event MON 89034 corn using restriction enzyme
digests and Southern blot analysis as well as DNA sequencing indicates that the DNA was
inserted in the corn genome at a single locus, and the insert contains one copy each of the
cryl A. 105 and cry2Ab2 expression cassettes. There were no other detectable elements other than
those associated with the respective cassettes. No backbone sequences from plasmid PV-
ZMIR245 or nptll coding sequences were detected in the corn genome. Southern blot analysis
also demonstrated the stability of the insert over multiple generations. DNA sequencing
indicated that the genetic elements were present in the inserted DNA as expected except that the
e35S promoter was modified, and the right border sequence present in PV-ZMIR245 was
replaced by a left border sequence in MON 89034.

Protein Characterization:

Protein characterization data demonstrate that the plant-produced Cryl A. 105 and Cry2Ab2
proteins have biochemical and functional activities that are similar to those of the E. coli-
produced proteins that were used in several toxicity studies. The following techniques were used
to characterize and compare the plant-produced and the E. co//-produced proteins: sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis,
densitometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass

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spectrometry, glycosylation analysis, N-terminal amino acid sequencing, and insecticidal activity
assays. Glycoslyation analysis indicated that the proteins are not glycoslyated. These analyses
demonstrated the structural and functional similarity between the plant-produced and the E. coli-
produced Cry 1 A. 105 and Cry2Ab2 proteins and justified the use of E. co/z'-produced proteins in
toxicity studies. Monsanto also provided information showing the similarity between
Cry 1 A. 105, CrylAb, and Cry 1 Ac.

In addition, Monsanto provided information comparing the expected (deduced) amino acid
sequence of Cry2Ab2 expressed in MON 89034 corn, the native form of the protein in B.
thuringiensis, and in Bollgard II cotton. Monsanto stated that Cry2Ab2 in MON 89034 and
Bollgard II are identical. Different chloroplast transit peptide sequences were used in the
different products; however, these are expected to be cleaved and degraded in the plants upon
uptake into the chloroplasts. When Monsanto attempted to determine the N-terminal sequence of
Cry2Ab2 from MON 89034 or Bollgard II, the results indicated that the N-terminus is blocked in
both. Therefore, Monsanto was unable to determine the cleavage site of the chloroplast transit
peptide. Because the chloroplast transit peptides used in MON 89034 and Bollgard II have
potential cleavage sites (methionine) three amino acids upstream from the start of the Cry2Ab2
protein sequence, the Cry2Ab2 produced in Bollgard II and in MON 89034 may differ by one
amino acid (leucine vs. glutamine) if the cleavage site is within the transit peptide. The E. coli-
produced Cry2Ab2 protein used in the toxicity studies for MON 89034 includes the three
additional amino acids from the chloroplast transit peptide at the N-terminus. Monsanto stated
that the Cry2Ab2 proteins produced in MON 89034 and Bollgard II are variants of the wild type
Cry2Ab2 protein produced in B. thuringiensis. The /^/-produced protein was used in some of the
previously submitted studies that are cited to support the ecological risk assessment for MON
89034. Monsanto therefore submitted a study demonstrating that the E. co//-produced Cry2Ab2
and the /^/-produced Cry2Ab2 have equivalent biological activity (EC50 values and rates of
growth inhibition) in a larval corn earworm diet-incorporation bioassay.

Analytical Detection Methods:

Short descriptions of enzyme-linked immunosorbent assay (ELISA) methods for detecting and
quantifying Cry 1 A. 105 and Cry2Ab2 as well as standard operating procedures for the methods
were provided with the registration application. Monsanto stated that these methods have been
validated and provided validation results in an appendix to MRID 46951403; but an independent
lab validation study was not provided for either method. In addition, Monsanto did not indicate
whether the ELISA method for Cry 1 A. 105 will distinguish between Cry 1 A. 105, CrylAb,
Cry 1 Ac, and Cry IF. Since Cry 1 A. 105 contains portions of all three proteins, there may be
cross-reactivity in the assay. Monsanto also provided a study demonstrating that a commercially
available qualitative immunochromatographic test strip can detect Cry2Ab2 in MON 89034 corn.
Since event MON 89034 is the only product that expresses Cry 1 A. 105 and the only corn product
that expresses Cry2Ab2, the detection method for Cry2Ab2 can be used for detecting both
Cry2Ab2 and Cry 1 A. 105. The presence of Cry2Ab2 in corn should also indicate the presence of
CrylA.105.

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Bacillus thuringiensis Cry 1 A. 105, and Cry2Ab2 Protein in Corn
Biopesticide Registration Action Document

When Bt Cry 1 A. 105, and Cry2Ab2 Protein in corn (EPA Reg. No. 524-575, and 524-576) were
initially registered, the Agency issued registration notices to Monsanto that contained the
following requirement for further product characterization information:

"For event MON 89034 corn, an independent lab validation of the
analytical method for the detection of Cry2Ab2 and/or Cry 1 A. 105 [is
required]. You must also agree to provide to the EPA laboratory (Ft.

Meade, MD) methodology and/or reagents necessary for validation of
such analytical method within 6 months from the date that the Agency
requests them."

Monsanto has provided an independent lab validation of this method (MRID 47731601) as
required in the conditions of registration. When the evaluation of the independent lab validation
of an analytical detection method for Cry2Ab2 protein in corn was conducted, it was determined
that the Cry2Ab2 protein can be detected at a level of detection (LOD) of 1.0%. The study
effectively demonstrated that performance based on the number of blinded samples tested and
also confirmed that at the 1.0% LOD the dipstick reagents show zero false positive and false
negative results. This indicates acceptable performance standards for a rapid analytical method
but significantly does not address several performance criteria required by GIPSA for its dipstick
test kit validation. The use of the kit manufacturer for independent validation is also
questionable.

Conclusion: The test verified the claim that the EnviroLogix QuickStix™ Kit for Cry2Ab2 Bulk
Grain can consistently detect Cry2Ab2 present in corn at a concentration of >1%. This study
was deemed acceptable.

Protein Expression:

Expression level data were provided for Cry 1 A. 105 and Cry2Ab2 in different plant tissues and at
different growth stages. Both proteins are expressed at relatively low levels in event MON
89034 corn. The data were produced using ELISA methods for each protein. Summary results
are provided below in Table 1. Table 2 provides summaries of the product characterization
studies and data provided.

Table 1. Mean Expression Levels of CrylA.105 and Cry2Ab2 from MON 89034 Plant
Tissues

Tissue Type

CrylA.105
(|ig/g dry weight + standard
deviation)*

Cry2Ab2
(|ig/g dry weight + standard
deviation)*

Leaf

72+ 14-520+ 130

130 + 34- 180 + 59

Root

11 + 1.4 -79+ 17

21 + 5.9 -58 + 18

Whole Plant

100 +26-380 + 90

39+ 16- 130 + 51

Pollen

12+ 1.7

0.64 + 0.091

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Bacillus thuringiensis Cry 1 A. 105, and Cry2Ab2 Protein in Corn
Biopesticide Registration Action Document

Silk

26 + 3.9

71 +35

Forage

42 + 9.4

38 + 14

Grain

5.9 + 0.77

1.3 + 0.36

*Ranges reflect means at different growth stages for the first three tissue types

Table 2. Product Characterization Data Submitted

Study Type/Title

Summary

MRID#

Characterization of the
inserted DNA/ Summary
of Southern Blot
Analyses of MON
89034 and MON 89697
Corn2

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 cry 1 A. 105 and cry 1 A. 105 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
nptll coding sequences were detected in the corn genome.

Classification: ACCEPTABLE

46694501

Analytical detection
method/Qualitative
Detection Method for
the Cry2Ab2 Protein in
Corn Leaf and Seed of
MON 89034 and MON
895972

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.

The study (MRID #477316-01) verified the claim that the EnviroLogix

QuickStix Kit for Cry2Ab2 Bulk Grain can consistently detect Cry2Ab2
present in corn at a concentration of >1%.

Classification: ACCEPTABLE

46694503
47731601

Characterization of the
active ingredient/
Structural and
Functional Similarity of
the Cry 1 A. 105 Protein
to CrylA Class of
Bacillus thuringiensis
Proteins: Final Report2

A summary of current information about the structural and functional
similarities of the CrylA. 105 protein to other Bt Cryl proteins is presented
in this submission. The CrylA. 105 protein is chimeric, with overall amino
acid sequence identity to the CrylAc, CrylAb and Cry IF proteins of 93.6,
90.0 and 76.7%, respectively. A structural model of the CrylA. 105 protein
was developed using the X-ray crystal structure of the Cry lAa protein. The
model demonstrated high overall main chain structural similarity with
CrylAa. Models of CrylAb and CrylAc were also prepared using the
CrylA. 105 model. Comparison of the aligned folds of all three proteins
showed that CrylAb and CrylA. 105 have essentially the same main chain
structure, and that CrylAc differs slightly in its main chain structure from
the other two in domain III. Thus, comparison of the modeled crystal
structures of the CrylA.105, CrylAb, and CrylAc with that of the
experimental CrylAa X-ray crystal structure demonstrated high structure
similarity between the four proteins.

Monsanto also summarized results from bioactivity assays using

46694601

2 Study submitted with EUP request and reviewed in memorandum from R. Edelstein and I. Barsoum to M.
Mendelsohn dated June 16, 2006.

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Bacillus thuringiensis Cry 1 A. 105, and Cry2Ab2 Protein in Corn
Biopesticide Registration Action Document

Study Type/Title

Summary

MRU) #



CrylA.105, CrylAb, and CrylAc in this submission and indicates that
complete study reports will be submitted for registration. Monsanto states
that purified £.co//'-produced Cry 1 A. 105 protein had significant activity
against each representative lepidopteran insect larvae in laboratory diet
bioassays. Tests species included; black cutworm (Agrotis ipsilori), corn
earworm (Helicoverpa zea), fall armyworm (Spodoptera frugiperda) and
European corn borer (Ostrinia nubilalis). Cry 1 A. 105 insecticidal activity
was similar to other Cryl proteins (i.e., CrylAc, Cry IF, CrylAb).
Coleopteran and heteropteran larvae showed no indication of sensitivity to
the Cry 1 A. 105 protein. The results of tests with purified Cry 1 A. 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 for potential activity of the Cry 1 A. 105 and
Cry2Ab2 proteins against Collembola (Folsomia Candida), Daphnia magna
and bobwhite quail with results indicating no effect on the tested non-target
organisms.

Classification: ACCEPTABLE



Characterization of the
active

ingredient/Characteriza-
tion of the Cry 1A. 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 Cry 1 A. 105
Proteins2

The physicochemical properties and functional properties of the plant-
produced Cryl A. 105 were analyzed and compared with the properties of the
E. coli produced Cryl A. 105 using sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE), western blot analysis, densitometry,
matrix assisted laser desorption/ionization time of flight (MALDI-TOF)
mass spectrometry, glycosylation analysis, and a Cryl A. 105 insecticidal
activity assay. Similar immunoreactive bands migrating between
approximately 85 and 130 kDa were observed in the plant-produced
Cry 1 A. 105 and E. coli-produced reference samples, and the full-length
Cry 1 A. 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 Cryl A. 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 Cry 1 A. 105 protein was
intact. Glycosylation analysis demonstrated that neither the plant-produced
nor the E. coli- produced Cryl A. 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 EC50 values for the
plant-produced Cryl A. 105 protein and it. coli-produced reference standard
were determined to be 0.0074 and 0.012 |ig Cryl A. 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
Cryl A. 105 proteins.

Classification: ACCEPTABLE

46694604

16

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Bacillus thuringiensis Cry 1 A. 105, and Cry2Ab2 Protein in Corn
Biopesticide Registration Action Document

Study Type/Title

Summary

MRU) #

Characterization of the
inserted DNA/ Amended
Report for MSL-20072:
Molecular Analysis of
Corn MON 89034

The DNA inserted in event MON 89034 was characterized by Southern blot
analysis and DNA sequencing. Southern blot analysis indicates that MON
89034 contains a single copy of the cry 1A. 105 and cry2Ab2 expression
cassettes at a single locus. No backbone sequences from plasmid PV-
ZMIR245 or nptll coding sequences were detected in the corn genome.
Southern blot analysis of DNA from several generations of MON 89034
demonstrated the stability of the insert over seven generations. In addition,
the DNA sequence of the insert and surrounding genomic sequences was
determined using PCR and DNA sequencing; this analysis confirmed the
organization of the elements within the insert and identified the 5' and 3'
insert-to-genomic DNA junctions.

Classification: ACCEPTABLE

46951402

Expression

levels/Assessment of the
Cry 1 A. 105 and
Cry2Ab2 Protein Levels
in Tissues of Insect-
protected corn MON
89034 Produced in 2005
U.S. Field Trials

The levels of Cry 1A. 105 and Cry2Ab2 in corn tissues collected from MON
89034 plants grown at five field sites in the U.S. were determined using
enzyme-linked immunosorbent assays (ELISA). The means for Cry 1 A. 105
protein levels across all sites were 5.9 |ig/g dry weight (dwt) in grain, 42
|ig/g dwt in forage, 12 |ig/g dwt in pollen, 520 |ig/g dwt in over season leaf
collected at growth stage V2-V4 (OSL-1), 120 |ig/g dwt in leaves OSL-4
(collected at growth stage pre-VT), 12 |ig/g dwt in forage root, and 50 |ig/g
dwt in stover. In tissues harvested throughout the growing season, mean
Cry 1A. 105 protein levels across all sites ranged from 72-520 |ig/g dwt in
leaf, 42-79 |ig/g dwt in root, and 100-380 |ig/g dwt in whole plant. The
means for Cry2Ab2 protein levels across all sites were 1.3 |ig/g dwt in
grain, 38 |ig/g dwt in forage, 0.64 |ig/g dwt in pollen, 180 |ig/g dwt in OSL-
1, 160 |ig/g dwt in OSL-4, 21 |ig/g dwt in forage root, and 62 |ig/g dwt in
stover. In tissues harvested throughout the growing season, mean Cry2Ab2
protein levels across all sites ranged from 130-180 |ig/g dwt in leaf, 26-58
|ig/g dwt in root, and 39-130 |ig/g dwt in whole plant.

Classification: ACCEPTABLE

46951403

17

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Bacillus thuringiensis Cry 1 A. 105, and Cry2Ab2 Protein in Corn
Biopesticide Registration Action Document

Study Type/Title

Summary

MRU) #

Characterization of the
active ingredient/
Characterization of the
Cry2Ab2 Protein
Purified from the Corn
Grain of MON 89034
and Comparison of the
Physicochemical and
Functional Properties of
the Plant-Produced and
E. coli-produced
Cry2Ab2 Proteins

The physicochemical and functional properties of the plant-produced
Cry2Ab2 were analyzed and compared with the properties of the E. coli
produced Cry2Ab2 using sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE), western blot analysis, densitometry, matrix
assisted laser desorption/ionization time of flight (MALDI-TOF) mass
spectrometry, glycosylation analysis, and a diet-incorporation corn earworm
(CEW) bioactivity assay. Similar immunoreactive bands migrating at
approximately 61 kDa were observed in the plant-produced Cry2Ab2 and E.
coli-produced reference samples. The plant-produced protein sample had an
additional immunoreactive band migrating at approximately 50 kDa; N-
terminal amino acid analysis of this protein indicated that it is a truncated
Cry2Ab2 protein with its N-terminus starting at amino acid 145. MALDI-
TOF mass spectrometry analysis of the ~61 and 50 kDa bands after trypsin
digestion yielded peptide masses consistent with peptide masses of the
predicted sequence of the Cry2Ab2 protein. Glycosylation analysis
indicated that the Cry2Ab2 protein is not glycosylated. The plant-produced
and E. co//'-produced proteins gave similar results in the bioactivity assay:
the mean EC50 values for the plant-produced Cry2Ab2 protein and E. coli-
produced reference standard were both determined to be 0.16 |ig Cry2Ab2
per mL diet, with standard deviations of 0.04 and 0.01 |ig Cry2Ab2 per mL
diet, respectively. The results of this study demonstrate the structural and
functional similarity between the plant-produced and the E. coli -produced
Cry2Ab2 proteins.

Classification: ACCEPTABLE

46951404

Characterization of
active

ingredient/Evaluation of
the Functional
Equivalence of the
Cry2Ab2 Protein
Produced in E. coli and
Bt Against a Sensitive
Lepidopteran Species

The functional activity of purified Cry2Ab2 produced from E. coli and
Cry2 Ab2 produced from Bt was evaluated using a corn earworm larvae diet
incorporation bioassay. There was no significant difference between the
EC50 values (the effective concentration to inhibit growth of the target
insect by 50%) for the two proteins, as shown by the large overlap in the
95% confidence intervals and the nearly identical dose response curves. In
addition, the two proteins showed the same rates of concentration-dependent
growth inhibition, indicating that the proteins have the same mechanism of
insecticidal action.

Classification: ACCEPTABLE

46951405

Response to EPA
Questions/ Responses to
EPA Questions
Regarding Applications
524-LTL and 524-LTA
to Register Insect-
protected Corn MON
89034 and MON 89034
x MON 88017

In an email message from S. Cerrelli to N. Bogdanova dated April 23, 2007,
EPA identified some deficiencies in the applications to register MON 89034
and MON 89034 x MON 88017 and requested some additional information
from Monsanto. In MRIDs 47127501-47127505, Monsanto responds to the
questions and supplies the requested additional information. Monsanto's
responses are adequate.

Classification: ACCEPTABLE

47127501-
47127505

18

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Bacillus thuringiensis Cry 1 A. 105, and Cry2Ab2 Protein in Corn
Biopesticide Registration Action Document

B. HUMAN HEALTH ASSESSMENT OF CrylA.105

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

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

1. Toxicological Profile

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

Mammalian Toxicity and Allergenicity Assessment

Monsanto submitted acute oral toxicity data demonstrating the lack of mammalian toxicity at
high levels of exposure to the pure Cry 1 A. 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).

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An acute oral toxicity study in mice (MRID 46694603) indicated that Cry 1 A. 105 is non-toxic to
humans. Cry 1 A. 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 LD50 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 Cry 1 A. 105, even at relatively high dose levels, the
Cry 1 A. 105 protein is not considered toxic. Further, amino acid sequence comparisons showed
no similarities between the Cry 1 A. 105 and known toxic proteins in protein databases that would
raise a safety concern.

Since Cry 1 A. 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 comparison with known allergens; and biochemical properties of the
protein, including in-vitro digestibility in simulated gastric fluid (SGF) and glycosylation. This
approach is consistent with the approach outlined in the Annex to the Codex Alimentarius
"Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-
DNA Plants." The allergenicity assessment for Cry 1 A. 105 follows:

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 Cry 1 A. 105 with
known allergens showed no overall sequence similarity or identity at the level of
eight contiguous amino acid residues.

3. Digestibility. The Cryl A. 105 protein was digested within 30 seconds in simulated
gastric fluid containing pepsin.

4. Glycosylation. Cry 1 A. 105 expressed in corn was shown not to be glycosylated.

5. Conclusion. Considering all of the available information, EPA has concluded that the
potential for Cry 1 A. 105 to be a food allergen is minimal.

Although Cryl A. 105 was only shown not to be glycosylated in corn, it is unlikely to be
glycosylated in any other crops because in order for a protein to be glycoslyated, it needs to
contain specific recognition sites for the enzymes involved in glycosylation, and the mechanisms

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Bacillus thuringiensis Cry 1 A. 105, and Cry2Ab2 Protein in Corn
Biopesticide Registration Action Document

of protein glycosylation are similar in different plants (Lerouge, P. Cabanes-Macheteau, M.,
Rayon, C., Fichette-Laine, A-C., Gomord, V., and Faye, L., "N-Glycoprotein biosynthesis in
plants: recent developments and future trends," Plant Molecular Biology 38: 31-48, 1998).

2. Aggregate Exposures

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 residues of the plant-incorporated protectants,
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 Cry 1 A. 105 to be an allergen is low, as
discussed previously. Although the allergenicity assessment focused on the Cry 1 A. 105 protein's
potential to be a food allergen, the data also indicated a low potential for Cry 1 A. 105 to be an
inhalation allergen. Exposure to infants and children via residential or lawn use is not expected,
because the use sites for the Cry 1 A. 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 in mammals showed no adverse effects.

3. Cumulative Effects

Pursuant to FFDCA section 408(b)(2)(D)(v), EPA has considered available information on the
cumulative effects of such residues and other substances that have a common mechanism of
toxicity. These considerations included the cumulative effects on infants and children of such
residues and other substances with a common mechanism of toxicity. Because there is no
indication of mammalian toxicity from the plant-incorporated protectant, EPA concluded that
there are no cumulative effects for the Cry 1 A. 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 Cry 1 A. 105 protein
included the characterization of the expressed Cry 1 A. 105 protein in corn, as well as the acute
oral toxicity study, amino acid sequence comparisons to known allergens and toxins, and in vitro

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Bacillus thuringiensis Cry 1 A. 105, and Cry2Ab2 Protein in Corn
Biopesticide Registration Action Document

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 Cry 1 A. 105 test material derived from
microbial culture was biochemically and functionally equivalent to the protein produced by the
plant-incorporated protectant ingredient in the plant. Microbially produced protein was used in
the studies so that sufficient material for testing was available.

The acute oral toxicity data submitted support the prediction that the Cry 1 A. 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. Given that no treatment-related adverse effects
were shown to be caused by the Cry 1 A. 105 protein, even at relatively high dose levels, the
Cry 1 A. 105 protein is not considered toxic. Basing this conclusion on acute oral toxicity data
without requiring further toxicity testing or residue data is similar to the Agency position
regarding toxicity and the requirement of residue data for the microbial Bacillus thuringiensis
products from which this plant-incorporated protectant was derived (See 40 CFR
158.740(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 acute 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. Data
submitted by the applicant, however, demonstrated low levels of Cry 1 A. 105 in corn tissues.

Since Cry 1 A. 105 is a protein, potential allergenicity is also considered as part of the toxicity
assessment. Considering all of the available information (1) Cry 1 A. 105 originates from a non-
allergenic source; (2) Cry 1 A. 105 has no sequence similarities with known allergens; (3)

Cry 1 A. 105 is not glycosylated; and (4) Cry 1 A. 105 is rapidly digested in simulated gastric fluid;
EPA has concluded that the potential for Cry 1 A. 105 to be a food allergen is minimal.

The Agency did not evaluate information concerning the dietary consumption patterns of
consumers (and major identifiable subgroups of consumers including infants and children) or
apply safety factors that are generally recognized as appropriate when animal experimentation
data are used to assess risks to humans. The lack of mammalian toxicity at high levels of
exposure to the Cry 1 A. 105 protein, as well as the minimal potential to be a food allergen,
satisfactorily demonstrated the safety of the products at levels well above the anticipated
maximum exposure levels.

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Bacillus thuringiensis Cry 1 A. 105, and Cry2Ab2 Protein in Corn
Biopesticide Registration Action Document

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 Cry 1 A. 105
protein has been exempted from the requirement of a tolerance under 40 CFR 174.507 "Nucleic
acids that are part of a plant-incorporated protectant."

b) Infants and Children Risk Conclusions

FFDCA section 408(b)(2)(C) provides that EPA shall assess the available information about
consumption patterns among infants and children, special susceptibility of infants and children to
pesticide chemical residues, and the cumulative effects on infants and children of the residues
and other substances with a common mechanism of toxicity. In addition, FFDCA section
408(b)(2)(C) also provides that EPA shall apply an additional tenfold margin of safety for infants
and children in the case of threshold effects to account for prenatal and postnatal toxicity and the
completeness of the database unless EPA determines that a different margin of safety will be safe
for infants and children.

In this instance, based on all the available information, the Agency concluded that there is a
finding of no toxicity for the Cry 1 A. 105 protein. Thus, there are no threshold effects of concern
and, as a result, the provision requiring an additional margin of safety does not apply. Further,
the considerations of consumption patterns, special susceptibility, and cumulative effects do not
apply.

c) Overall Safety Conclusion

There is a reasonable certainty that no harm will result from aggregate exposure to the U.S.
population, including infants and children, to the Cry 1 A. 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 previously discussed, no toxicity to mammals has been observed, nor any indication
of allergenicity potential for this plant-incorporated protectant.

5. Other Considerations

a) Endocrine Disruptors

As required under FFDCA section 408(p), EPA has developed the Endocrine Disruptor
Screening Program (EDSP) to determine whether certain substances (including pesticide active
and other ingredients) may have an effect in humans or wildlife similar to an effect produced by
a "naturally occurring estrogen, or other such endocrine effects as the Administrator may
designate." The EDSP employs a two-tiered approach to making the statutorily required
determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a
chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal

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Bacillus thuringiensis Cry 1 A. 105, and Cry2Ab2 Protein in Corn
Biopesticide Registration Action Document

systems. Chemicals that go through Tier 1 screening and are found to have the potential to
interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP where EPA
will determine which, if any, of the Tier 2 tests are necessary based on the available data. Tier 2
testing is designed to identify any adverse endocrine related effects caused by the substance, and
establish a quantitative relationship between the dose and the E, A, or T effect.

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

Bt Cry 1 A. 105, and Cry2Ab2 Protein in corn is not among the group of 58 pesticide active
ingredients on the initial list to be screened under the EDSP. Under FFDCA § 408(p) the
Agency must screen all pesticide chemicals. Accordingly, EPA anticipates issuing future EDSP
orders/data call-ins for all Registration Review cases, including those for which EPA has already
opened a Registration Review docket for a pesticide active ingredient.

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

b) Analytical Method(s)

A standard operating procedure for an enzyme-linked immunosorbent assay for the detection and
quantification of Cry 1 A. 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 Cry 1 A. 105 protein.

The human health studies submitted for Cry 1 A. 105 are summarized in Table 3 below.

Table 3. Summary of

CrylA.105 Human Health Data

Study Type/Title

Summary

mrii) #

Acute oral toxicity
(OPPTS 870.1100)/
Acute Oral Toxicity
Study in Mice with
Cry 1 A. 105 Protein2

The Cry 1 A. 105 test protein (2072 mg/kgbody 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.

46694603

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Biopesticide Registration Action Document

Study Type/Title

Summary

mrii) #



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 LD50 for males, females, and combined mice was
greater than 2072 mg/kg. This places Cry 1 A. 105 Protein in TOXICITY
CATEGORY III due to dose amounts only; no signs of toxicity were
observed.

Classification: ACCEPTABLE



Amino acid sequence
comparison/
Bioinformatics Analysis
of the Cry 1 A. 105
Protein Utilizing the
AD6, Toxin5, and
Allpeptides Databases2

Bioinformatic analyses were used to search for sequence similarities
between the Cry 1 A. 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
Cry 1 Ac protein; this alignment is not surprising, since the Cry 1 A. 105
protein contains a significant portion of the Cry 1 Ac protein. The
Cry 1 A. 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 Cry 1 A. 105 protein in the AD5 database.
No similarities between Cry 1 A. 105 protein and known allergens, human or
animal toxins, or pharmacologically active proteins were found in the study.
Classification: ACCEPTABLE

46694605

In vitro digestibility/
Assessment of the In
Vitro Digestibility of
the Cry 1 A. 105 Protein
in Simulated Gastric
Fluid2

No bands representative of intact Cry 1 A. 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 Cry 1 A. 105 protein. Both the pepsin stability
and test material stability controls gave appropriate responses. In the
Western Blot assay, Cry 1 A. 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 Cry 1 A. 105 protein. The pepsin
stability and test material stability controls gave appropriate responses.
Classification: ACCEPTABLE

46694606

Heat stability/

Immunodetection of

Cry2Ab2 and

Cry 1 A. 105 Proteins in

Corn Grain from MON

89034 Following Heat

Treatment2

The immunodetectability of the Cry 1 A. 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 °C 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 Cry 1 A. 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

46694607

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Biopesticide Registration Action Document

Study Type/Title

Summary

MRU) #



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 Cry 1 A. 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



Amino acid sequence
comparison/
Bioinformatics Analysis
of the Cry 1 A. 105
Protein Utilizing the
AD6, Toxin5, and
Allpeptides Databases

The amino acid sequence of the Cry 1 A. 105 protein was compared to the
sequences of known allergens and toxins using allergen (AD6), toxin
(TOXIN5), and public domain (ALLPEPTIDES) databases and the FASTA
algorithm. In addition to the FASTA comparisons, the Cry 1 A. 105 protein
sequence was compared to the AD6 database using 8 amino acid sliding
blocks and the ALLERGENSEARCH algorithm. No proteins were
identified with an E score of less than lxlO"5 from the search using the AD6
database, indicating that Cry 1 A. 105 has no structural similarity to any
known allergens, gliadins, or glutenins. In addition, no matches of 8 or
more amino acids were found between the Cry 1 A. 105 protein and sequences
in the AD6 database. In the comparisons using the TOXIN5 and
ALLPEPTIDES databases, the highest similarity identified was with
Bacillus thuringiensis pesticidal protein Cry 1 Ac (92% identity over an
1,182 aa window and an E score of 0). This result is not surprising, given
that the CrylA. 105 protein contains a significant portion of the Cry lAc
protein. No significant similarities between CrylA. 105 protein and known
allergens, human or animal toxins, or pharmacologically active proteins
were found in this study.

Classification: ACCEPTABLE

46951410

In vitro digestibility/
Assessment of the in
vitro Digestibility of the
Cry 1 A. 105 Protein in
Simulated Intestinal
Fluid

The in vitro digestibility of CrylA. 105 in simulated intestinal fluid
containing pancreatin was investigated using western blot analysis. The
band for the full-length CrylA. 105 was below the LOD in the 5 minute
time-point sample and in the later time-point samples. The LOD for the
full-length CrylA. 105 protein by western blot analysis was estimated to be
0.1 ng, which was 0.5 % of the total protein loaded. Therefore, at least
99.5% of the full-length protein was digested within 5 minutes. Bands from
proteolytic fragments with approximate molecular weights of 60, 32, and 30
kDa were visible in the five-minute time-point sample. The ~32 kDa
fragment was digested and undetectable at the 2-hour time-point and after.
The ~30 kDa fragment, which appears as a doublet, was still visible in the
24-hour time-point sample, but its intensity decreased substantially. The
-60 kDa fragment, which also appears as a doublet, represents the trypsin-
resistant core and appears to be fairly stable throughout the 24-hour
digestion experiment.

Classification: ACCEPTABLE

46951408

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Biopesticide Registration Action Document

C. HUMAN HEALTH ASSESSMENT Cry2Ab2

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

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

1. Toxicological Profile

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

Mammalian Toxicity and Allergenicity Assessment

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 position regarding toxicity testing and the requirement of
residue data for the Agency's 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 (Tiers II & III) and residue data are triggered by significant adverse acute
effects in studies such as the acute oral toxicity study, to verify the observed adverse effects and
clarify the source of these effects.

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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 body weight 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). 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 body weight.

When proteins are toxic, they are known to act via acute mechanisms and at very low dose
levels. Therefore, given that 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 comparison with known allergens; and biochemical properties of the
protein, including in vitro digestibility in simulated gastric fluid (SGF) and glycosylation. This
approach is consistent with the approach outlined in the Annex to the Codex Alimentarius
"Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-
DNA Plants." The allergenicity assessment for Cry2Ab2 follows:

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

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

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

Although Cry2Ab2 was only shown not to be glycosylated in corn, it is unlikely to be
glycosylated in any other crops because in order for a protein to be glycoslyated, it must contain
specific recognition sites for the enzymes involved in glycosylation, and the mechanisms of
protein glycosylation are similar in different plants (Lerouge, P. Cabanes-Macheteau, M., Rayon,
C., Fichette-Laine, A-C., Gomord, V., and Faye, L., "N-Glycoprotein biosynthesis in plants:
recent developments and future trends," Plant Molecular Biology 38: 31-48, 1998).

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Biopesticide Registration Action Document

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 considered
available information on the aggregate exposure levels of consumers (and major identifiable
subgroups of consumers) to the pesticide chemical residue and to other related substances.

These considerations include dietary exposure under the tolerance exemption and all other
tolerances or exemptions in effect for the Plant Incorporated Protectant's chemical residue, and
exposure from non-occupational sources. Exposure via the skin or inhalation is not likely since
the plant incorporated protectant is contained within plant cells, which essentially eliminates
these exposure routes or reduces exposure by these routes to negligible. In addition, even if
exposure can occur through inhalation, the potential for Cry2Ab2 protein to be an allergen is
low, as previously discussed. Although the allergenicity assessment focused on Cry2Ab2
protein's potential to be a food allergen, the data also indicated a low potential for Cry2Ab2 to be
an inhalation allergen. Exposure to infants and children via residential or lawn use 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 in laboratory mammals showed no adverse effects.

3. Cumulative Effects

Pursuant to FFDCA section 408(b)(2)(D)(v), EPA has considered available information on the
cumulative effects of such residues and other substances that have a common mechanism of
toxicity. These considerations included the cumulative effects on infants and children of such
residues and other substances with a common mechanism of toxicity. Because there is no
indication of mammalian toxicity from the plant incorporated protectant, we conclude that there
are no cumulative effects for the Cry2Ab2 protein.

4. Determination of Safety for U.S. Population, Infants and Children
Toxicity and Allergenicity Conclusions

The data submitted and cited regarding potential health effects for the Cry2Ab2 protein included
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 in the plant.

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Bacillus thuringiensis Cry 1 A. 105, and Cry2Ab2 Protein in Corn
Biopesticide Registration Action Document

Microbially produced protein was used in the safety studies so that sufficient material for testing
was available.

The acute oral toxicity data submitted by Monsanto 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. Given that 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 acute oral toxicity study. Further
studies verify the observed adverse effects and clarify the source of these effects.

Residue chemistry data were not required for a human health effects assessment of the subject
plant-incorporated protectant ingredients because of the lack of mammalian toxicity. Data
submitted by the applicant however, demonstrated low levels of the Cry2Ab2 in corn tissues.

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

The Agency did not consider information concerning the dietary consumption patterns of
consumers (and major identifiable subgroups of consumers including infants and children) or
safety factors that are generally recognized as appropriate when animal experimentation data are
used to assess risks to humans. 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 from the requirement of a tolerance under 40 CFR 174.507 "Nucleic acids
that are part of a plant-incorporated protectant."

a) 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

30

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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 concluded that there is a
finding of no toxicity for the Cry2Ab2 protein. Thus, there are no threshold effects of concern
and, as a result, the provision requiring an additional margin of safety does not apply. Further,
the considerations of consumption patterns, special susceptibility, and cumulative effects do not
apply.

b) 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 protocol for 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.

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Bacillus thuringiensis Cry 1 A. 105, and Cry2Ab2 Protein in Corn
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Table 4. Summary of

Cry2Ab2 Human Health Data

Study Type/Title

Summary

mrii) #

Acute oral toxicity
(OPPTS 870.1100)/An
Acute Oral Toxicity
Study in Mice with
Cry2Ab2 Protein

The acute oral toxicity of the Cry2Ab2 protein was assessed in CD-I mice.
Ten male and 10 female mice received E. coli-produced Cry2Ab2 protein at
a dose of 2198 mg/kg by oral gavage in two doses (test protein group); ten
male and 10 female mice were treated with 2 mM carbonate-bicarbonate,
2mM reduced glutathione buffer (vehicle control group); and ten male and
10 female mice received bovine serum albumin at a dose of 2424 mg/kg
(protein control group). 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 daily for 14 days. A general
health/mortality check was done twice daily. All animals were euthanized
and necropsied on day 14. All 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 LD50 for males, females, and
combined mice was greater than 2198 mg/kg. This places Cry2Ab2 protein
in TOXICITY CATEGORY III because of dose amounts only; no signs of
toxicity were observed.

Classification: ACCEPTABLE

46951406

In vitro

digestibility/Assessment
of the in vitro
digestibility of the
Cry2Ab2 protein in
simulated gastric fluid

The in vitro digestibility of Cry2Ab2 in simulated gastric fluid was
investigated. No bands representative of intact Cry2Ab2 protein were
identified by SDS-PAGE or western blot analysis after > 30 seconds
incubation with simulated gastric fluid containing pepsin. In the stained gel,
a faint band with molecular weight ~5 kDa was visible in the 30-second
time-point, but not in any other samples. No proteolytic fragments were
observed in the western blot. The limit of detection (LOD) for the full-
length Cry2Ab2 protein by SDS-PAGE with staining was determined to be
5 ng or approximately 0.6% of the total protein loaded. Therefore, at least
99.4% of the full-length Cry2Ab2 protein was digested within 30 seconds.
The LOD for the full-length Cry2Ab2 protein by western blot analysis was
determined to be 0.2 ng or 1% of the total protein loaded. Based on the fact
that no band was observed for the Cry2Ab2 protein in the 30 second time-
point sample in the western blot and the LOD for the protein using this
method, it was concluded that at least 99% of the Cry2Ab2 protein was
digested within 30 seconds.

Classification: ACCEPTABLE

46951407

In vitro digestibility/
Assessment of the in
vitro Digestibility of the
Cry2Ab2 Protein in
Simulated Intestinal
Fluid

The in vitro digestibility of Cry2Ab2 in simulated intestinal fluid containing
pancreatin was investigated using western blot analysis. The band for the
full-length Cry2Ab2 was below the LOD in the 15 minute time-point sample
and in the later time-point samples. The LOD for the full-length Cry2Ab2
protein by western blot analysis was estimated to be 0.5 ng, which was 2.5%
of the total protein loaded. Therefore, at least 97.5% of the full-length
protein was digested within 15 minutes. Bands from proteolytic fragments
with approximate molecular weights of 60, 55, 50, 40, 12, and 10 kDa were
visible in the five-minute time-point sample. The bands for all of these
proteolytic fragments except for the 50 kDa fragment were undetectable at

46951409

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Study Type/Title

Summary

mrii) #



the 24-hour incubation time-point.
Classification: ACCEPTABLE



Heat stability/

Immunodetection of

Cry2Ab2 and

Cry 1 A. 105 Proteins in

Corn Grain from MON

89034 Following Heat

Treatment2

The immunodetectability of the Cry 1 A. 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 °C 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 Cry 1 A. 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 Cry 1 A. 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

46694607

Amino acid sequence
comparison/ Bioinformatic
Evaluation of the Cry2Ab2
Protein Utilizing the AD6,
TOXIN5, and
ALLPEPTIDES Databases

The amino acid sequence of the Cry2Ab2 protein was compared to the
sequences of known allergens and toxins using allergen (AD6), toxin
(TOXIN5), and public domain (ALLPEPTIDES) databases and the FASTA
algorithm. In addition to the FASTA comparisons, the Cry2Ab2 protein
sequence was compared to the AD6 database using 8 amino acid sliding
blocks and the ALLERGENSEARCH algorithm. No proteins were
identified with an E score of less than lxlO"5 from the search using the AD6
database, indicating that Cry2Ab2 has no structural similarity to any known
allergens, gliadins, or glutenins. In addition, no matches of 8 amino acids
were found between the Cry2Ab2 protein and sequences in the AD6
database. In the comparisons using the TOXIN5 and ALLPEPTIDES
databases, the highest similarities identified were with Cry protein
homologues derived from Bacillus thuringiensis, Clostridium bifermentans,
Paenibacillus popilliae, and Paenibacillus lentimorbus. The results indicate
that the Cry2Ab2 protein does not share sequence homology with any
proteins that may have adverse effects in humans or animals.

46951411

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D. ENVIRONMENTAL ASSESSMENT for MON 89034

Background

Monsanto has requested a registration for Bacillus thuringiensis Cry 1 A. 105 and Cry2Ab2
proteins and the genetic material necessary for their production in all corn lines and varieties.
The nptll selectable marker gene was used in the transformation process, but was isolated and
removed from transformed plants via traditional breeding. The result is marker-free MON 89034
corn. The Cry proteins expressed in this event are intended to control the lepidopteran pests
European corn borer (ECB, Ostrinia nubilalis), corn ear worm (CEW, Helicoverpa zed), fall
army worm (FAW, Spodopterafrugiperda), and black cutworm (BCW, Agrotis ipsilon) which
are primary pests of corn in the United States. These pests feed on the base of seedlings and on
the stalk, leaf, and ear tissue of corn plants, thereby destroying the entire plant, weakening the
stalk, and/or damaging the ear. In areas where one or more of these pests is prevalent, significant
financial losses are realized from decreased corn yields and increased expenditures on chemical
pest control agents, including organophosphate, carbamate and pyrethroid insecticides.

EPA has conducted an environmental risk assessment of Cry 1 A. 105, Cry2Ab2 and MON 89034
when expressed in corn. General topics covered in this assessment include effects on wildlife,
gene flow to related wild plants and its potential effects, and fate of these Cry proteins in the
environment. This assessment is based on data submitted to EPA during the development of
Event MON 89034 corn lines, additional data submitted for registration, Federal Insecticide
Fungicide and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) recommendations,
consultations with scientific experts, and public comments on Plant-incorporated Protectant
(PIP) regulation.

1. Tiered Testing Hazard and Risk Assessment Process

To minimize data requirements and avoid unnecessary tests, risk assessments are structured such
that risk is determined first from estimates of hazard under "worst-case" exposure conditions. A
lack of adverse effects under these conditions would provide enough confidence that there is no
risk and no further data would be needed. Hence, such screening tests conducted early in an
investigation tend to be broad in scope but relatively simple in design, and can be used to
demonstrate acceptable risk under most conceivable conditions. When screening studies suggest
potentially unacceptable risk, additional studies are designed to assess risk under more realistic
field exposure conditions. These later tests are more complex than earlier screening studies. Use
of this "tiered" testing framework saves valuable time and resources by organizing the studies in
a cohesive and coherent manner and eliminating unnecessary lines of investigation. Lower tier,
high dose screening studies also allow tighter control over experimental variables and exposure
conditions, resulting in a greater ability to produce statistically reliable results at relatively low
cost3.

3 Non-target invertebrate hazard tests often are conducted at exposure concentrations several times higher than the

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Tiered tests are designed to first represent unrealistic worst case scenarios and ONLY progress to
real world field scenarios if the earlier tiered tests fail to indicate adequate certainty of acceptable
risk. Screening (Tier I) non-target organism hazard tests are conducted at exposure
concentrations several times higher than the highest concentrations expected to occur under
realistic field exposure scenarios. This has allowed an endpoint of 50% mortality to be used as a
trigger for additional higher-tier testing. Less than 50% mortality under these conditions of
extreme exposure suggest that population effects are likely to be negligible given realistic field
exposure scenarios.

EPA uses a tiered (Tiers I-IV) testing system to assess the toxicity of a PIP to representative non-
target organisms that could be exposed to the toxin in the field environment. Tier I high dose
studies reflect a screening approach to testing designed to maximize any toxic effects of the test
substance on the test (non-target) organism. The screening tests evaluate single species in a
laboratory setting with mortality as the end point. Tiers II - IV generally encompass definitive
hazard level determinations, longer term greenhouse or field testing, and are implemented when
unacceptable effects are seen at the Tier I screening level.

Testing methods that utilize the tiered approach were last published by EPA as Harmonized
OPPTS Testing Guidelines, Series 850 and 885 (EPA 712-C-96-280, February 1996)4. These
guidelines, as defined in 40 CFR 152.20, apply to microbes and microbial toxins when used as
pesticides, including those that are naturally occurring, and those that are strain-improved, either
by natural selection or by deliberate genetic manipulation. EPA has determined that it is
appropriate to utilize these testing guidelines in the context of PIPs.

The Tier I screening maximum hazard dose (MHD) approach to environmental hazard
assessment is based on some factor (whenever possible >10) times the maximum amount of
active ingredient expected to be available to terrestrial and aquatic non-target organisms in the
environment (EEC)5. Tier I tests serve to identify potential hazards and are conducted in the
laboratory at high dose levels that increase the statistical power to test the hypotheses. Elevated
test doses, therefore, add certainty to the assessment, and such tests can be well standardized.
The Guidelines call for initial screening testing of a single group or several groups of test
animals at the maximum hazard dose level. The Guidelines call for testing of one treatment
group of at least 30 animals or three groups of 10 test animals at the screening test concentration.
The Guidelines further state that the duration of all Tier I tests should be approximately 30 days.

maximum concentrations expected to occur under realistic exposure scenarios. This has customarily allowed an endpoint of 50%
mortality to be used as a trigger for additional higher-tier testing. Lower levels of mortality under these conditions of extreme
exposure suggest that population effects are likely to be negligible given realistic exposure scenarios. Thus, it follows that the
observed proportion of responding individuals can be compared to a 50% effect to determine if the observed proportion is
significantly lower than 50%. For example, using a binomial approach, a sample size of 30 individuals is sufficient to allow a
treatment effect of 30% to be differentiated from a 50%) effect with 95%o confidence using a one-sided Z test. A one-sided test is
appropriate because only effects of less than 50% indicate that further experiments are not needed to evaluate risk.

4 http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/885_Microbial_Pesticide_Test_Guidelines/Series/

5 The dose margin can be less than lOx where uncertainty in the system is low or where high concentrations of test
material are not possible to achieve due to test organism feeding habits or other factors. High dose testing also may not be
necessary where many species are tested or tests are very sensitive, although the test concentration used must exceed IX EEC.

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Some test species, notably non-target insects, may be difficult to culture and the suggested test
duration has been adjusted accordingly. Control and treated insects should be observed for at
least 30 days, or in cases where an insect species cannot be cultured for 30 days, until negative
control mortality rises above 20 percent.

Failing the Tier I (10 X EEC) screening does not necessarily indicate the presence of an
unacceptable risk in the field but it triggers the need for additional testing.6 A less than 50%
mortality effect at the MHD is taken to indicate minimal risk. Greater than 50% mortality does
not necessarily indicate the existence of unacceptable risk in the field, but it does trigger the need
to collect additional dose-response information and a refinement of the exposure estimation
before deciding if the risk is acceptable or unacceptable. Where potential hazards are detected in
Tier I testing (i.e. mortality is greater than 50%), additional information at lower test doses is
required which can serve to confirm whether any effect might still be detected at more realistic
field [IX EEC] concentrations and routes of exposure7.

When screening tests indicate a need for additional data, the OPPTS Harmonized Guidelines call
for testing at incrementally lower doses in order to establish a definitive LD50 and to quantify the
hazard. In the definitive testing, the number of doses and test organisms evaluated must be
sufficient to determine an LD50 value and, when necessary, the Lowest Observed Effect
Concentration (LOEC), No Observed Adverse Effect Level (NOAEL), or reproductive and
behavioral effects such as feeding inhibition, weight loss, etc. In the final analysis, a risk
assessment is made by comparing the NOAEL to the EEC; when the EEC is lower than the
NOAEL, a no risk conclusion is made. These tests offer greater environmental realism, but they
may have lower statistical power. Appropriate statistical methods, and appropriate statistical
power, must be employed to evaluate the data from the definitive tests. Higher levels of
replication, test species numbers or repetition are needed to enhance statistical power in these
circumstances.

Data that show less than 50 % mortality at the maximum hazard dosage level - (i.e., LC50, ED50,
or LD50 >10 X EEC) is sufficient to evaluate adverse effects, making lower field exposure dose
definitive testing unnecessary. It is also notable that the recommended >10X EEC maximum
hazard dose level is a highly conservative factor. The published EPA Level of Concern [LOC] is
50% mortality at 5X EEC 8.

6 It is notable that that the 10 X EEC MUD testing approach is not equivalent to what is commonly known as "testing at
a 10X SAFETY FACTOR" where any adverse effect is considered significant. Tier I screen testing is not 'safety factor testing'.
In a "10X safety factor" test any adverse effect noted is a "level of concern", whereas in the EPA environmental risk assessment
scenario any adverse effect is viewed as a concern only at IX the field exposure.

7 The IX EEC test dose is based on plant tissue content and is considered a high worst case dose (sometimes referred to
as HEEC). This IX EEC is still much greater than any amount which any given non-target organism may be ingesting in the field
because most non-target organisms do not ingest plant tissue.

8 Environmental Protection Agency (USEPA) (1998). "Guidelines for Ecological Risk Assessment." EPA 630/R-95-
002F. Washington, DC, USA. [Federal Register, May 14, 1998. 63(93): 26846-26924.] The established peer and EPA Science
Board reviewed guidance on screening test levels of concern is 50% mortality at 5X environmental concentration. The
appropriate endpoints in high dose limit/screening testing are based on mortality of the treated, as compared to the untreated
(control) non-target organisms. A single group of 30 test animals may be tested at the maximum hazard dose.

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Validation: The tiered hazard assessment approach was developed for EPA by the American
Institute of Biological Sciences (AIBS) and confirmed, in 1996, as an acceptable method of
environmental hazard assessment by a FIFRA Scientific Advisory Panel (SAP) on microbial
pesticides and microbial toxins. The December 9, 1999, SAP agreed that the Tiered approach
was suitable for use with plant-incorporated protectants; however, this panel recommended that,
for PIPs with insecticidal properties, additional testing of beneficial invertebrates closely related
to target species and/or likely to be present in GM crop fields should be conducted. Testing of Bt
Cry proteins on species not closely related to the target insect pest was not recommended,
although it is still performed to fulfill the published EPA non-target species data requirements. In
October 2000, another SAP also recommended that field testing should be used to evaluate
population-level effects on non-target organisms. The August 2002 SAP, and some public
comments, generally agreed with this approach, with the additional recommendation that
indicator organisms should be selected on the basis of potential for field exposure to the subject
protein.9

Chronic studies: Since delayed adverse effects and/or accumulation of toxins through the food
chain are not expected to result from exposure to proteins, protein toxins are not routinely tested
for chronic effects on non-target organisms. The 30 day test duration requirement does,
however, amount to subchronic testing when performed at field exposure test doses. Proteins do
not bioaccumulate. The biological nature of proteins makes them readily susceptible to
metabolic, microbial, and abiotic degradation once they are ingested or excreted into the
environment. Although there are reports that some proteins (Cry proteins) bind to soil particles,
it has also been shown that these proteins are degraded rapidly by soil microbial flora upon
elution from soil particles.

Conclusion: The tiered approach to test guidelines ensures, to the greatest extent possible, that
the Agency requires the minimum amount of data needed to make scientifically sound regulatory
decisions. EPA believes that maximum hazard dose Tier I screening testing presents a
reasonable approach for evaluating hazards related to the use of biological pesticides and for
identifying negative results with a high degree of confidence. The Agency expects that Tier 1
testing for short-term hazard assessment will be sufficient for most studies submitted in support
of PIP registrations. If long range adverse effects must be ascertained, then higher-tier longer-
term field testing will be required. The Agency has been frequently asking the registrants to

9 EPA-SAP. February 4, 2000. Characterization and non-target organism data requirements for protein plant-pesticides.
SAP report No. 99-06A for FIFRA Scientific Advisory Panel Meeting held December 8, 1999, held at the Sheraton Crystal City
Hotel, Arlington, VA.

EPA-SAP. November 6, 2002. Corn rootworm plant-incorporated protectant insect resistance management and non-
target insect issues. Transmittal of meeting minutes of the FIFRA Scientific Advisory Panel Meeting held August 27-29 at the
Marriott Crystal City Hotel, Arlington, VA.

EPA-SAP. March 12, 2001. Bt plant-pesticides risk benefit assessments. SAP report No. 2000-07 for FIFRA Scientific
Advisory Panel Meeting held October 18-20, 2000 at the Marriott Crystal City Hotel, Arlington, VA.

EPA-SAP. August 19, 2004. Product characterization, human health risk, ecological risk, and insect resistance
management for Bacillus thuringiensis (Bt) cotton products. Transmittal of meeting minutes of the FIFRA Scientific Advisory
Panel Meeting held June 8-10 at the Holiday Inn Ballston, Arlington, VA.

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conduct post-registration long term invertebrate population/community and Cry protein
accumulation in soils studies as a condition of registration. As noted above, the October 2000
SAP and the National Academy of Sciences10 (NAS 2000) recommended testing non-target
organisms directly in the field. This approach, with an emphasis on testing invertebrates found
in crop fields, was also recommended by the August 2002 SAP and was supported by several
public comments. The issue of long range effects of cultivation of currently registered Cry
proteins on the invertebrate community structure in Bt crop fields has since been adequately
addressed by a meta analysis of field studies performed during the last 10 years. No unexpected
adverse effects on invertebrate community structure were reported.11 The meta analysis of short
term and long term field study effects on invertebrate populations in Bt corn and cotton fields
indicate that no unreasonable adverse effects are taking place as a result of wide scale Bt crop
cultivation. The Agency is in agreement with these conclusions. Slight reductions in some
invertebrate predator populations are an inevitable result of all pest management practices which
result in reductions in the abundance of the pests as prey. Based on these considerations,
regulatory testing of the specialist predators and parasitoids of target pests may eventually be
considered unnecessary.

2. Environmental Exposure Assessment

The EPA risk assessment is centered only on adverse effects at the field exposure rates (IX
EEC), and not on adverse effects at greater concentrations. The dose margin can be less than
lOx where uncertainty in the system is low or where high concentrations of test material are not
possible to achieve due to test organism feeding habits. High dose testing also may not be
necessary where many species are tested or tests are very sensitive, although the concentration
used must exceed IX EEC. It is important to note that Tier I screen testing is not "safety factor
testing." In a traditional "10X safety factor" test any adverse effect noted is a "level of concern",
whereas in the EPA environmental risk assessment scenario any adverse effect is viewed as a
concern only at IX the field exposure.

For the purposes of the nontarget organism (NTO) studies submitted in support of the MON
89034 registration, test material dose levels were based on the estimated concentration of
Cry 1 A. 105 and/or Cry2Ab2 protein expressed in the tissue(s) that NTO would most likely be
exposed to in the environment (see Edelstein, 2007 for protein expression levels). Whenever
possible, a targeted margin of exposure (MOE) of greater than 10X the maximum environmental
exposure was used in the tests. The primary route of Cry 1 A. 105 and Cry2Ab2 protein exposure
for honeybee, ladybird beetle, parasitic wasp, and minute pirate bug is corn pollen.

Consequently, test material dose levels were based on the maximum level of measured protein

10 Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation is available from the National
Academy Press, 2101 Constitution Avenue, N.W., Lockbox 285, Washington, DC 20055; (800) 624-6242 or (202) 334-3313 (in
the Washington metropolitan area); http://www.nap.edu.

11 Marvier, M., McCreedy, C., Regetz, J. & Kareiva, P. A meta-analysis of effects of Bt cotton and maize on nontarget
invertebrates. Science 316, 1475-1477 (2007).

Sanvido,0., Romeis, J., Bigler, F. (2007). Ecological Impacts of Genetically Modified Crops: Ten Years of Field
Research and Commercial Cultivation. Adv Biochem Engin/Biotechnol 107: 235-278

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Biopesticide Registration Action Document

expression in pollen (8.8 ug/g fwt for Cry 1 A. 105 and 0.47 ug/g fwt for Cry2Ab2). The principal
route of Cry 1 A. 105 and Cry2Ab2 protein exposure for soil-dwelling organisms, such as
Collembola and earthworms, is assumed to be from decomposing plant tissue and plant exudates
in soil. Consequently, the test material dose levels were based on the maximum level of
estimated protein concentration in the soil environment.

3. Non-Target Wildlife Hazard Assessment for MON 89034 corn

Two separate SAP reports (October 2000 and August 2002) recommended that non-target testing
of Bt Cry proteins should focus on invertebrate species exposed to the crop being registered.
Following the SAP recommendations, EPA determined that non-target organisms with the
greatest exposure potential to Cry protein in transgenic corn fields are beneficial insects, which
feed on corn pollen and nectar, and soil invertebrates, particularly Coleopteran species.

Therefore, maximum hazard dose toxicity testing on representative beneficial organisms from
several taxa was performed in support of this Section 3 FIFRA registration. The toxicity of the
Cry 1 A. 105 and Cry2Ab2 proteins has been evaluated on several species of invertebrates
including the lady beetle, minute pirate bug, parasitic hymenoptera, collembola, daphnia, honey
bee, and earthworm. Developmental observations were also made in the lady beetle, minute
pirate bug and honeybee studies. Observations of possible reproductive effects were made in the
collembola studies.

Although the Cry 1 A. 105 and Cry2Ab2 proteins in MON 89034 are known to be very host
specific, conferring toxic effects on ECB, CEW, FAW, BCW and closely related species, and
despite the October 2000 and August 2002 SAP's recommendations against testing of non-target
species not related to susceptible target pests, EPA has done a risk assessment on a range of non-
target wildlife to comply with the Agency's published non-target data requirements (in the
absence of PIP-specific risk assessment guidance, EPA requires applicants for PIP registrations
to meet the 40 CFR Part 158 data requirements for microbial toxins). These requirements
include birds, mammals, plants and aquatic species. In addition, an earthworm study was
voluntarily submitted to the Agency to ascertain the potential effects of the Cry 1 A. 105 and
Cry2Ab2 proteins on beneficial decomposer species.

Test substances used for studies submitted in support of the event MON 89034 registration
included bacterially produced purified Cry 1 A. 105 and Cry2Ab2 proteins and MON 89034 corn
grain. The October 2000 SAP recommended that while actual plant material is the preferred test
material, bacterially-derived protein is also a valid test substance, particularly in scenarios where
test animals do not normally consume corn plant tissue and where large amounts of Cry protein
(Cry protein concentrations that exceed levels present in plant tissue) are needed for maximum
hazard dose testing. An insect feeding study, which compared the relative potency of plant
produced Cry 1 A. 105 and Cry2Ab2 proteins to the microbe produced proteins, indicated that
plant produced protein was similar in toxicity to the microbe produced protein (Edelstein Memo,
November 7, 2007).

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Potential Interaction between the Cry 1 A. 105 and Cry2Ab2 proteins was addressed in a
memorandum for the MON 89034 Experimental Use Permit accompanying the Agency's
review of "Evaluation of the Potential for Interactions Between the Bacillus thuringiensis
Proteins Cry 1 A. 105 and Cry2Ab2," (Hunter, M., July 6, 2006). The purpose of this study was
to characterize the potential for interaction between the lepidopteran-active proteins Cry 1 A. 105
and Cry2Ab2. The study provides evidence that the proteins do not interact in an antagonistic,
or synergistic manner and that there will not be any unexpected interaction with regard to target
and non-target insects. New data on the potential interaction between combined Cry 1 A. 105,
Cry2Ab2 with the Cry3Bbl protein was submitted. The results from the study demonstrated
that combined Cry 1 A. 105 and Cry2Ab2 activity was not affected by the Cry3Bbl protein and
that Cry3Bbl activity was not affected by combined Cry 1 A. 105 and Cry2Ab2 activity (MRID
469513-05 & 469513-06).

The results of ecological effects studies submitted in support of the MON 89034 Section 3
FIFRA registration are summarized in Table 5 and presented in a more descriptive format in
subsequent sections of this risk assessment document. Full reviews of each study can be found
in the individual Data Evaluation Reports (DERs/MRID#s) and accompanying memos.

Table 5. Summary of environmental effects studies and waiver justifications submitted to

comply with t

ata requirements published in 40 CFF

t § 158.2150(d).

Data
Requirement

Guideline

Classification

Test
Substance

Results Summary

MRID#

Avian oral

885.4050*
154-16**

Acceptable

MON 89034
corn grain^

A 42-day dietary study showed that
Event MON 89034 did not
adversely affect broiler chickens.

469514-12

Avian injection

885.4100
154-17

Acceptable waiver
rationale

N/A

Avian acute oral

850.2100

Acceptable

MON 89034
corn grain

An eight-day dietary study showed
that the LC50 for MON 89034 is
>500,000 ppm in the diet northern
bobwhite quail.

469514-27

Wild mammal

885.4150
154-18

Acceptable waiver
rationale

N/A

Freshwater fish

885.4200
154-19

Acceptable waiver
rationale

N/A

Freshwater fish studies were not
required because of the low
potential that fish will be exposed to
high levels of the Cry 1A.105 and
Cry2Ab2 proteins

N/A

Freshwater
aquatic
invertebrate
Daphnia magna

885.4240
154-20

Unacceptable

[The 885 Series
Guidelines call for
a 7-14 day study.
The submitted 48
hour acute study is
inadequate.]

MON 89034

corn

Pollen

A 48-hour static renewal limit
bioassay resulted in 17% mortality
compared with 0% mortality in the
control groups (120 mg/L). A 48-
hour static renewal dose-response
bioassay was conducted and no
mortality or adverse effects were
observed at any concentration (6.3-
120 mg/L). The acute EC50 was
estimated to be >120 mg/L and the
NOEC was 100 mg/L.

469514-17

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Biopesticide Registration Action Document

Data
Requirement

Guideline

Classification

Test
Substance

Results Summary

mrii) #

Freshwater
aquatic
invertebrate
Daphnia magna

885.4240
154-20

Acceptable

MON 89034

corn

Pollen

It was determined that the study
is acceptable and satisfies the
condition of registration for
additional aquatic invertebrate
acute toxicity testing. No
unreasonable adverse effects to
aquatic invertebrates are
expected from exposure to
MON 89034 corn.

478388-01

Estuarine and
marine animal

885.4280
154-21

Acceptable waiver
rationale

N/A

N/A

N/A

Non-target plant

885.4300
154-22

Acceptable waiver
rationale

N/A

N/A

N/A

Non-target
insect testing,
minute

pirate/insidious
flower bug
Orius insidiosus

885.4340
154-23

Acceptable

Cry2Ab2
protein
(Lot No.
20-100071)

Orius nymphs were fed a pollen diet
containing 100 \ig Cry2Ab2
protein/diet for 14 days. No adverse
effects were observed.

469514-24

Non-target
insect testing,
minute

pirate/insidious
flower bug
Orius insidiosus

885.4340
154-23

Acceptable

Cry 1A.105
protein
(Lot No.
20-100073)

Orius nymphs were fed a diet
containing 30 to 240 \ig
CrylA.105/g diet for 14 days. In an
initial maximum dose test (240 |ig)
the survival rate was 47% compared
to 88% in the control groups. In the
three subsequent dose-response
tests, the mean survival rate of the
240 \ig group was 55% compared to
91%) and 89%o in the control groups.
No statistically significant effects on
survival or development were seen
at concentrations less than or equal
to 120 \ig CrylA.105/g diet.

469514-23

Non-target
insect testing,
parasitic wasp,
Ichneumon
promissorius

885.4340
154-23

Acceptable

Cry2Ab2
protein
(Lot No.
20-100071)

Adult female wasps were fed a
sucrose solution containing 100 \ig
Cry2Ab2 protein/mL for 21 days.
Mortality in the Cry2Ab2 group was
3%o and the LC50 was determined to
be >100 ng/L.

469514-26

Non-target
insect testing,
parasitic wasp
Ichneumon
promissorius

885.4340
154-23

Acceptable

Cry 1A.105
protein
(Lot No.
20-100073)

Adult female wasps were fed a
sucrose solution containing 240 \ig
Cry 1A. 105 protein/mL for 21 days.
Mortality in the Cry 1 A. 105 group
was 7%o and the LC50 was
determined to be >240 |ig/L.

469514-25

Non-target
insect testing,
ladybird beetle
Coleomegilla
maculata

885.4340
154-23

Acceptable

Cry2Ab2
protein
(Lot No.
20-100071)

C. maculata larvae were fed a diet
containing 120 |ig Cry2Ab2
protein/g diet for 17-20 days. No
statistically significant difference in
survival or development to adult
was found between the test and
control groups. A slight (~5%>)
statistical decrease in mean adult

469514-22

41

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Data
Requirement

Guideline

Classification

Test
Substance

Results Summary

mrii) #









body weight was found between the
test and buffer control groups;
however, this difference was not
observed between the test and assay
control group.



Non-target
insect testing,
ladybird beetle
Coleomegilla
maculata

885.4340
154-23

Acceptable

Cry 1A.105
protein
(Lot No.
20-100073)

Ladybird beetle larvae were fed a
diet containing 240 \ig Cry 1A. 105
protein/g diet for 14 days. No
statistically significant differences
in survival, development, or adult
beetle weight were found between
the test and control groups.

469514-21

Non-target
insect testing,
collembolan
Folsomia
Candida

885.4340
154-23

Acceptable

MON 89034
Leaf Tissue
(80 ng
Cry 1A.105
and 70 \ig
Cry2Ab2/g
diet)

Collembola were fed a diet
containing 50% Brewer's yeast and
50% lyophilized leaf tissue for 28
days. No statistically significant
effects on survival or reproduction
were found among the test and
negative control groups.

469514-16

Honeybee
testing, Adult
Honeybee, Apis
mellifera

885.4380
154-24

Acceptable

Cry 1A.105
protein
(Lot No.
20-100073)

Adult honeybees were fed a 30%
sucrose solution containing 550 \ig
Cry 1A. 105 protein/mL for 19 days.
No statistically significant
differences in mortality were
observed between the test group and
negative controls. The NOEC was
determined to be at least 550 \ig
Cry 1A.105 protein/mL.

469514-20

Honeybee
testing,
Honeybee
larvae, Apis
mellifera

885.4380
154-24

Acceptable

Cry 1A.105
protein
(Lot No.
20-100073)

Two-to-three day old honeybee
larvae in brood frames were
administered a single 10 |xL dose of
Cry 1A. 105 protein per brood cell
(equivalent to 12 (rg total
protein/cell). On day 18 after dosing
mean survival of the test group was
95%). The NOEC was determined
to be at least 12 \ig Cry 1A. 105
protein per brood cell

469514-20

Earthworm
subchronic
toxicity, Eisenia
fetida

850.620

Acceptable

Cry 1A.105
protein
(Lot No.
20-100073)

Adult earthworms were exposed to
artificial soil containing 178 mg
Cry 1A. 105 protein/kg dry soil for
14 days. No mortality was observed
in the test group. The LC50 was
determined to be >178 mg
CrylA.105/kg dry soil and the
NOEC was 178 mg CrylA.105
mg/kg dry soil.

469514-18

42

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Data
Requirement

Guideline

Classification

Test
Substance

Results Summary

MRID#

Soil fate

885.5200

Acceptable

Purified
Cry 1A.105
(Lot No.
20-100073)
and

Cry2Ab2
protein
(Lot No.
20-100071)

Results of this degradation study
indicate that Cry 1 A. 105 and
Cry2Ab2 proteins do not persist in
soil beyond approximately three
weeks.

469514-28

Note: Earthworm and honey bee studies for Cry2Ab2 protein were submitted and reviewed with previously registered products.
The interaction study between Cry 1A.105 and Cry2Ab2 was reviewed for the MON 89034 Experimental Use Permit

OPPTS Microbial pesticide test guidelines
**Microbial pesticide test guidelines identified in the 40 CFR data tables.

^ Cry 1 A. 105 and Cry2Ab2 are the active ingredients (a.i.) in MON 89034 corn.

a) Non-target Wildlife Study Summaries
i. Avian species

Published data and studies on file at EPA show that consumption of Bt corn has no measurable
deleterious effects on avian species. To comply with published data requirements, the following
studies were submitted to EPA in support of the MON 89034 product registration. These studies
were GLP compliant and, when considered together, meet EPA data requirements for avian
species.

(a) Broiler (MRID 469514-12)

For the first 42 days of life, commercial broiler chickens (Gallus domesticus) were fed a corn
and soybean diet that contained up to 59% ground corn grain. Treatments consisted of soybean
meal with MON 89034, a similar isoline (negative control), or one of four different commercial
hybrid corn varieties. At test end, chickens were processed in order to obtain performance and
carcass yield data. Breast and thigh meat were also analyzed for moisture, protein, and fat
content. Among treatments, there were no biologically significant differences in broiler
performance, carcass, or meat quality.

(b) Northern Bobwhite Quail (MRID 469514-27)

In this eight-day dietary study, 10-day-old northern bobwhites (Colinus virginianus) were fed a
corn and game bird ration containing 50% ground corn grain. Treatments consisted of game bird
ration with MON 89034, a similar isoline (negative control), or one of three different commercial
hybrid corn varieties. At test end, no mortality was seen in the MON 89034 treatment group, all
birds appeared normal for test duration, and feed consumption was comparable to that of the
control group. The dietary LC50 of MON 89034 corn grain was determined to be >500,000 ppm
in the diet.

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ii. Wild mammalian species

Mammalian wildlife exposure to Cry 1 A. 105 and Cry2Ab2 proteins is considered likely;
however, mammalian toxicology information gathered to date on Bt Cry proteins does not show
a hazard to wild mammals. In addition, acute oral toxicity studies submitted to EPA in support
of the MON 89034 registration indicated that no significant toxicity was seen when rodents were
exposed to Cry 1 A. 105 or Cry2Ab2 at the maximum hazard dose level. Therefore, no hazard to
mammalian wildlife is anticipated and data on wild mammal testing is not required for this
registration.

iii. Aquatic species

There is no evidence for sensitivity of aquatic species to anti-coleopteran Cry proteins. A
published laboratory study with lepidopteran-active Cry proteins has revealed that the leaf
shredding (caddis fly) trichopteran, Lepidostoma liba, had 50% lower growth rate when fed Bt
corn litter (Rosi-Marshall, et al. 2007). Two previous field study reports by the same authors did
not find adverse effects on head stream invertebrates. The Agency's position on this matter is
that until Tier III and Tier IV field studies are performed, there is not enough information to
assert that sufficient corn plant litter enters streams to cause unreasonable adverse effects on
stream invertebrate populations or communities (See Section D. 1. above - Tiered Hazard and
Risk Assessment Process). Two years ago the Iowa State University and the University of
Maryland received Research grants to study the effects of Bt corn cultivation on streams and to
develop methods for aquatic hazard assessment. The results of these studies are pending. When
the study reports are reviewed the Agency will respond with action commensurate with the
outcome of the studies.

The Agency's current position is that there is no evidence to conclude that there is sufficient
aquatic exposure to Cry proteins in corn plant litter to result in adverse effects on stream
invertebrate populations or communities. Aquatic animal exposure to Bt crops is extremely
small.

(a) Freshwater fish -Waiver granted

Freshwater fish studies were not required for this product, because of the low potential that
aquatic systems will be exposed to the Cry 1 A. 105 and Cry2Ab2 proteins produced in MON
89034 corn plant tissues.

(b) Freshwater aquatic invertebrates (MRID 469514-17)

The objective of this study was to determine the potential for acute effects to the aquatic
organism, Daphnia magna, during a static renewal exposure to MON 89034 corn pollen. The
test was initially conducted as a limit test using one test concentration. Slight effects were noted
at the limit concentration. In response, a dose-response test was conducted. The test substance,
MON 89034 pollen expressing Cry 1 A. 105 and Cry2Ab2 proteins, was evaluated for potential

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adverse effects to neonate Daphnia. Test organisms were < 24 hours old at test initiation and
came from in-house cultures at the test facility.

Limit Test

In the initial test, daphnids were exposed to a single nominal test concentration of 120 mg
pollen/L for 48 hours with renewal of the test solution at approximately 24 hours. Two control
groups were included: a group in well water exposed to pollen (120 mg/L) from conventional
corn with a genetic background similar to MON 89034, and an assay control group exposed to
well water only. Each treatment was replicated three times and each replicate contained 10
neonate daphnids. Test chambers consisted of 600-mL glass beakers containing 300 mL of the
appropriate treatment solution. Observations of mortality, immobility and other clinical signs
were made at approximately 3.5, 24 and 48 hours after test initiation. At test termination there
was 17% immobility in the 120 mg/L treatment group, with two daphnids exhibiting lethargy.
All daphnids in the assay control group and 120 mg pollen/L control group appeared normal
throughout the testing period.

Dose-Response Test

In the dose-response test, daphnids were exposed to six concentrations of MON 89034 pollen for
48 hours. The concentrations tested were 6.3, 13, 25, 50, 100, and 120 mg pollen/L. Two
control groups were included: a group in well water exposed to pollen (120 mg pollen/L) from
conventional corn with a genetic background similar to MON 89034, and an assay control group
exposed to well water only. The test and control solutions were renewed at approximately 24
hours. Each treatment was replicated two times and each replicate contained 10 neonate
daphnids. Test chambers consisted of 600-mL glass beakers containing 300 mL of the
appropriate treatment solution. Observations of mortality, immobility and other clinical signs
were made at approximately 5, 24, and 48 hours after test initiation. The NOEC was estimated
by visual interpretation of the mortality, immobility and observation data. At test termination
there were no mortalities, immobile daphnids or signs of toxicity noted in any control or test
substance group during the 48 hour exposure period.

Conclusions: Based on the results of the dose-response test, the 48-hour EC50 was estimated to
be greater than 120 mg MON 89034 pollen/L. Based on the results of both studies, the 48-hour
NOEC was 100 mg MON 89034 pollen/L. This study is unacceptable because it is an 850 Series
OPPTS Guideline study. The 48 hour test duration is not sufficient to show mortality for Bt
toxins. The 48 hours test duration is not considered to be sufficient duration to assess the
potential for adverse effects to non-target organisms. Consistent with the 885 Series OPPTS
Guidelines, a 7 to 14 day Daphnia study is necessary. The study may be submitted as a condition
of registration. Alternatively, a dietary study of the effects on an aquatic invertebrate,
representing the functional group of a leaf shredder in headwater streams, may be performed and
submitted in lieu of the Daphnia study.

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When Bt Cry 1 A. 105, and Cry2Ab2 Protein in corn (EPA Reg. No. 524-575, and 524-576) were
initially registered, the Agency issued registration notices to Monsanto Company that contained
the following requirement for further Environmental Assessment information:

"A 7-14 day Daphnia study as per the 885 Series OPPTS Guidelines needs
to be performed. Alternatively, a dietary study of the effects on an aquatic
invertebrate, representing the functional group of a leaf shredder in
headwater streams, can be performed and submitted in lieu of the Daphnia
study."

Due to the fact that there was a statistically significant reduction in survival rate in the pollen
control group when compared to the assay control group, the study author concluded the
exposure to high concentrations of conventional corn pollen resulted in impacts on the overall
health of daphnids. It was therefore considered most appropriate to compare performance by the
MON 89034 treatment groups to the pollen control group. Based on the immobility and
sublethal signs of toxicity noted in the pollen control group, effects on survival noted in the 8.3
and 42 mg/L pollen from MON 89034 treatment groups were not considered to be related to
toxicity of MON 89034, but were considered to be due to physical toxicity caused by high
concentrations of pollen in the solutions. Reductions in survival in the 8.3 and 42 mg/L pollen
from MON 89034 treatment groups at the Day 14 interval when compared to the pollen control
group were not statistically significant (p > 0.05). The 14-day median effect concentration (EC50)
and no observed effect concentration (NOEC) values for adult immobility when compared to the
pollen control were determined to be >42 mg/L and 42 mg/L pollen from MON 89034,
respectively.

From the results, there was no indication of a delay in the onset of reproduction in any of the
treatment groups. Observations of immobile neonates in the 42 mg/L pollen control and 42
mg/L treatment group may have been related to high pollen concentrations in the test solutions,
but were not considered by the study author to be related to toxicity of MON 89034. The NOEC
for reproduction was considered to be 42 mg/L pollen from MON 89034 when compared to the
pollen control group.

Conclusions: It was determined that the study is acceptable and satisfies the condition of
registration for additional aquatic invertebrate acute toxicity testing. No unreasonable adverse
effects to aquatic invertebrates are expected from exposure to MON 89034 corn.

(d) Estuarine and marine animals- Waiver granted

Estuarine and marine animal studies were not required for this product, because of the low
probability that estuarine or marine systems will be exposed to the Cry 1 A. 105 and Cry2Ab2
proteins produced in MON 89034 corn plant tissues.

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iv. Terrestrial and aquatic plant species-Waiver granted

Plant toxicity studies were not required for this product because the active ingredient is an insect
toxin (Bt endotoxin) that has never shown any toxicity to plants.

v. Terrestrial Invertebrate species

The Cry 1 A. 105 and Cry2Ab2 proteins are meant to target species within the order Lepidoptera
(moths and butterflies). Bt toxins are known to have a limited host range, however, to address
any unforeseen change in activity spectrum and to fulfill the published registration data
requirements EPA requires that test species used for non-target insect evaluations should include
several species that are not related to the target pests. Earthworm studies are also recommended.

(a) Ladybird beetle
MRID 469514-21

The purpose of this study was to determine the potential dietary effects of the Cry 1 A. 105 protein
on the mortality and development of the ladybird beetle, Coleomegilla maculata. The test
substance, Cry 1 A. 105 protein, was produced by a recombinant E. coli fermentation system. The
test substance was incorporated at 240 |ig Cry 1 A. 105 protein/g of diet. The diet consisted of an
artificial agar-based diet. Three control treatments were included in the experiment. The buffer
control contained 25 mM CAPS buffer, which was the buffer used for storage of the test
material. The assay control (purified water) was used to generate a diet-only treatment and a
positive control treatment was also tested, containing potassium arsenate. Ladybird beetle larvae
were less than 48 hours old at test initiation. The larvae were contained in individual test arenas
(inverted 60 x 15 mm Petri dishes) and were allowed to feed ad libitum on the appropriate test
diet. Each treatment was replicated six times and each replicate contained 15 or 16 larvae. All
six replicates met the acceptance criteria of less than or equal to 20% mortality; the mortality of
larvae ranged from 0 to 18.8% in the assay control treatment groups. The diet treatments were
replaced with fresh diet approximately every 48 to 72 hours. The larvae were monitored every
24 to 72 hours for survival and development to the adult stage. Adults were weighed within 30
hours of eclosion and each adult was dissected and sexed. Any abnormal behavior or
development was noted during feedings and observational evaluations. The study duration
ranged from 17 to 20 days depending on adult emergence. Samples were taken to test the
biological activity, homogeneity, and stability of the Cry 1 A. 105 protein. Results showed that
there were no differences in the mean survival percentage of C. maculata between the
Cry 1 A. 105 protein, buffer control, and assay control treatments (88.5%, 87.5%, 91.6%). The
survival rate was 2.08% for the positive control treatment. There were no significant differences
in the mean percent of C. maculata that developed to adults when comparing the Cry 1 A. 105,
buffer control, and assay control treatments (88.5%, 85.4%, 90.6%). None of the larvae
developed to adults in the positive control treatment. Further, there were no significant
differences in the mean weight of C. maculata adults between the Cry 1 A. 105, buffer control, and

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assay control. No significant interaction was found between insect sex and treatment. In the
bioactivity confirmation assay, results indicated equivalent biological activity among the
Cry 1 A. 105 used in the test diets and the reference standard. In addition, the homogeneity and
stability study confirmed that Cry 1 A. 105 was homogeneous in the test substance diet and was
stable under the storage conditions employed in the study.

Conclusions: This study is acceptable. The results indicate that Cry 1 A. 105 protein had no
adverse effect on the survival, development, and growth of the ladybird beetles at a dietary
concentration of 240 |ig/g of diet.

MRID 469514-22

The objective of this study was to determine the potential dietary effects of Cry2Ab2 protein on
the mortality and development of the ladybird beetle, Coleomegilla maculata. The test
substance, Cry2Ab2, was produced by recombinant E. coli fermentation system. The endpoints
evaluated were survival and development through 20 days (some replicates were completed
before 20 days if all insects had developed to adults). The Cry2Ab2 protein was incorporated in
to an agar-based artificial diet at a concentration of 120 |ig Cry2Ab2/g diet. Three control
treatments were included in the study: 1) buffer control, 2) assay control (purified water), and 3)
positive control (potassium arsenate). The ladybird beetle larvae were less than 48 hours old
when testing began and the larvae were allowed to feed ad libitum. The diet treatments were
replaced approximately every 48 to 72 hours. Each treatment was replicated six times and each
replicate included 14 to 16 ladybird beetle larvae. Each larva was contained in its own test arena
which consisted of an inverted 60 x 15 mm Petri dish. The larvae were monitored every 24 to 72
hours for survival and development to the adult stage. Adults were weighed within 30 hours of
eclosion and adults were sexed. The biological activity, homogeneity, and stability of the
Cry2Ab2 protein in the diet were confirmed in a separate bioassay using Helicoverpa zea. The
mean survival for C. maculata was 94.7% for the Cry2Ab2 treatment, 88.8% for the buffer
control treatment, 91.6% for the assay control, and 2.08% for the positive control. The mean
percent of larvae that developed to adults was 92.6% in the Cry2Ab2 treatment, 85.3% for the
buffer control treatment, and 90.6% for the assay control. None of the larvae developed to adults
in the positive control treatment. The mean adult weights for the test material and groups were
about 5%> lower buffer control than those of the assay control group, which was a statistically
significant difference. However, there was no significant difference in adult weight of the test
material and buffer control groups.

Conclusions: This study is acceptable. No adverse effects were seen in C. maculata fed 120 |ig
Cry2Ab2 protein/g diet. Although the mean adult weight of the Cry2Ab2 protein treatment
group was statistically significantly lower than that of the assay control group, the difference was
slight (~5%>) and there was no significant difference between the weight of the Cry2Ab2 protein
group and buffer control groups.

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(b) Minute pirate bug
MRID 469514-23

The purpose of this study was to determine the potential dietary effects of Cry 1 A. 105 protein on
mortality and development of Orius insidiosus, the minute pirate bug or insidious flower bug.
The Cry 1 A. 105 protein (E. co/z'-produced) was incorporated into a pollen-based diet for
treatment of the test group. Both a buffer control diet and an assay control diet (pollen diet only)
were included in the study. A positive control group was fed a diet treated with potassium
arsenate. The initial test involved dosing the insects with a single maximum dose level (240 |ig
Cry 1 A. 105/g diet) for 14 days, resulting in 47% survival. The assay and buffer control resulted
in 88% survival. A total of 75 Orius were tested in each treatment. Based on the results of the
maximum hazard dose assay, three 14-day dose-response tests were conducted with test
substance exposure levels of 30, 60, 120 and 240 |ig Cry 1 A. 105/g diet. Again, a buffer control,
assay control, and positive control were included in each of the three tests. Each exposure test
was conducted independently at a different time using separate groups of Orius. During the test,
Orius were supplied with a capsule (50 |iL) of the appropriate diet and the capsules were
replaced every other day. The test arenas consisted of 1-ounce plastic cups with plastic covers
and each cup contained one Orius. For each dose-response exposure, 25 test arenas were
included for each diet treatment. Observations and feeding behavior were recorded each feeding
day for each test arena. Results of the first replicate resulted in percent survival in the 30, 60,
120 and 240 |ig Cry 1 A. 105 protein/g diet was 88, 84, 88 and 56%, respectively. Percent survival
in the assay, buffer, and positive control was 92, 88, and 36%, respectively. The percent of
nymphs developing to adults for the 30, 60, 120 and 240 |ig Cry 1 A. 105 protein/g diet, assay
control, buffer control and potassium arsenate control organisms was 92, 100, 96, 92, 96, 96 and
40%, respectively. No statistically significant differences were detected between the 30, 60, 120
and 240 |ig Cry 1 A. 105 protein/g diet. Percent survival in the second replicate in the 30, 60, 120
and 240 |ig Cry 1 A. 105 protein/g diet was 88, 92, 92, and 52%, respectively. Percent survival in
the assay and buffer controls was 88% and percent survival in the positive control was 32%. The
percent of nymphs developing to adults for the 30, 60, 120 and 240 |ig Cry 1 A. 105 protein/g diet,
assay control, buffer control and positive control organisms was 100, 92, 96, 92, 96, 92 and 96%,
respectively. The mean number of days to development for all treatments was 6.0 days. Percent
survival in the third replicate in the 30, 60, 120 and 240 |ig Cry 1 A. 105 protein/g diet treatments
was 92, 80, 80, and 56%, respectively. Percent survival in the assay, buffer and positive controls
was 92 and 28%, respectively. The percent of nymphs developing to adults for the 30, 60, 120
and 240 |ig Cry 1 A. 105 protein/g diet, assay control, buffer control and positive control
organisms was 100, 96, 100, 92, 100, 100 and 80% respectively. The mean number of days to
development for all treatments was 6.0 days. Throughout the study samples of the test and
control substances were taken to be used in a bioassay with Helicoverpa zea to confirm the
presence of the test substance, homogeneity of the test substance in the diet, diet stability and
bioactivity of the test material. The bioassay confirmed that the test substance was stable
throughout the study, was biologically active at anticipated levels and was appropriately mixed
in the test diet.

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Conclusions: This study is acceptable. Orius insidiosus were exposed for 14 days to a range of
dietary concentrations of Cry 1 A. 105. For the three dose-response replicates the mean survival
for the 240 |ig CrylA.105/g diet treatments was 55%. Therefore, the LC50 value was empirically
determined to be greater than 240 |ig Cry 1 A. 105/g diet. No adverse effects were observed at
concentrations less than or equal to 120 |ig Cry 1 A. 105/g diet.

MRID 469514-24

The purpose of this study was to determine the potential dietary effects of Cry2Ab2 protein on
mortality and development of Orius insidiosus. The Cry2Ab2 protein was incorporated into a
pollen-based diet at a concentration of 100 |ig Cry2Ab2 protein/g diet. The protein was
produced by a recombinant E. coli fermentation system. Both a buffer control treatment and an
assay control (pollen-based diet only) were included in the study. In addition, a positive control
was included which consisted of the pollen-diet treated with potassium arsenate. The duration of
the experiment was 14 days which was long enough to observe the Orius develop from nymph to
adult. The test insects were approximately 3 days old at test initiation. Each treatment contained
seventy-five insects and each insect was contained in its own test arena. During the exposure
period, one capsule of approximately 50 |iL of the appropriate test diet was provided in each test
arena on Day 0 and every other day thereafter for the duration of the test. Observations and
feeding behavior were recorded each day for each test arena. The biological activity,
homogeneity, and stability of Cry2Ab2 protein in the test diet were tested and confirmed in a
separate bioassay using Helicoverpa zea. The percent survival of insects exposed to the 100 |ig
Cry2Ab2 protein/g diet treatment was 91%, which was similar to the percent survival of the
insects in the buffer and assay control groups. The percent of nymphs developing to adults in the
100 |ig Cry2Ab2 protein/g diet treatment, assay control, buffer control and positive control was
93, 95, 91, and 73%, respectively. The mean number of days to develop to adult for insects
exposed to the 100 |ig Cry2Ab2 protein/g diet, assay control, buffer control and positive control
treatments was 6.1, 7.1, 8.0 and 6.0 days, respectively.

Conclusions: This study is acceptable. No adverse effects were observed for Orius insidiosus at
the concentration level of 100 |ig Cry2Ab2 protein/g diet. Therefore, the LC50 is greater than
100 |ig Cry2Ab2 protein/g diet.

This study was conducted to evaluate the potential effects of acute exposure of Cry 1 A. 105
protein to the parasitic wasp, Ichneumon promissorius. The Cry 1 A. 105 protein was
administered to the wasps at a concentration of 240 |ig/mL in a 30% sucrose solution. The
protein was produced by a recombinant E. coli fermentation system. Three control treatments
were included in the experiment: 1) buffer control, 2) assay control (sucrose solution only), and
3) positive control (potassium arsenate). The positive control substance was tested at two
concentrations (100 and 400 ppm). There were three replications per treatment and each
replication contained 10 female wasps. The wasps were 3 to 6 days old at the time of test

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initiation. The test chambers were disposable 64 ounce containers. Observations of mortality
and clinical signs were conducted once within the hour of test initiation and then continued daily
until Day 21 of the test. Samples of the assay control, control substance and protein group diets
were collected for analysis by bioassay to test for bioactivity and stability of the Cry 1 A. 105
protein. Mortality in the assay control, buffer control, and test material treatments was 10%, 8%,
and 7% respectively. All surviving wasps in those groups appeared normal in appearance and
behavior. There was no statistically significant difference in mean mortality in the Cry 1 A. 105
treatment and buffer control treatments. The biological activity and stability of the Cry 1 A. 105
protein was confirmed in a seven-day bioassay using the corn earworm (Helicoverpa zea).

Conclusions: This study is acceptable. The LC50 for Ichneumonpromissorius was greater than
240 |ig Cry 1 A. 105 protein/mL and the NOEC was at least 240 |ig Cry 1 A. 105 protein/mL.

MRID 469514-26

A laboratory bioassay was conducted to evaluate the potential effects of acute exposure to
Cry2Ab2 protein to the parasitic wasp Ichneumon promissorius. The Cry2Ab2 protein used was
produced by an E. coli fermentation system. Wasps were exposed to the Cry2Ab2 at a
concentration of 100 |ig/L in a 30% sucrose solution. Three control groups were utilized,
including: 1) buffer control, 2) negative assay control group (sucrose solution only) and 3) two
positive controls using two concentrations of potassium arsenate. The test diets were prepared
by diluting 60% (w:v) sucrose solution with equal amounts of solutions containing test and
control substances in deionized water to o/^/ain diets with approximately 30% sucrose. Test diet
containing Cry2Ab2 protein was prepared weekly. The wasps were given fresh diet daily. Three
replicate test chambers were used for each treatment and control group and 10 female wasps
were contained in each test chamber. The test chambers consisted of disposable 64 oz.
polypropylene containers. The wasps were approximately 3 to 6 days old at test initiation.
Observations were made once during the hour of test initiation and once daily until Day 21 of the
test. Samples of the assay control, control substance and protein group diets were collected for
analysis by bioassay. The biological activity relative to a reference standard and stability of the
Cry2Ab2 protein in the test diet was confirmed in a seven-day corn earworm (Helicoverpa zea)
bioassay. At test termination (Day 21), mortality in the assay control, buffer substance, and test
substance groups was 10%, 3%, and 3%, respectively. All surviving wasps were normal in
appearance and behavior. No statistically significant differences in mean mortality were found
between the Cry2Ab2 treatment group and the negative control group.

Conclusions: This study is acceptable. The LC50 for Ichneumon promissorius was determined to
be >100 |ig Cry2Ab2 protein/mL and the NOEC was at least 100 |ig Cry2Ab2 protein/mL.

(d) Collembola (MRID 469514-16)

The objective of this study was to determine the potential effect of chronic exposure of
lyophilized corn leaf tissue from MON 89034 maize on the survival and reproduction of
Folsomia Candida. The study sponsor verified the identity and the concentrations of Cry 1 A. 105

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and Cry2Ab2 in the lyophilized leaf material. The concentration of Cry 1 A. 105 was 160 |ig/g
lyophilized leaf dry weight and the concentration of Cry2Ab2 was 140 |ig/g lyophilized leaf dry
weight. The lyophilized test material was incorporated in to a diet containing 50% Brewer's
yeast and 50% test material (0.500g leaf tissue with 0.500 g yeast). Therefore, the test diet
contained Cry 1 A. 105 at a nominal concentration of 80 |ig/g lyophilized leaf dry weight and
Cry2Ab2 at a nominal concentration of 70 |ig/g lyophilized leaf dry weight. A control diet was
prepared by mixing 50% control leaf tissue, by weight, with 50% Brewer's yeast. An additional
control treatment consisted of a test diet containing only Brewer's yeast. Three positive control
treatments were included. The three treatments included three treatments of thiodicarb,
representing nominal concentrations of 1.0, 10 and 100 mg a.i./kg. Collembola were provided
enough food such that an excess was always available. Each treatment contained four replicates
and each replicate initially contained 10 juvenile Collembola (12 days old). Each replicate was
contained in a glass jars containing a water saturated substrate consisting of plaster of Paris and
charcoal at a ration of 8:1 by weight. The biological activity and concentration of Cry 1 A. 105
protein were confirmed in samples collected at the end of dosing. The bioassay confirmed that
the test material was biologically active against CEW and the level of activity was not
significantly different from that of the reference standard. Mortality and observations of
sublethal effects of the surviving Collembola were recorded on day 28 (test termination).
Collembola were removed from the test arenas and the number of adult and young Collembola
were counted. Among the yeast-only diet control organisms, mean survival was 98% and mean
reproduction was 170 offspring per arena. Mean survival of Collembola exposed to the control
diet (control leaf tissue) and MON 89034 diet was 100 and 98%, respectively. The mean number
of offspring produced in the control substance diet (control leaf tissue) and the MON 89034 diet
was 260 and 257 offspring per arena, respectively. Mean survival in the positive control
substance treatments 1.0, 10 and 100 mg thiodicarb/kg diet was 95, 63 and 35% respectively.
The mean number of offspring produced in the 1.0, 10 and 100 mg thiodicarb/kg diet treatments
was 136, 93, and 16 offspring per arena, respectively. Statistical analysis demonstrated no
significant reduction in survival or reproduction among Collembola exposed to the MON 89034
diet when compared to either negative control.

Conclusions: The study is acceptable. The NOEC for Folsomia Candida is at least 80 |ig
Cry 1 A. 105 and 70 |ig Cry2Ab2 per gram of diet.

(e) Honeybee
MRID 469514-19

The objective of this study was to evaluate potential dietary effects of Cry 1 A. 105 protein when
administered to honeybee larvae (Apis mellifera). Honeybees were approximately 2 to 3 days
old during the experiment. The test substance was Cry 1 A. 105 protein produced by an E. coli
fermentation system. The protein was used to prepare a test solution at a concentration of 1200
Hg/mL. This concentration is equivalent to 12 |Lxg total protein per cell. Additional treatment
groups included an assay control, buffer control, and two reference substance concentrations
(potassium arsenate at low and high doses). Each treatment included four replications of 20 bees

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for a total of 80 bees per treatment. The larvae were exposed to a single dose (10 mL) of the
appropriate dosing solution at initiation and observed during larval and pupal development.
Survival of larvae was assessed at study completion (Day 18) by observing adult emergence.
The Cry 1 A. 105 treatment group resulted in 95% survival. Survival in the assay control and
buffer control was 92.5%. Adult emergence in the low and high dose potassium arsenate
treatments was 26.5% and 5.0%, respectively. To verify test concentration and bioactivity of the
Cry 1 A. 105 protein, samples of the test material were taken at test initiation. A bioassay using
Helicoverpa zea was conducted to test for bioactivity and no significant difference was observed
between the test substance and the Cry 1 A. 105 reference standard.

Conclusions: This study is acceptable. The NOEC for CrylA.105 protein to honey bee larvae
was determined to be at least 12 |Lxg Cry 1 A. 105 protein/cell.

MRID 469514-20

An acute bioassay was conducted to determine the effects of Cry 1 A. 105 protein on adult
honeybees (Apis mellifera). The study was initiated a total of five times, with the first four
attempts resulting in early termination (high control mortality). Adult bees were approximately
five days old at the start of the bioassay. The test material was E. co/z'-produced Cry 1 A. 105
protein supplied by the study sponsor. The protein was used to prepare a 30% sucrose solution
containing 550 |ig Cry 1 A. 105 protein/mL. A buffer control diet was prepared by combining the
buffer solution with stock sucrose solution producing a 12.5 mM buffer in a 30% sucrose
solution. An assay control diet was also prepared and consisted of only the 30% sucrose
solution. A positive control diet was prepared and contained 100 |ig/mL potassium arsenate in a
solution of 30%) sucrose. Honeybees were maintained in cages that were approximately 12.7 cm
on each side. To induce clustering, a small cone of beeswax was attached to the cage cover and
extended down into the cage. The diet was provided via an inverted 12 mL glass vial fitted with
a plastic screw cap containing two -1.0 mm holes. Each treatment group included 270 adult
honeybees in six replicates of 45 adult honeybees per replicate. The number of dead bees in each
cage was assessed on a daily basis. The study acceptance criteria stipulated that the assay be
terminated at 30 days or when the adult control mortality reached 30%. The 30% criterion was
met between Day 18 and 19 and the study was terminated on Day 20. On Day 18, the buffer
control treatment produced significantly higher mortality (37.41%) than either the sucrose
(20.00%) or the Cry 1 A. 105 treatments (20.37%). Mortality in the Cry 1 A. 105 treatment was not
statistically different than the sucrose treatment on Day 18. On Day 19, no significant
differences were detected among the three treatment groups (buffer, sucrose, and Cry 1 A. 105
protein) with mortalities of 52.22%, 51.48%), and 47.04%, respectively. The potassium arsenate
treatment resulted in 100% mortality by Day 2 of the study. A diet incorporation assay using the
Cry 1 A. 105 test diet was conducted to confirm the bioactivity of the protein. The biological
activity of the test substance was evaluated using Helicoverpa zea and compared with the
biological activity of a Cry 1 A. 105 reference standard. In addition, the control substance and the
assay control substance were evaluated in the diet incorporation assay. The bioassay confirmed
the test diet contained the expected level of Cry 1 A. 105 activity.

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Conclusions: This study is acceptable. The NOEC for Cry 1 A. 105 protein fed to adult honey
bees is at least 550 |ig/mL.

(f) Earthworm (MRID 46954-18)

The objective of this study was to evaluate the potential effects of acute exposure of Cry 1 A. 105
protein administered to earthworms (Eisenia fetida) during a 14-day exposure period. In the test,
earthworms were exposed to a single concentration of Cry 1 A. 105 protein that was incorporated
into an artificial soil substrate. The Cry 1 A. 105 protein used in this study was E. coli produced.
The concentration of the test substance was 178 mg Cry 1 A. 105 protein/kg soil dry weight. A
total of four control treatments were included in the study, including: 1) buffer solution control,
2) assay control group containing neither test substance or buffer solution, 3) positive control
group exposed to 15 mg chloroacetamide/kg dry soil and 4) additional positive control group
exposed to 30 mg chloroacetamide/kg dry soil. Each treatment was replicated four times and
each replicate contained 10 earthworms. Test chambers consisted of one-liter glass beakers
covered with plastic wrap what was perforated for air exchange. The artificial soil was prepared
in bulk by blending 70% sand, 20% kaolin clay and 10% sphagnum peat. Each test container
contained 750 grams of prepared soil. The worms were not provided food during the test period.
At test initiation (Day 0), the worms were placed on the surface of the soil and observed for 30
minutes to assess burrowing behavior. On Days 7 and 14, the contents of each test chamber
were removed to determine the number of surviving worms. On Day 7, following observations,
the test soil was returned to the test chambers and the worms were placed on the soil surface in
order to observe burrowing behavior. On Day 14, following observations and body weight
determinations, surviving earthworms were euthanized. Samples of soil were collected from
each treatment (except positive controls) and saved to verify the presence or absence of
biological activity. This was done by conducting a bioassay with Helicoverpa zea. The
bioactivity of the Cry 1 A. 105 treated soil was also compared against a reference standard of
Cry 1 A. 105 provided by the study sponsor. There were no mortalities in the assay control group,
buffer control group, or CrylA.105 protein group. In the 15 mg chloroacetamide/kg reference
group there was 48% mortality and in the 30 mg chloroacetamide/kg reference group there was
100%) mortality at test termination. A slight loss in average individual body weight from test
initiation to test termination was noted in all test groups and was expected since the worms were
not fed during the 14-day test. Losses in body weight in the Cry 1 A. 105 protein test substance
group were not statistically significant when compared to the control substance group. Analysis
of the test soil showed that Cry 1 A. 105 was present in the soil and was biologically active against
Helicoverpa zea.

Conclusions: This study is acceptable. The 14-day LC50 for earthworms was determined to be
greater than 178 mg Cry 1 A. 105 protein/kg dry soil. The NOEC was determined to be greater
than 178 mg Cry 1 A. 105 protein/kg dry soil.

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vi. Soil Fate (MRID 469514-28)

Soil organisms may be exposed to Cry 1 A. 105 and Cry2Ab2 protein through contact with corn
plant roots (by direct feeding), corn plant root exudates, incorporation of above-ground plant
tissues into soil following harvest, or by soil-deposited pollen. Some evidence suggests that soils
which are high in clays and humic acids are more likely to bind Cry protein. However, neutral
pH soils (above pH 5.6), that are typical of corn production sites, tend to have high microbial
activity and microbes contribute to Cry protein degradation. Despite evidence that soils high in
clay and humic acids may bind Cry proteins, and thus interfere with the microbial degradation
processes, the weight of evidence indicates that Cry proteins do not accumulate in soil to
arthropod-toxic levels. Nonetheless, the Agency required the following soil fate evaluations to
support the MON 89034 Bt corn registration.

A study of Cry protein degradation in soil evaluated clay, silt loam, and loamy sand soils that
were spiked with Cry2Ab2 (0.60 jug/g) or Cry 1 A. 105 (0.062 jug/g) protein and incubated under
controlled conditions for four months. The soils were dosed with an approximately 500-fold
excess of the maximum calculated protein concentrations in the field. Samples of the treated soils
were collected eleven times during the incubation period and analyzed for protein content using
western blot analysis (Cry2Ab2 only) and a corn earworm bioassay. Results indicated that
Cry2Ab2 protein concentration decreased by 50% in about 1 to 6 days, and by 90% in about 3 to
14 days in the three soils. The amount of Cry 1 A. 105 protein decreased by 50% in about 2 to 7
days and by 90% in about 7 to 19 days in the same soils.

This study utilized field soil spiked with purified insecticidal protein. This approach is useful
because dose responses can be easily quantified. But, the degradation and accumulation of Cry
proteins found within decaying plant tissue may behave differently than proteins in artificially
spiked soil. Thus, the presence of low levels of Cry protein in the soil (at or below the level of
detection) is anticipated until all plant tissue is 'mineralized'. The data reviewed here do,
however, show that Cry proteins will be quickly degraded upon release from decaying plant
tissue. More specifically, a study that evaluated Cryl Ab protein accumulation in a field with
three years of continuous Cryl Ab field corn production showed that the protein had not
accumulated in soil to a level that would elicit a toxic response from ECB larvae, a species that is
highly susceptible to CrylAb protein (Milofsky, 2006).

As a result of FIFRA Scientific Advisory Panel recommendations and public comments, the
Agency has been receiving three year soil fate studies for the currently registered Cry protein
producing crops grown in a variety of soils and environmental conditions. The results of these
studies show that there is no detectable Cry protein accumulation in agricultural soils during
commercial planting of currently registered Cry protein producing crops. Therefore no additional
long term soil degradation studies are required for Cry2Ab2 or Cryl A. 105 proteins.

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vii. Effects on Soil Microorganisms

Numerous published studies indicate that exposure to Cry protein produced in Bt PIP crop plants
does not adversely affect soil microorganisms (Sanvido etal. 2007; Oliveira eial. 2008). In
addition, Bt toxin released from root exudates and biomass of Bt corn has no apparent effect on
earthworms, nematodes, protozoa, bacteria, and fungi in soil (Saxena and Stotzky 2001). Other
research findings conclude no ^-related risks have evolved from the decomposition of Bt- corn
leaves for the meso- and macrofauna soil community (Honemann et al. 2008). Although a
minimal transient increase and shift in microbial populations may result from the presence of
transgenic plant tissue in soil, no adverse effects have been attributed to the Cry protein.

In addition, there are several ongoing U.S. Department of Agriculture and EPA Office of
Research and Development funded research projects evaluating the effects of Bt crops on soil
microbial flora. If adverse effects are seen from this or any other research, the Agency will take
appropriate action to mitigate potential risks.

With regard to the impact of genetically engineered crops on soil, it is important to note that
agricultural practices themselves cause large changes in soil and soil microbial composition.
Furthermore, factors such as variations in seasons and weather, plant growth stage, and plant
varieties, independent of being genetically engineered, are also responsible for significant shifts
in soil microbial communities. To date, most studies with genetically engineered crops have
shown minor or no effects on soil microbes beyond the variation caused by the factors listed
above.

4. Horizontal Transfer of Transgenes from Bt Crops

EPA has evaluated the potential for horizontal gene transfer (HGT) from Bt crops to soil
organisms and has considered possible risk implications if such a transfer were to occur. Genes
that have been engineered into Bt crops are mostly found in, or have their origin in, soil-
inhabiting bacteria. Soil is also the habitat of anthrax, tetanus, and botulinum toxin-producing
bacteria. Transfer of these genes and/or toxins to other microorganisms or plants has not been
detected. Furthermore, several experiments (published in scientific journals), that were
conducted to assess the likelihood of HGT, have been unable to detect gene transfer under
typical environmental conditions. Horizontal gene transfer to soil organisms has only been
detected with very promiscuous microbes under laboratory conditions designed to favor transfer.

As a result of these findings, which suggest that HGT is at most an artificial event, and the fact
that the Bt toxins engineered into Bt Cry 1 A. 105, and Cry2Ab2 Protein in corn are derived from
soil-inhabiting bacteria, EPA has concluded that there is a low probability of risk from HGT of
transgenes found in Bt Cry 1 A. 105, and Cry2Ab2 Protein in corn.

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5. Gene Flow and Weediness Potential

The movement of transgenes from the host plant into weeds has been a significant concern for
the Agency due to the possibility of novel exposures to the pesticidal substance. The Agency has
determined that there is no significant risk of gene capture and expression of Cry34/35Abl
protein by wild or weedy relatives of corn in the U.S., its possessions, and/or its territories. In
addition, the Animal and Plant Health Inspection Service (APHIS) of the United States
Department of Agriculture (USDA) has made this same determination under its statutory
authority under the Plant Pest Act.

Under FIFRA, the Agency has reviewed the potential for gene capture and expression of Bt
endotoxins by wild or weedy relatives of corn, cotton, and potatoes in the U.S., its possessions,
and/or its territories. Bt plant-incorporated protectants that have been registered to date have been
expressed in agronomic plant species that, for the most part, do not have a reasonable possibility
of passing their traits to wild native plants. Feral species related to these crops, as found within
the United States, cannot be pollinated by these crops (corn, potato, and cotton) due to
differences in chromosome number, phenology (i.e., periodicity or timing of events within an
organism's life cycle as related to climate, e.g., flowering time) and habitat. The only exception,
however, is the possibility of gene transfer from Bt cotton to wild or feral cotton relatives in
Hawaii, Florida, and the Caribbean.

The Scientific Advisory Panel meeting held on October 18-20, 2000 further discussed the matter
of gene flow and offered some issues for consideration in this matter. The panel agreed that the
potential for gene transfer between corn (maize) and any receptive plants within the U.S., its
possessions, and/or its territories was of limited probability and nearly risk free.

Concern over the potential for species related to maize (Zea mays ssp. mays), such as Tripsacum
species and the teosintes, as potential recipients of gene flow from genetically modified Zea
mays indicated a need for review of what is known related to gene flow potential of Z. mays.
Some Zea species, such as the teosintes, are known to be interfertile with maize and are
discussed as potential recipients of pollen-directed gene flow from maize. This issue is of
particular concern based upon the increased planting of genetically modified maize. Therefore,
the Agency conducted a reevaluation in early 2000, the results of which are reported here.

a. Zea mays ssp. mays - Maize - General Biology

Zea mays is a wind-pollinated, monoecious, annual species with imperfect flowers. This means
that spatially separate tassels (male flowers) and silks (female flowers) are found on the same
plant, a feature that limits inbreeding. A large variety of types are known to exist (e.g., dent, flint,
flour, pop, sweet) and have been selected for specific seed characteristics through standard
breeding techniques. Maize cultivars and landraces are known to be diploid (2n = 20) and
interfertile to a large degree. However, some evidence for genetic incompatibility exists within
the species (e.g., popcorn x dent crosses; Mexican maize landraces x Chalco teosinte). Zea mays

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has been domesticated for its current use by selection of key agronomic characters, such as a
non-shattering rachis, grain yield, and resistance to pests. The origin of corn is thought to be in
Mexico or Central America, based largely on archaeological evidence of early cob-like maize in
indigenous cultures approximately 7,200 years ago.

A recent study has indicated that cross-pollination of commercial maize cultivars at 100 feet
downwind from the source of genetically modified maize was 1%, and this proportion declined
exponentially to 0.1% at 130 feet and further declined to 0.03% at 160 feet. At 1,000 feet, the
farthest distance measured, no cross-pollination was detected (Jemison and Vayda 2000). For
production of Foundation Seed, a distance of 660 feet has been generally required to mitigate
outcrossing between different genotypes. The relatively large size of corn pollen and its short
viability period under most conditions reduce long distance transfer for purposes of outcrossing
(Schoper, personal communication, 1999). Under conditions of high temperature or low
humidity, corn pollen may only survive for a matter of minutes. Under more favorable conditions
in the field or with controlled handling in the laboratory, pollen life may be extended to several
hours.

b. Triysacum species - Gama Grass - General Biology

Close relatives of corn or maize are found in the genus Tripsacum. Sixteen species of Tripsacum
are known worldwide and generally recognized by taxonomists and agrostologists; most of the
16 different Tripsacum species recognized are native to Mexico, Central America, and South
America, but three occur within the U.S. Hitchcock (1971) reports the presence of three species
of Tripsacum in the continental United States: Tripsacum dactyloides, Tripsacum floridanum,
and Tripsacum lanceolatum. Of these, T. dactyloides, Eastern Gama Grass, is the only species of
widespread occurrence and of any agricultural importance. It is commonly grown as a forage
grass and has been the subject of some agronomic improvement (i.e., selection and classical
breeding). T. floridanum is known from southern Florida, and T. lanceolatum is present in the
Mule Mountains of Arizona and possibly southern New Mexico.

For the species occurring in the United States, T. floridanum has a diploid chromosome number
of 2n = 36 and is native to Southern Florida; T. dactyloides includes 2n = 36 forms, which are
native to the central and western U.S., and 2n = 72 forms, which extend along the Eastern
seaboard and along the Gulf Coast from Florida to Texas but which have also been found in
Illinois and Kansas; these latter forms may represent tetraploids (x = 9 or 18)(Lambert, personal
communication, 1999); and T. lanceolatum (2n = 72), which occurs in the southwestern U.S.
Tripsacum differs from corn in many respects, including chromosome number (T. dactyloides n
= 18; Z. mays n = 10). Many species of Tripsacum can cross with Zea, or at least some
accessions of each species can cross, but only with difficulty and the resulting hybrids are
primarily male and female sterile (Duvick, personal communication, 1999; Galinat 1988; Wilkes
1967). Tripsacum!maize hybrids have not been observed in the field but have been accomplished
in the laboratory using special techniques under highly controlled conditions.

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Eastern Gama Grass is considered by some to be an ancestor of Z. mays or cultivated maize
(Mangelsdorf 1947), while others dispute this (Galinat 1983; litis 1983; Beadle 1980), based
largely on the disparity in chromosome number between the two species (maize n = 10; Gama
Grass x = 9 or 18, with diploid, triploid, and tetraploid races existing; 2n = 36 or 72), as well as
radically different phenotypic appearance. Albeit with some difficulty, hybrids between the two
species have been made (Mangelsdorf and Reeves 1939; DeWald, personal communication,
1999). In most cases, these progeny have been sterile or viable only by culturing with in vitro
"embryo rescue" techniques.

Even though some Tripsacum species occur in areas where maize is cultivated, gene
introgression from maize under natural conditions is highly unlikely, if not impossible (Beadle
1980). Hybrids of Tripsacum species with Z. mays are difficult to n outside of the
controlled conditions of laboratory and greenhouse. Seed obtained from such crosses are often
sterile or progeny have greatly reduced fertility. Approximately 10-20% of maizQ-Tripsacum
hybrids will set seed when backcrossed to maize, and none are able to withstand even the mildest
winters. The only known case of a naturally occurring Zea - Tripsacum hybrid is a species native
to Guatemala known as Tripsacum andersonii. It is 100% male and nearly 99% female sterile
and is thought to have arisen from gene flow to teosinte, but the lineage is uncertain (Doebley,
personal communication, 2000). Z. mays is not known to harbor properties that indicate it has
weedy potential and, other than occasional volunteer plants in the previous season's corn field,
maize is not considered as a weed in the U.S.

In a telephone conversation with Dr. Chester "Chet" DeWald (Agricultural Research Service of
the USD A; Woodward, Oklahoma), a geneticist working on improvement of grasses, he stated
that relatively few accessions of T. dactyloides will cross with maize, and the majority of
progeny are not fertile or viable even in those that do. In controlled crosses, if the female parent
is maize, there is a greater likelihood of obtaining viable seed. When these hybrids have been
backcrossed to maize in attempts to introgress Tripsacum genes for quality enhancement or
disease resistance, the Tripsacum chromosomes are typically lost in successive generations. In
many instances where hybridization has been directed between these two species, the resultant
genome is lacking in most or all of the chromosomal complements of one of the parent species in
subsequent generations.

Only recently has Dr. DeWald (or anyone else) succeeded in obtaining a true Tripsacum
cytoplasm with a maize nuclear background. This was done by using gama grass as the female
parent and maize as the male or pollen donor. Numerous accessions were tested and crosses
made before this came to fruition. The Tripsacum-derived mitochondrial chondrome and
chloroplast plastome in these hybrids contribute to the seed qualities of the plants, but the nuclear
genome appears to be totally maize in origin (DeWald et al. 1999).

Dr. DeWald concluded that the possibility of maize contributing genetic material to Eastern
Gama Grass through random pollen flow in agricultural or natural situations is extremely remote
based upon his experience trying to create hybrids under the best of conditions. He also felt that

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no other known grass species present in the continental U.S. would interbreed with commercial
maize populations (i.e., be recipients of pollen-directed gene flow). This is in agreement with
Holm et al. (1979), who determined that none of the sexually compatible relatives of corn in the
U.S. are considered to be serious, principal, or common weeds in the U.S.

c. Zea species - Teosintes - General Biology

Teosintes—specifically Zea mays ssp. mexicana (Schrader) litis, Zea mays ssp. parviglumis litis
and Doebley, Zea mays ssp. huehuetenangensis (litis and Doebley) Doebley, Zea luxurians
(Durieu and Ascherson) Bird, Zeaperennis (Hitchc.) Reeves and Mangelsdorf, and Zea
diploperennis litis, Doebley and Guzman—have co-existed and co-evolved in close proximity to
maize in the Americas over thousands of years; however, maize and teosinte maintain distinct
genetic constitutions despite sporadic introgression (Doebley 1990).

The teosintes retain a reduced cob-like fruit/inflorescence that shatters more than cultivated
maize but still restricts the movement of seeds as compared to more widely dispersed weedy
species. Hence, the dispersal of large numbers of seeds, as is typical of weeds, is not
characteristic of teosintes or maize. In their native habitat, some teosintes have been observed to
be spread by animals feeding on the plants. Teosintes and teosinte-maize hybrids do not survive
even mild winters and could not propagate in the U.S. Corn Belt. Additionally, some types have
strict day length requirements that preclude flowering within a normal season (i.e., they would be
induced to flower in November or December) and, hence, seed production under our temperate
climate (Beadle 1980; litis, personal communication, 2000; Wilkes, personal communication,
2000; Wilkes 1967).

Since both teosinte and Tripsacum are included in botanical gardens in the U.S., the possibility
exists (although unlikely) that exchange of genes could occur between corn and its wild relatives.
The Agency is not aware, however, of any such case being reported in the United States. Gene
exchange between cultivated corn and transformed corn would be similar to what naturally
occurs at the present time within cultivated corn hybrids and landraces. Plant architecture and
reproductive capacity of the intercrossed plants will be similar to normal corn, and the chance
that a weedy type of corn will result from gene flow with cultivated corn is extremely remote.

Like corn, Z. mays ssp. mexicana (annual teosinte) and Z. diploperennis (diploid perennial
teosinte) have 10 pairs of chromosomes, are wind pollinated, and tend to outcross but are highly
variable species that are often genetically compatible and interfertile with corn, especially when
maize acts as the female parent. Z. perennis (perennial teosinte) has 20 pairs of chromosomes
and forms less stable hybrids with maize (Edwards et al. 1996; Magoja and Pischedda 1994).
Corn and compatible species of teosinte are capable of hybridization when in proximity to each
other. In Mexico and Guatemala, teosintes exist as weeds around the margins of corn fields. The
Fi hybrids have been found to vary in their fertility and vigor. Those that are fertile are capable
of backcrossing to corn. A few isolated populations of annual and perennial teosinte were said to
exist in Florida and Texas, respectively (USDA APHIS 1997). The Florida populations were

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presumably an escape from previous use of Z. mays ssp. mexicana as a forage grass, but local
botanists have not documented any natural populations of this species for approximately twenty-
five years (Bradley, personal communication, 2000; Hall, personal communication, 2000;
Wunderlin, personal communication, 2000).

Consultation with botanists and agronomists familiar with Texas flora suggested that no teosinte
populations exist in the state (Benz, personal communication, 2000; Read, personal
communication, 2000; Orzell, personal communication, 2000; Wilson, personal communication,
2000). Further, given the day length characteristics of Z. diploperennis, it is highly unlikely a
sustaining population would result from introduction of this species. Z. mays ssp. mexicana, Z.
mays ssp. parviglumis, Z. luxurians, and Z. diploperennis may cross with maize to produce
fertile hybrids in many instances (Wilkes 1967). None of these teosinte species have, however,
been shown to be aggressive weeds in their native or introduced habitats (Schoper, personal
communication, 1999). Except for special plantings as noted above, teosinte is not present in the
U.S. or its territories. Its natural distribution is limited to Mexico, Honduras, Nicaragua, El
Salvador, and Guatemala.

Given the cultural and biological relationships of various teosinte species and cultivated maize
over the previous two millennia, it would appear that significant gene exchange has occurred
(based upon morphological characters) between these two groups of plants, and that no weedy
types have successfully evolved as a result. More recent cytogenetic, biochemical, and molecular
analyses have indicated that the degree of gene exchange is far less than previously thought
(Doebley 1984; Doebley etal. 1987; Kato 1997a; Kato 1997b; Smith etal. 1985). Partial and
complete gametophytic incompatibility has been documented among cultivated maize, landraces,
and teosinte (Kermicle 1997). The former is demonstrated by differential pollen growth and a
skewed recovery of alleles linked to incompatibility genes. Complete incompatibility
mechanisms serve to isolate a species or subspecies and are evidenced as pollen exclusion or
non-functioning of pollen types on certain genotypes. Attempts to cross six collections of Z.
mays ssp. mexicana with U.S. maize cultivars (W22, W23) yielded no or few seeds in five of the
six groups (Kermicle and Allen 1990).

Based on the ability of maize to hybridize with some teosintes, the suggestion of previous
genetic exchange amongst these species over centuries, and their general growth habits, any
introgression of genes into wild teosinte from Z. mays is not considered to be a significant
agricultural or environmental risk. The growth habits of teosintes are such that the potential for
serious weedy propagation and development is not biologically plausible in the United States.

d. Conclusion

The potential for pollen-directed gene flow from maize to Eastern Gama Grass is extremely
remote. This is evidenced by the difficulty with which T. dactyloides x Z. mays hybrids are
produced in structured breeding programs. Additionally, the genus does not represent any species
considered as serious or pernicious weeds in the United States or its territories. Any introgression

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of genes into this species as a result of cross fertilization with genetically modified maize is not
expected to result in a species that is weedy or difficult to control. In many instances where
hybridization has been directed between these two species, the resultant genome is lacking in
most or all of the maize chromosomal complement in subsequent generations.

Many of the Zea species loosely referred to as "teosintes" will produce viable offspring when
crossed with Z. mays ssp. mays. None of these plants are known to harbor weedy characteristics
and none of the native teosinte species, subspecies, or races are considered to be aggressive
weeds in their native or introduced habitats. In fact, many are on the brink of extinction where
they are indigenous and will be lost without human intervention (i.e., conservation measures).
Further, none of the landraces or cultivated lines of Z. mays are considered to have weedy
potential and are generally considered to be incapable of survival in the wild as a result of
breeding practices (i.e., selection) during domestication of the crop.

6. Impacts on Endangered Species

The primary route of exposure to Cry 1 A. 105 and Cry2Ab2 proteins in corn is through ingestion
of corn tissue. There are no reports of threatened or endangered species feeding on corn plants,
therefore such species would not be exposed to corn tissue containing Cry protein. Since
Cry 1 A. 105 and Cry2Ab2 proteins have not been shown to have toxic effects on mammals, birds,
plants, aquatic species, insects outside the order Lepidoptera and other invertebrate species at the
Estimated Environmental Concentration (EEC), a "may affect" situation for endangered land and
aquatic species is not anticipated. In addition, EPA does not expect that any threatened or
endangered plant species will be affected by outcrossing to wild relatives or by competition with
such entities. Hybrid corn does not exist in the wild, nor are there wild plants that can interbreed
with corn in the United States.

Because of the selectivity of Cry 1 A. 105 and Cry2Ab2 proteins for lepidopteran species,
endangered species concerns are mainly restricted to the order Lepidoptera. Examination of an
overlay map showing the county level distribution of endangered/threatened lepidopteran species
(currently listed by the U.S. Fish and Wildlife Service) relative to corn production counties in the
United States clearly indicated that any potential concern regarding range overlap with corn
production was mainly restricted to the Karner blue butterfly (Lycaeides melissa samuelis).
Research demonstrates that the Cry 1 A. 105 and Cry2Ab2 proteins are selectively toxic to
lepidopteran larvae at field concentrations and that the Karner Blue butterfly is the only
endangered lepidopteran species that may be exposed to MON 89034 (via pollen). A model
developed to assess the risk of Bt corn to Monarch butterfly larvae was used to assess the risk of
MON 89034 to Karner blue larvae. Based on the LC50 value for larvae of the most sensitive
known lepidopteran species (ECB) and the maximum estimated level of Cry protein in pollen-
contaminated food (9.27 |ig/g fresh weight), the margin of safety was calculated to be >10X
maximum estimated exposure of Karner blue larvae to corn pollen. These results indicate that
cultivation of MON 89034 is not likely to pose a risk to endangered species.

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After careful review of available data, EPA determined that exposure of the Karner blue butterfly
to harmful levels of MON 89034 corn plant tissues is not expected. Likewise, a review of the
preferred habitats of other lepidopteran species listed as endangered by the U.S. Fish and
Wildlife Service indicated that exposure to harmful levels of Cry 1 A. 105 or Cry2Ab2 protein
would not take place. The main reasons for the lack of exposure are geographical and habitat
limitations. These species are located in non-corn production areas and/or their habitat does not
encompass agricultural areas.

Likewise, other insect species in the orders Diptera, Hemiptera, Lepidoptera, Odonata and
Orthoptera that are listed as endangered/threatened species are found in dune, meadow/prairie or
open forest habitats and are not closely associated with row crop production, often times due to
the specificity of the habitat of their host plants. The reviewed toxicological data shows the
relative insensitivity of a range of insects in non-1 epidopteran orders to the Cry 1 A. 105 and
Cry2Ab2 proteins, indicating that MON 89034 maize hybrids are not likely to have detrimental
effects on non-1 epidopteran insects included on the endangered/threatened species list.

In light of the above considerations (based on no spatial and temporal overlap), the Agency has
determined that registered uses of MON 89034 corn will have No Effects (NE), direct or
indirect, on endangered and threatened species or their habitat as listed by the United States Fish
and Wildlife Service (USFWS) and the National Marine Fisheries Services (NMFS), including
mammals, birds or terrestrial and aquatic plants and invertebrate species. Therefore, no
consultation with the USFWS is required under the Endangered Species Act.

Current ecological effects data and EPA reviews of Bt Cry 1 A. 105, and Cry2Ab2 Protein in corn
support the Agency's determination that adverse effects will not occur to nontarget organisms.
Due to a demonstrated lack of toxicity and/or exposure, no effects from Bt Cry 1 A. 105, and
Cry2Ab2 Protein in corn are anticipated for any nontarget species, including federally-listed
threatened and endangered ("listed") lepidopteran and coleopteran species and their designated
critical habitats. EPA has also determined that there are no indirect effects on endangered and
threatened plant species, such as impacts on lepidopteran pollinators that are important and/or
essential to an endangered or threatened plant. The Agency is therefore upholding its
determination that the registered uses of Bt Cry 1 A. 105, and Cry2Ab2 Protein in corn will have
"No Effect," direct or indirect, on endangered or threatened terrestrial or aquatic species as listed
by the U.S. Fish and Wildlife Service (USFWS) and the National Marine Fisheries Services
(NMFS).

7. MON 89034 Corn Environmental Risk Assessment Conclusions

The EPA uses a Maximum Hazard Dose Tiered system for biopesticide non-target wildlife
hazard assessment. When no adverse effects at the maximum hazard screening dose are
observed on representative non-target species, the Agency concludes that there are no
unreasonable adverse effects on non-target populations from the use of the pesticide.

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a) Direct Effects

At present, the Agency is aware of no identified significant adverse effects of Cry protein on the
abundance of non-target organisms in any population in the aquatic or terrestrial field
environment, whether they are animals, plants, pest parasites, pest predators, or pollinators.
Further, EPA believes that cultivation of MON 89034 corn may have fewer adverse impacts on
non-target organisms than use of chemical pesticides for corn production, because under normal
circumstances, MON 89034 corn requires substantially fewer applications of chemical
pesticides, compared to production of non-Bt corn. Fewer chemical insecticide applications
generally result in increased populations of beneficial organisms that control secondary pests,
such as aphids and leafhoppers. In addition, no adverse effect on Federally listed endangered
and threatened species is expected from the proposed lepidopteran-resistant corn registration.
Further, EPA has determined that there is no significant risk of gene capture and expression of
Cry 1 A. 105 or Cry2Ab2 proteins by wild or weedy relatives of corn in the U.S., its possessions,
or territories (see Section 5. "Gene Flow and Weediness Potential" above), available data do not
indicate that Cry proteins have any measurable adverse effect on microbial populations in the
soil (see Section vii. "Effects on Soil Microorganisms" above), nor has horizontal transfer of
genes from transgenic plants to soil bacteria been demonstrated (see Section 4. "Horizontal
Transfer of Transgenes from Bt Crops" above). In conclusion, this risk assessment finds no
hazard to the environment at the present time from cultivation of MON 89034 corn for a time-
limited registration.

b) Indirect Effects:

The purpose of using PIP plants is the same as for any other pest management tactic, i.e., to
reduce pest populations below economic injury levels. As a result the abundance of pest insects
should be significantly reduced and this will have corresponding implications for those
organisms that exploit these pests as prey and hosts. Thus, the potential for these indirect
ecological effects on biological control organisms should not be regarded as a unique ecological
risk associated with the PIP crop. Some reductions, however, should be expected if the pest
management strategy is effective. Since PIP crops are often grown in vicinity with conventional
crops to prevent resistance build-up by the target pest(s), specialist antagonists can persist in
these 'refuges', in other crops and in non-crop habitats and retain the potential for recolonization
of the PIP crop area. Based on these considerations, regulatory testing of the specialist predators
and parasitoids of target pests may eventually be considered unnecessary.

c) Supplemental Data Needed to Confirm MON 89034 Non-Target Hazard Assessment

The Agency has sufficient information to believe that there is no risk from the proposed uses of
MON 89034 corn to non-target terrestrial wildlife, aquatic, and soil organisms. The Agency has
been frequently asking the registrants to conduct post-registration long term invertebrate
population/community and Cry protein accumulation in soils studies as a condition of

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registration. The issue of long range effects of cultivation of these Cry proteins on the
invertebrate community structure in corn fields has since been adequately addressed by the
analysis of field studies performed during the last 10 years (Marvier, et al. 2007; Sanvido, et al.
2007). No unexpected adverse effects on invertebrate community structure were reported. The
Agency is in agreement with these conclusions. Similarly, no unexpected accumulation of Cry
proteins in agricultural soils was seen in published studies (Icoz and Stotzky 2007; Sanvido, et
al. 2007) and in numerous studies submitted directly to the EPA for the currently registered Cry
proteins. (Milofsky, 2006; See Section vi. "Soil Fate" above).

In light of published laboratory studies showing reduced growth in shredding caddis flies
exposed to anti-lepidopteran Cryl A protein corn litter (Rosi-Marshall, et al. 2007), additional
aquatic invertebrate data were required when these products were initially registered The
submitted study (MRID 478388-01) satisfies this requirement. As discussed earlier, no
unreasonable adverse effects to aquatic invertebrates are expected from exposure to MON 89034
corn.

8. Potential Interaction Between CrylA.105, Cry2Ab2 and Cry3Bbl Proteins
(MRID 469513-05 & 469513-06)

The purpose of these studies was to characterize the potential for interaction between the
lepidopteran-active proteins Cryl A. 105 and Cry2Ab2 and the coleopteran-active protein
Cry3Bbl. The Cryl A. 105 and Cry2Ab2 proteins were tested alone and in combination with
either the Cry3Bbl protein against European corn borer (ECB, Ostrinia nubilalis) and corn ear
worm (CEW, Helicoverpa zea) in diet incorporation studies. Also, the Cry3Bbl protein was
tested alone and with the Cryl A. 105 and/or the Cry2Ab2 proteins, against the Colorado potato
beetle (CPB, Leptinotarsa decemlineata). The activity of Cry 1 A. 105 and Cry2Ab2 proteins
was not significantly altered by the presence of Cry3Bbl, and the activity of Cry3Bbl was not
significantly altered by the presence of Cryl A. 105 and/or Cry2Ab2. Collectively these data
provide evidence that the proteins do not interact in an antagonistic or synergistic manner. This
study, along with the interaction study between Cry 1 A. 105 and Cry2Ab2 reviewed for the
MON 89034 Experimental Use Permit indicate that MON 89034 x MON 88017 maize will not
result in any unexpected interaction in an antagonistic, or synergistic manner with regards to
target insects. It is therefore extremely unlikely that the CrylA.105, Cry2Ab2 and Cry3Bbl
proteins contained in a single plant will impart any hazard to non-target organisms exposed to
these hybrids in the environment.

E. INSECT RESISTANCE MANAGEMENT (IRM)

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1. IRM Assessment for the Initial Registration of MON 89034 and MON 89034 x MON
88017

This section presents the original assessment for MON 89034 (based on BPPD's review — see
BPPD, 2007). Subsequent to registration, Monsanto submitted an amendment request with
additional IRM data to support a 5% lepidopteran refuge. This amendment and BPPD's
assessment are detailed in section 2 of the Insect Resistance Management Assessment. Please
note that the assessment in section 1 contains some IRM program elements that have been
superseded by the amendment discussed in section 2.

Monsanto developed through the use of genetic engineering, MON 89034, a corn product that
produces the Bacillus thuringiensis (7?/)-derived insecticidal proteins Cry 1 A. 105 and Cry2Ab2.
The Cry 1 A. 105 toxin is a "chimeric" protein containing domains I and II and the C-terminal
from CrylAc and domain III from CrylFa (domain III). The Cry2Ab2 protein is exactly the
same as that currently expressed in Monsanto's Bollgard II cotton. MON 89034 is protected
from damage caused by larval feeding of Ostrinia nubilalis (European corn borer; ECB),
Diatraea grandiosella (southwestern corn borer; SWCB) and Diatraea saccharalis (sugarcane
borer; SCB), Spodopterafrugiperda (fall armyworm; FAW), and Helicoverpa zea (corn
earworm; CEW). Monsanto presented data to support its proposed IRM plan for MON 89034.
Monsanto wished to demonstrate that: (1) resistance to Cry 1 A. 105 and Cry2Ab2 proteins in
MON 89034 is expected to be at least partially recessive; (2) the probability of cross-resistance
between Cry 1 A. 105 and Cry2Ab2 is low; and (3) the level of both Cry 1 A. 105 and Cry2Ab2
produced in MON 89034 confer high level of control of susceptible target pests {in vitro and in
planta). Monsanto originally proposed a 5% structured refuge in the U.S. Corn Belt (currently,
a 20% structured refuge) and a 20% structured refuge in cotton growing regions (currently, a
50% structured refuge) to mitigate insect resistance to the Cry 1 A. 105 and Cry2Ab2 proteins.
Simulation modeling was provided to support this plan. BPPD's technical analysis of
Monsanto's proposed IRM plan for MON 89034 is described below.

a) Assessment of the Probability of Cross-Resistance to the CrylA.105 and Cry2Ab2
Proteins

The Cry 1 A. 105 toxin is a "chimeric" protein containing domains I and II and the C-terminal
from CrylAc and domain III from CrylFa (domain III). The Cry2Ab2 protein is exactly the
same as that currently expressed in Monsanto's Bollgard II cotton. There are a number of Bt
corn products on the market that produce the insecticidal proteins, CrylAb and CrylFa
(potential cross resistance with CrylA.105/Cry2Ab2 and CrylAb is discussed in section c,
"Impact of Prior Use of Cry 1 Ab-Expressing Bt Corn Products on MON 89034"). There are
also Bt cotton products that produce the CrylAc, Cry IF, and Cry2Ab2 insecticidal proteins.
Mathematical models indicate that the IRM values of a Bt corn product with two insecticidal
proteins, like MON 89034, would be the greatest if there is a low probability of cross-
resistance (See Roush 1998). Cross-resistance is most likely when proteins share key

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structural features, which allows one resistance mechanism to confer resistance to more than
one protein (Tabashnik, 1994; Gould et al., 1995).

There are three models that have been proposed to explain the mode of action of Cryl A toxin
mode of action (see discussion in Piggott and Ellar, 2007). The most accepted Bravo model
proposes that both the cadherin and aminopeptidase (APN) receptors are required for full
Cryl A toxicity. This model suggests that receptor binding is sequential: 1) ingestion of the
protein inclusions by a susceptible insect larva, 2) solubilization of the protein in the insect
midgut, 3) cleavage of the protoxin by host proteases and release of the active toxin, 4)
binding of the active toxin to specific receptors on the midgut epithelieum, 5)
oligomerization of toxin subunits to form pore structures that inject into the membrane, 6)
passage of ions and water through the pores, resulting in swelling, lysis, and the eventual
death of the host. Differences in any of these steps will reduce the probability of cross-
resistance between any two Cry proteins. The more controversial Zhang model suggests that
receptor binding activates an Mg+-dependent signaling cascade that promotes cell death. The
Jurat-Fuentes model suggests that cytotoxicity is due to the combined effects of osmotic lysis
and cell signaling. The latter two models are, at present, more speculative.

Resistance associated with modification of the binding site receptor has been the primary Bt
resistance mechanism reported to date (reviewed in Ferre & Van Rie 2002). Other Bt
resistance mechanisms have been reported that are based on alterations in the proteases that
cleave the protoxin, processing it into a smaller active toxin (Candas et al. 2003) and most
recently, the discovery that esterases can bind and detoxify Bt toxins (Gunning et al. 2005).
Only the binding reduction mechanism has a demonstrated causal link between the
biochemical modification and resistance (Ferre and Van Rie 2002). Ferre and Van Rie
(2002) indicate that in all cases of binding site modification, resistance is due to a recessive
or partially recessive mutation in a major autosomal gene, and cross-resistance extends only
to Cry proteins sharing binding sites. Cry proteins that do not share high levels of sequence
similarity tend to have different binding sites and different modes of action. Analyses of
resistance to Bt Cry proteins indicate that cross-resistance occurs most often with proteins
that are similar in structure (Tabashnik, 1994; Gould et al., 1995).

With this information in mind, Monsanto has assessed the probability of cross-resistance
between Cry 1 A. 105 and Cry2Ab2 on three levels: 1) structural similarity between the
proteins, which is indicative of mode of action; 2) characterization of elements of the mode
of action, such as the biophysical nature of binding of the Bt proteins to the target insect
midgut; and 3) demonstration that the individual proteins are effective in controlling
resistance to the other protein. Results of these efforts are discussed below.

The first piece of the analysis relates to whether the Cry 1 A. 105 protein has high sequence
similarity with the Cry2Ab2 protein. Monsanto provided BPPD with a summary of current
information about the structural and functional similarities of the Cry 1 A. 105 protein to other
Bt Cryl proteins. The Cry 1 A. 105 protein is a chimeric protein with overall amino acid

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sequence identity to the CrylAc, CrylAb and CrylFa proteins of 93.6, 90.0 and 76.7%,
respectively. The Cry 1 A. 105 protein expressed in MON 89034 corn plants results in
increased activity against FAW, SCB, and CEW compared to CrylAb expressed in MON
810 corn plants (see BPPD review of efficacy data, Matten, 2007; Monsanto study MRID#
46951415). A structural model of the CrylA.105 protein was developed using the X-ray
crystal structure of the CrylAa protein. This model demonstrated high overall main chain
structural similarity with CrylAa. Models of CrylAb and CrylAc were also prepared using
the Cry 1 A. 105 model. Comparison of the aligned folds of all three proteins showed that
CrylAb and Cry 1 A. 105 have essentially the same main chain structure (i.e., similar three
domain structures) and that CrylAc differs slightly in its main chain structure from the other
two in domain III. Thus, comparison of the modeled crystal structures of the Cry 1 A. 105,
CrylAb, and CrylAc with the experimental CrylAa X-ray crystal structure demonstrated
high three-dimensional structural similarity between the four proteins (i.e., Cry 1 A. 105,
CrylAb, CrylAc, and CrylFa).

In the case of Cry 1 A. 105 and Cry2Ab2 proteins, however, there is only a 14% amino acid
sequence similarity. Based on the available data, Monsanto has sufficiently demonstrated
that there is low sequence similarity between the Cry 1 A. 105 protein and the Cry2Ab2
protein. Lack of sequence similarity would suggest that cross-resistance between the
Cry 1 A. 105 and Cry2Ab2 proteins would be unlikely. On the other hand, high sequence
similarity between the CrylA.105 protein and CrylAa, CrylAb, CrylAc, and CrylFa
proteins is one indicator that cross-resistance may be a concern for these proteins. This is
important because CrylA.105 is composed of domains I and II and the C-terminus of
CrylAc and domain III of CrylFa. This subject will be discussed further in the review.

Previous studies have shown that Cryl A proteins are activated by proteolytic cleavage of the
C-terminal domain and the N-terminus of domain I in the insect gut. In contrast, Cry 2A
proteins are activated by cleavage of the N-terminus of domain I and the C-terminal part of
domain III. Different activation mechanisms would tend to decrease the likelihood of cross-
resistance between the CrylA and Cry2A proteins.

Assessment of binding characteristics is one way of determining the potential for cross-
resistance between the two proteins. As noted above, changes in the nature of protein
binding to the insect midgut is the mode of action step that has most often been associated
with insect resistance to Bt Cry proteins (for reviews, see Tabashnik, 1994; Baxter et al.,
2005). Biacore is used to quantify the interaction kinetics of Bt proteins with the insect brush
border membranes (BBM). Competitive and non-competitive binding may not always be
distinguished by Biacore and other analyses, such as ligand blotting, may be used. Ligand
blotting is a qualitative tool used to identify protein bands that have the specific secondary
modification to bind Bt proteins. Monsanto used both Biacore and ligand blotting to
characterize Cry 1 A. 105 and Cry2Ab2 binding to ECB brush border membranes (studies by

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Li and Guzov, 2006 were provided in Appendix 1 of Monsanto submission, MRID# 469514-
30, Head, 2006 and are discussed below).

Binding constants for the interaction of Cry 1 A. 105 and Cry2Ab2 with immobilized BBMV
(ECB) differed by more than an order of magnitude with essentially no BBMV-specific
binding being observable for Cry2Ab2. The Biacore system could not distinguish unique
aspects of non-competitive binding for Cry 1 A. 105 and Cry2Ab2 on BBMV. This result
suggests that there are very different binding sites for Cry 1 A. 105 and Cry2Ab2 in the ECB
midgut. Additional Biacore analyses indicated that Cry 1 A. 105 and Cry2Ab2 bound to
different glycosyl moieties linked to bovine serum albumin (BSA). Cry 1 A. 105 preferentially
bound to galactosamine (KD=1.5xl0"8M and Rmax=2419 RU). Cry2Ab2 preferentially bound
to N-acetyl glucosamine (KD=7.0xl0"nM and Rmax=32 RU), but also bound galactosamine
with a Kd =2.0 xlO"8M and Rmax=625 RU. Furthermore, Cry 1 A. 105 binding to
galactosamine filled a two-binding-site model as evidenced by the reduction in the Chi2 value
from 2583 to 53, but the fit of Cry2Ab2 binding was similar for both models suggesting that
the Cry2Ab2 and Cry 1 A. 105 proteins not only bind to different sugars but also differ in their
binding kinetics.

The ligand blotting analysis demonstrated that the Cry 1 A. 105 and Cry2Ab2 proteins bound
to different components on ECB brush border membrane filaments (BBMF) separated by
SDS-Page and immobilized on a nitrocellulose membrane. Trypsin-treated Cry 1 A. 105
protein was shown to bind to a -150 kDa band while the trypsin-treated Cry2Ab2 protein
was shown to a -130 kDa band, but weakly to a -150 kDa band. The trypsin-treated
Cry2Ab2 protein had a greater rate of binding than the Cry 1 A. 105 protein. Overall these
results support the conclusion, as Monsanto has described, that the Cry2Ab2 and Cry 1 A. 105
proteins displayed different binding components and different kinetics in binding to ECB
BBMF. These results are consistent with the differences in binding affinity for Cry 1 A. 105
and Cry2Ab2 proteins observed with Biacore. In addition, Monsanto noted that Cry2Aa did
not bind to a specific, high affinity Cryl Ac receptor in work performed by English et al.
(1994).

In conclusion, Biacore and ligand blotting analyses demonstrate that Cryl A. 105 and
Cry2Ab2 proteins bind to some unique components on ECB brush border membranes. They
also share many common binding sites. Screening a limited number of glycosylated BSAs,
indicated that galactosamine is recognized by Cryl A. 105 only, while Cry2Ab2 demonstrated
a high affinity for both N-acetylglucosamine and galactosamine. These data support the
conclusion that Bt protein binding to carbohydrate moieties is the principal basis of the
specific interactions between the Cryl A. 105 and Cry2Ab2 proteins and the ECB brush
border membrane. Specific binding of Bt proteins to the target insect gut membrane is a key
step in their mode of action. Differences in the Cryl A. 105 and Cry2Ab2 protein interactions
with the BBM suggest that these two proteins have differences in mode of action. BPPD

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agrees with Monsanto that these differences should minimize the development of cross-
resistance by the target insect pests to these two proteins.

Monsanto also provided evidence to show that there is a lack of cross-reactivity between
Cry 1 A. 105 and Cry2Ab2 antibodies. The homologous primary-secondary antibody pairs
recognized only their corresponding antigens (i.e., trypsin-treated Cry 1 A. 105 or Cry2Ab2)
with no cross-reactivity. Similarly, Monsanto previously demonstrated that anti-Cry2Ab
antibodies do not cross-react with the Cry 1 Ac proteins, nor do the anti-Cry 1 Ac antibodies
cross-react with the Cry2Ab2 protein (Head and Reding 2001, MRID# 455457-01). The lack
of cross-reactivity shows that the epitope binding sites for antibody recognition are different
and therefore the tertiary structure is different. Lack of similar tertiary structure supports the
conclusion that there will be a very low likelihood of high levels of cross-resistance in the
target insect pests for the Cry 1 A. 105 (and all Cry 1A proteins) and Cry2Ab proteins.
Monsanto provided indirect information (i.e., there are no colonies of lepidopteran corn pests
resistant to either Cry 1 A. 105 or Cry2Ab2 proteins) to indicate that insects resistant to one of
the two insecticidal proteins, Cry 1 A. 105 or Cry2Ab2, will be controlled by the other
insecticidal protein. First, Monsanto cited to studies provided in support of the Bollgard II
cotton registration (i.e, Cry2Ab2 and Cryl Ac Bt plant-incorporated protectants as expressed
in cotton) that indicated that Cryl Ac-resistance did not confer Cry2Ab2 resistance to tobacco
budworm, cotton bollworm, and pink bollworm (Head and Reding 2001; EPA 2007). In
addition, Monsanto shared information that a Cry2Ab2-resistant colony (called SP15) of
Helicoverpa armigera (Dr. Rod Mahon, CSIRO, Australia) showed little or no cross-
resistance to Cryl Ac and the microbial insecticide, DiPel®, that contains the Cryl Ab,
CrylAc, and Cry2Aa proteins. Monsanto tested this Cry2Ab2-(SP15) resistant colony
against purified Cryl A. 105, CrylAc, and Cry2Ab2 protein relative to a susceptible
laboratory colony of H. armigera. The SP15 colony was found to be highly resistant to the
Cry2Ab2 protein, but showed little or no cross-resistance to the CrylAc and Cryl A. 105
proteins. Other published research indicates that there is evidence for broad cross-resistance
(low levels of resistance) to CrylA and Cry2A proteins in laboratory-selected strains of beet
armyworm (Moar et al. 1995) and tobacco budworm (Gould, et al., 1992). Collectively,
results of resistant colony studies indicate that there is some low potential for cross-
resistance, but that high levels of cross-resistance to CrylA. 105 and Cry2Ab2 is unlikely. In
the field, this would translate to the efficacy of MON 89034 being maintained even though
resistance might occur to one of the proteins.

b) Dose

The determination of dose, or the amount of toxin expressed by the transgenic crop relative
to the susceptibility of the target pests, is a critical component of IRM. Models have shown
that a high dose of toxin, coupled with a non-transgenic refuge to provide a supply of
susceptible insects, is the most effective strategy for delaying resistance in Bt crops. The
high dose/refuge strategy assumes that resistance to Bt is recessive and is conferred by a

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single locus with two alleles resulting in three genotypes: susceptible homozygotes (SS),
heterozygotes (RS), and resistant homozygotes (RR). It also assumes that there will be a low
initial resistance allele frequency and that there will be extensive random mating between
resistant and susceptible adults. In practice, a high dose PIP should express sufficient
quantities of toxin to kill all susceptible insects (SS) as well as heterozygous insects with one
resistance allele (RS). Lower dose PIPs might allow for survival of insects with at least one
susceptibility allele (SS or RS), although effective IRM may still be possible with a suitable
refuge strategy.

The 1998 Science Advisory Panel (SAP) defined high dose as a level of toxin 25 times
greater than is needed to kill all susceptible insects. The SAP also outlined five techniques to
determine high dose: 1) Serial dilution bioassay with artificial diet containing lyophilized
tissues of Bt plants using tissues from non-Bt plants as controls; 2) Bioassays using plant
lines with expression levels approximately 25-fold lower than the commercial cultivar
determined by quantitative ELISA or some more reliable technique; 3) Survey large numbers
of commercial plants in the field to make sure that the cultivar is at the LD99.9 or higher to
assure that 95% of heterozygotes would be killed (see Andow & Hutchison 1998); 4)

Similar to #3 above, but would use controlled infestation with a laboratory strain of the pest
that had an LD50 value similar to field strains; and 5) Determine if a later larval instar of the
targeted pest could be found with an LD50 that was about 25-fold higher than that of the
neonate larvae. If so, the later stage could be tested on the Bt crop plants to determine if 95%
or more of the later stage larvae were killed.

It must be noted that both the high dose definition and verification techniques were
developed in 1998 when all of the registered Bt crops were single toxin products targeted
against lepidopteran pests. In recent years, PIPs (in Bt cotton) have been approved that
contain two genes targeted at the same insect pest. These "pyramided" products can be
beneficial for IRM, since target pests must overcome two toxins to develop field resistance to
the PIP. The benefits are greatest for two toxins with unrelated modes of action (i.e. binding
to different Bt receptor sites in the midgut) that are expressed at high doses in the plant
(Roush 1994).

For pyramided products, the dose of each toxin should be evaluated separately. This can be
easily accomplished if the pyramided product is created through conventional breeding — in
this case, the dose of the single toxin products has already been established and the combined
dose in the pyramided PIP can be determined with comparative efficacy studies. But, for
pyramids created by non-conventional breeding (e.g. recombinant DNA techniques), defining
the dose can be more complicated since single toxin lines may not be available (or
commercialized) for comparisons. The dual toxins can also be evaluated collectively to
determine an "effective" high dose. In some examples, each toxin by itself may not supply a
high dose, but in combination a sufficient control (>95% of heterozygotes) is provided to be
considered high dose.

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MON 89034 was created with recombinant DNA technology (and not conventional breeding)
to express the Cry 1 A. 105 and Cry2Ab2 toxins. Both of the toxins are located on the same
plasmid in the MON 89034 plant genome. Because of this, there are no originating single
gene lines (i.e., expressing Cry 1 A. 105 or Cry2Ab2 only) for dose comparisons, although
single gene events were separately engineered. The Cry 1 A. 105 toxin is a "chimeric" protein
containing domains I and II and the C-terminal from CrylAc and domain III from CrylFa
(domain III). By creating this chimera, Monsanto hoped to improve efficacy against several
target pests including fall armyworm and corn earworm. The Cry2Ab2 protein is exactly the
same as that currently expressed in Monsanto's Bollgard II cotton.

To evaluate dose, Monsanto conducted a number of laboratory and field studies with diet
bioassays and MON 89034 plant material. Three sets of experiments were conducted: 1)
bioassays with purified toxin incorporated into artificial diet to determine pest susceptibility,
2) leaf disk or kernel testing conducted in the laboratory, and 3) field tests with whole plants
(artificial infestation of small corn plots) compiled over a several year period. Four target
pests were evaluated including European corn borer (ECB), southwestern corn borer
(SWCB), fall armyworm (FAW), and corn earworm (CEW). A description of the test
procedures is included in Monsanto's submission (Head 2006; MRID# 469514-30). Toxin
expression data was also obtained from MON 89034 leaf tissue and other tested lines.

Laboratory bioassays (Head 2006; MRID# 469514-30, section 2.2.1) were conducted using
purified protein in diet to determine susceptibility (molting inhibitory concentration, MIC90)
to the MON 89034 toxins. Molting inhibition is often used instead of straight mortality (i.e.
an LC50 or LC90) because it can be assumed that insects that fail to develop as larvae will be
functionally dead in the field. A MIC90 bioassay can also reduce the amount of purified toxin
needed for the testing relative to an LC90 determination, though it is unclear whether
Monsanto had insufficient purified protein to determine LC90 values. The MIC90 tests
showed that all four target species were more susceptible to Cry 1 A. 105 than Cry2Ab2 (as
measured in ppm). ECB was more sensitive to both Cry 1 A. 105 and Cry2Ab2 than the other
tested lepidoptera by at least an order of magnitude. For Cry 1 A. 105, BPPD agrees with
Monsanto that the amount expressed in plant leaf tissue is high relative to the susceptibility
of the target insects. Toxin levels in leaf tissue measured throughout the growing season (V2
- Pre-VT) exceeded the MIC90 for all four species (both measured in ppm). On the other
hand, the amount of Cry2A2b expressed in MON 89034 exceeded the MIC90 value only for
ECB. For the other three pests, the level of Cry2Ab2 was at (for SWCB) or below (CEW
and FAW) the MIC90 level. These data suggest that the CrylA.105 component of MON
89034 may be expressed at a sufficient level for all four pests to be considered "high dose"
while the Cry2Ab2 expression is less certain. However, BPPD concurs with Monsanto's
contention that the results of laboratory bioassays are difficult to correlate with natural field
systems and larval survival on plant tissue is more challenging than on artificial diet.

Unlike the artificial diet bioassays, the tests with plant material (leaf disks and whole plant)
directly assessed the performance of MON 89034 against the target pests. Since MON

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89034 expresses both Cry 1 A. 105 and Cry2Ab2 simultaneously, the tests with MON 89034
plant material evaluate the "effective dose" of both toxins together. However, Monsanto was
also able to include single gene lines producing either Cry 1 A. 105 or Cry2Ab2, though none
of these were ultimately commercialized or used to create MON 89034. To relate the single
gene isolines to MON 89034, Monsanto supplied some plant expression data for the isolines
which could be compared to the known toxin expression of the stacked product. For the leaf
disk/kernel tests, two Cry 1 A. 105 isolines were used; one (LAJ138) with toxin expression
equivalent to that of MON 89034 and another (LAJ129) with less than half the expression.
Both of the Cry2Ab2 lines that were used (70774 and 67620) had less toxin expression than
in MON 89034. Other single gene lines were used for the field tests, although no expression
data were reported for those hybrids.

The results of the leaf disk tests generally supported the conclusions derived from the
susceptibility diet bioassays (i.e., high efficacy against the target pests). Two-toxin MON
89034 was highly effective against all four target pests with at least 90% mortality among
exposed larvae and significant growth inhibition in the survivors. On the other hand,
mortality was more variable for the single gene isolines that were also tested. For ECB, both
MON 89034 and the single gene (Cry 1 A. 105 and Cry2Ab2) isolines killed nearly all exposed
larvae. Low survival (4%) was noted only on a Cry2Ab2 isoline (67620) and on MON
89034, though the surviving larvae were stunted (< 41% the mass) relative to larvae on
control leaf disks. The highest level of survival was noted for SWCB with some survival (up
to 41%) of the control group) observed on both the isolines and MON 89034, although the
surviving larvae showed growth inhibition in all cases. For FAW and CEW, no survival was
noted on MON 89034 or the isolines (though CEW survival on the control was only 26%,
presumably due to CEW preference for feeding on corn ears instead of leaf tissue).

A second trial using kernels instead of leaf disks was performed for CEW. This test revealed
relatively high survival (up to 35%) on the lower expressing isolines (LAJ129 and 67620)
and 9% survival on MON 89034 (growth inhibition was not recorded).

Several sets of field tests (conducted in 2000 and 2002) showed high efficacy, though they
provided less information on dose. The field tests were targeted primarily at ECB (one study
was designed for SWCB) and assessed plant damage (as opposed to directly evaluating
mortality). Single gene isolines were used, but no expression data were given (they were
claimed to be lower than MON 89034) and MON 89034 was not included in the trials. The
trials showed that Cry 1 A. 105 and Cry2Ab2 isolines significantly reduced ECB and SWCB
leaf and tunneling damage relative to the non-Bt control groups. Feeding damage was
comparable to the commercial product MON 810, which is known to express a high dose for
ECB and SWCB. While these studies demonstrated field efficacy of the Cry 1 A. 105 and
Cry2Ab2 isolines, they provide limited information for the assessment of MON 89034 dose.
This is because 1) MON 89034 was not evaluated in any of the trials, 2) mortality was not
assessed, and 3) CEW and FAW were not included in the trials. Monsanto recognized the

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limitations of the field work, but indicated that they should be considered in toto with the
laboratory bioassays and leaf disk tests.

Overall, the dose studies presented a mixed picture of the dose profile for MON 89034. Dose
and efficacy data indicated that: (1) the Cry 1 A. 105 and Cry2Ab2 proteins in MON 89034
each provide essentially 100% control of ECB; (2) the CrylA. 105 protein in MON 89034
provides approximately 95% control of SWCB, while the Cry2Ab2 protein provides 80-90%)
control; (3) the CrylA. 105 and Cry2Ab2 proteins in MON 89034 each provide >95% control
of FAW; and (4) the CrylA. 105 and Cry2Ab2 proteins in MON 89034 each provide 90-95%
control of CEW. Clearly, the hybrid offers a high level of control against the four major
target pests including greater than 95% control of ECB and FAW and greater than 90%
control of CEW and SWCB. The actual level of control may be even higher due to growth
inhibition among survivors that would likely preclude developmental completion. As
demonstrated in the diet bioassays, the target pests appear to be somewhat more sensitive to
Cry 1 A. 105 than to Cry2Ab2. However, much of the dose information is circumstantial; the
leaf disk assays were the only trial phase that directly evaluated MON 89034. The other data
were obtained from susceptibility assays with purified protein (that were compared to MON
89034 expression data) and tests with (non-commercialized) single gene isolines.

BPPD agrees with Monsanto that MON 89034 provides strong control; these tests
demonstrate that MON 89034 will likely kill >90% of susceptible insects. On the other hand,
the data did not support a high dose under the definition put forth by the 1998 SAP (a level of
toxin 25 times greater than needed to kill susceptible larvae; i.e. a dose greater than the LC99
of the pest). Some survival of MON 89034 plant tissue was noted for ECB, SWCB, and
CEW. Monsanto assumed that the survivors would not reach adulthood due to growth
inhibition (and therefore are functionally dead), but that assumption was not tested due to the
short time frame of the experiment.

Monsanto's dose studies did not directly evaluate the effect of MON 89034 on potentially
heterozygous larvae (i.e. with one copy of a resistance allele). Since heterozygotes may be
more tolerant of Bt toxins than susceptible homozygous larvae, the 1998 SAP indicated that a
high dose product should kill at least 95% of homozygous susceptibles. Roush's modeling
(1998) specifies that 70% of heterozygotes should be killed by the toxins expressed in the
dual gene PIP. Monsanto assumes that MON 89034 meets the criteria for the Roush model
(i.e. 95%) susceptible and 70% heterozygote mortality), but no empirical evidence was
presented regarding potential heterozygote mortality. Given that the major support for
Monsanto's proposal to reduce corn refuge from 20% to 5% is the Roush model, BPPD
recommended that Monsanto further investigate whether MON 89034 consistently has high
mortality of susceptible homozygotes (>95%) and further investigate heterozygote mortality
for MON 89034. BPPD recognizes that direct evaluations of heterozygote effects can be
difficult, particularly if resistant colonies for the target pests are unavailable and given that
there is no field resistance to either protein. Monsanto has not provided enough information
to determine the "killing power" of each individual protein — it would be useful to assess

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whether CrylA.105 and Cry2Ab2, individually, will kill greater than 95% of the susceptible
homozygotes. The 1998 SAP suggested several ways to estimate mortality for less
susceptible larvae (i.e. heterozygotes) (EPA 1998). These techniques included testing larger,
later instar larvae that may be less susceptible to the toxins or with PIPs expressing lower
levels of toxin than the commercial event (see the discussion of the SAP recommendations at
the beginning of this section).

c) Impact of Prior Use of CrylAb-Expressing Bt Corn Products on MON 89034

Monsanto examined the impact of prior use of Cry 1 Ab-expressing Bt corn products on MON
89034 Bt corn. Cry 1 Ab-expressing Bt corn products have been on the U.S. market since
1997 and planted on millions of acres. This selection pressure could result in increased
Cryl Ab-resistant allele frequencies in lepidopteran corn pests, particularly those that are
more dependent on corn as a primary host such as ECB and SWCB. Should there be
Cryl Ab-resistant insects that are cross-resistant to either the Cryl A. 105 protein and/or
Cry2Ab2 protein then IRM value of MON 89034 would be significantly reduced. Given that
there is very high amino acid similarity between the CrylAb and Cryl A. 105 proteins, the
potential for cross-resistance between CrylAb and Cryl A. 105 is an important consideration.
In an earlier section of this assessment, BPPD concluded that there is a low likelihood of
cross-resistance between Cryl A. 105 (Cryl A proteins) and Cry2Ab2 proteins.

European corn borer (ECB) populations have been monitored for susceptibility to CrylAb
since the 1995 growing season (diagnostic concentration information has been collected
since 1999). Since 1998, monitoring has also been required for corn earworm (CEW),
southwestern corn borer (SWCB), and fall armyworm (FAW, sweet corn only) susceptibility
to CrylAb. All of the CrylAb monitoring data through the 2000 growing season were
reviewed by the Agency during the 2001 Bt crops reassessment (EPA 2001). Data for the
2001 through 2005 growing seasons were independently reviewed by BPPD (see Reynolds
2004a, 2004b, 2006; Milofsky 2007). Estimates of the frequency of CrylAb resistance in
ECB indicate that Cryl Ab-resistant alleles capable of conferring ECB survival on a Bt corn
plant are very rare (Andow et al. 2000; Bourguet et al. 2003, Stodola et al. 2006). The
highest estimate of CrylAb resistance allele frequency in U.S. ECB populations was
<4 X 10"4 at the 95% confidence level. The CrylAb resistance allele frequency has not
increased significantly in frequency even with the ten years of widespread use of Cryl Ab-
expressing corn products. There have been no instances of CrylAb resistance capable of
conferring survival on Cry 1 Ab-expressing Bt corn plants in annual monitoring of ECB,
SWCB, and CEW (see ABSTC 2006 and BPPD's technical review in Milofsky 2007).

BPPD agrees with Monsanto that the frequency of Cryl Ab-resistance is very low and that it
has not increased significantly in over ten years of widespread use of Cryl Ab-expressing
corn products.

Current knowledge about Bt toxin receptors is summarized in a recent review by Piggott &
Ellar (2007). By far the most studied receptors have been lepidopteran receptors associated

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with Cryl A toxins. These authors summarized Cry toxins for which a putative receptor has
been identified (see Table 2 in Pillar & Ellar 2007). The Cryl A proteins (Cryl Aa, Cryl Ab,
Cryl Ac, CrylBa, CrylCa, CrylFa) all have aminopeptidase N receptors (APNs) that can
serve as Cry-binding proteins that mediate pore formation, but their relevance to toxin
susceptibility has not been demonstrated. Cryl Aa, Cryl Ab, and Cryl Ac also have cadherin-
like receptors that have been shown to mediate Cryl A toxicity. Other putative receptors, i.e.,
alkaline phosphatases, glycolipids, 57R-270, P252, may also play a role in Cryl A toxicity,
but further study is needed. While there has been progress in what is known about Cryl A
toxicity, little is known about other Cry families, such as the Cry2A family. How pore
formation confers toxicity requires further study.

Monsanto characterized binding of Cryl A. 105 and CrylAb proteins to ECB brush border
membrane vesicles using Biacore. These studies (Li & English 2006) indicated that
Cryl A. 105 and CrylAb occupy different binding sites on the ECB midgut epithelial
membrane and therefore have distinct membrane binding mechanisms. CrylAb binding data
suggest that the binding patterns are much more complex for CrylAb than for Cryl A. 105
despite these two proteins having 90% amino acid sequence homology. BPPD agrees with
Monsanto that differences in binding mechanisms lessen the likelihood of CrylA.105 and
CrylAb cross-resistance.

Monsanto also summarized the laboratory studies examining ECB colonies selected for
resistance to the CrylAb protein. While these colonies are imperfect tools for predicting
what will happen in the field, they are the best tools available for looking at potential
resistance mechanisms. In particular, Monsanto discussed a series of studies conducted on
three ECB colonies selected for resistance to CrylAb by Blair Siegfried at the University of
Nebraska (Siegfried & Spencer 2001). Two of the colonies were created by laboratory
selection, the Europe colony was established from larvae collected in Lombardia region of
northern Italy and a second colony was created from larval collected in Nebraska. A third
colony was created from survivors of diagnostic bioassays from both the Europe and
Nebraska populations. All three colonies, along with two susceptible colonies, were assayed
for their response to purified CrylAb, Cry 1 Ac, Cry2Ab2, and a version of the Cry 1 A. 105
protein. Results of the bioassays indicated that all three Cryl Ab-resistant colonies were
resistant to CrylAb and Cryl Ac, but remained susceptible to Cryl A. 105 and Cry2Ab2 with
no evidence of cross-resistance.

As noted earlier, Cryl A. 105 is a chimeric protein consisting of domains I and II and the C-
terminus of Cryl Ac and domain III of CrylFa. Several pieces of evidence suggest that there
is at least some likelihood of cross-resistance of CrylFa and CrylAc/CrylAb. Denolf et al.
(1993) conducted CrylAb, CrylAc, and CrylB proteins binding experiments with isolated
brush border membrane vesicles (BBMV) and gut tissue sections from ECB. These studies
indicated that CrylAb and CrylAc proteins recognized the same membrane receptor with
different binding affinities while the CrylB protein recognized a separate receptor. More
recent binding studies with BBMV from ECB conducted by Hua et al. (2001) indicated that

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there was limited shared binding between CrylFa and Cryl Ab/Cryl Ac proteins. Pereira et
al. (2008) showed that there was little cross-resistance to Cryl Ac (6.9-fold) in a CrylFa-
resistant line of ECB (>3,000-fold). Jurat-Fuentes & Adang (2001) demonstrated that
CrylFa (and Cry 1 Ja) share the Receptor A binding site with the Cry 1A toxins in Heliothis
virescens (tobacco budworm), but they also have unique binding sites. These researchers
proposed a model that suggests that CrylFa, CrylAb, and CrylAc all bind to the CrylAa
binding site (called Receptor A, although with different binding affinities) as well as to
unique binding sites. An altered CrylAa binding site may cause resistance to CrylAa,
CrylAb, CrylAc, and CrylFa proteins, but the unique binding sites also play a role in
toxicity. Competition binding experiments performed by Hernandez & Ferre (2005) showed
the occurrence of a common receptor for CrylAc, CrylFa, and CrylJa in Helicoverpa
armigera, H. virescens, and Spodoptera exigua. So far, all available information on binding
site competition suggests that CrylAa, CrylAb, CrylAc, CrylFa, and CrylJa share a
common binding site in most, if not all, Lepidoptera. These authors suggest that CrylAa,
CrylAb, CrylAc, CrylFa, and CrylJa protein binding to a common site explains, perhaps,
the biochemical basis of multiple resistance and cross-resistances among these five proteins
in some insect species. Jurat-Fuentes & Adang (2006) recently demonstrated that a cadherin-
like protein, HevCaLP, is the functional receptor for CrylAc binding in a highly-resistant
(>300,000-fold) tobacco budworm colony (YHD2) although it is not a receptor for CrylFa
(130-fold resistant). These results suggest that the CrylFa and CrylAc shared binding site is
not a cadherin-like protein and that cross-resistance would be due to modification of some
other receptor. Collectively, the availability information indicates that there is some
likelihood of cross-resistance to both the CrylFa and Cryl Ab/Cryl Ac proteins through
modification of a single shared receptor site. Hernandez & Ferre (2005) suggest that neither
transgenic plants expressing stacked combinations of CrylAc (and by extension CrylAb),
CrylFa, and CrylJa nor rotations of Bt crops containing single genes of these three (four)
proteins would be a good resistance management strategy. In the case of corn, primary pests
susceptible to CrylAb and CrylFa, such as ECB (and SWCB and CEW), would necessitate
the importance of establishing the binding site model for this species in order to develop an
appropriate resistance management strategy.

Monsanto has shown, using the weight-of-evidence approach, that there is a low likelihood
of cross-resistance between Cryl A. 105 and Cry2Ab2 (see section a., "Assessment of the
Probability of Cross-Resistance to the CrylA.105 and Cry2Ab2 Proteins" of this IRM
assessment). It is assumed that the primary mechanism of resistance will be that of binding
site modification, which is a reasonable assumption based on studies with other ifr-resistant
insect populations (laboratory and field) (see Ferre & Van Rie 2002). Similarly, Monsanto
has adequately demonstrated that there is a low likelihood of cross-resistance of Cryl A. 105
and CrylAb. On the other hand, Monsanto has not addressed the likelihood of cross-
resistance of Cryl A. 105 and CrylFa and CrylAc. The Cryl A. 105 protein is a chimeric
protein consisting of Domains I and II and the C-terminus of CrylAc and Domain III of
CrylFa.

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It was recommended that Monsanto provide BPPD with additional information on cross-
resistance of Cry 1 A. 105 and CrylFa and Cry 1 Ac (including binding site models and use of
resistant colonies) for the target pests and determine how such cross-resistance may impact
the durability of MON 89034.

d) Proposed IRM Plan for MON 89034

Monsanto stated that the introduction of MON 89034 would significantly decrease the risk of
lepidopteran pests evolving resistance to Bt corn. Monsanto's Insect Resistance Management
(IRM) plan for MON 89034 focused on three key assumptions: (1) resistance to Cry 1 A. 105
and Cry2Ab2 is expected to be at least partially recessive; (2) the probability of cross-
resistance between Cry 1 A. 105 and Cry2Ab2 is low; and (3) the level of both Cry 1 A. 105 and
Cry2Ab2 produced in MON 89034 confer high level of control of susceptible target pests
(high dose defined as at least 90% and preferably >95% control). Should these assumptions
be met, MON 89034 would have significantly more durability than all existing single-gene
products for lepidopteran control in the U.S., including MON 810 (Cryl Ab), BTl 1
(CrylAb), Herculex I (CrylFa), and their respective stacked products. The primary focus of
the MON 89034 IRM plan was on management of ECB resistance and to a lesser extent,
SWCB and CEW in regions where these pests are economically important. FAW and
sugarcane (SCB) were not a focus of Monsanto's IRM for MON 89034.

Monsanto's proposed IRM plan for MON 89034 originally consisted of the following
elements.

1. A 5% structured non-1 epidopteran Bt corn refuge for the Corn Belt based on two
independent (minimal cross-resistance), highly effective modes of action of Cryl A. 105
and Cry2Ab2;

2. A 20% structured non-1 epidopteran Bt corn refuge for cotton-growing areas;

3. Annual resistance monitoring, grower education, and compliance monitoring programs;
and

4. A remedial action plan that describes a series of action to investigate suspected
resistance, confirms actual resistance, and mitigates the resistant population(s).

Each of these elements will be discussed below.

i. 5% Structured Refuge for Field Corn Uses of MON 89034 in the Corn Belt

The critical question is whether Monsanto has provided sufficient data/information to
indicate that the durability of a 5% structured refuge (as Monsanto has proposed) is equal to
or greater than durability of a 20% structured refuge (the current structured requirement for
lepidopteran-protected Bt corn products) for management of resistance to MON 89034.
Monsanto's MON 89034 IRM plan for field corn uses focused on ECB, though the issue of

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whether MON 89034 has consistently high mortality of susceptible homozygotes for all of
the primary target species also has to be considered.

In the case of MON 89034, two Bt genes, crylA. 105 and cry2Ab2, were engineered into Bt
corn plants to provide even better control (than first-generation, single Bt protein products) of
ECB, CEW, SWCB, and FAW. Two proteins are expressed in MON 89034 corn plants:
CrylA. 105, a chimeric protein consisting of domains of Cry 1 Ac and CrylFa; and Cry2Ab2,
the same protein that is expressed in Bollgard II cotton (a Monsanto product). Monsanto has
provided sufficient efficacy data to demonstrate that MON 89034 provides good control of
ECB, CEW, SWCB, FAW, and SCB (see Matten 2007 for BPPD's review of Monsanto's
submission, Headrick et al. 2006, MRID# 469514-15). The level of control of MON 89034
for these pests was equal to or greater than YieldGard (MON 810, Monsanto's single Bt
(CrylAb) trait corn product).

Monsanto's first assumption was that Cry2Ab2 and CrylA. 105 have different modes of
action and therefore the potential for cross-resistance is low. As discussed earlier in this
review, Cry2Ab2 and CrylA. 105 have low sequence homology (14%), different activation
mechanisms and binding characteristics, unique antibody binding sites, and resistant insects
to one protein will be controlled by the other protein. Therefore, it can be concluded that
Cry2Ab2 and Cry 1 A. 105 have different modes of action and therefore it is expected that
there will be a low likelihood of cross-resistance (see earlier discussion in Section a.
"Assessment of the Probability of Cross-Resistance to the CrylA.105 and Cry2Ab2
Proteins"). Lack of cross-resistance would increase the durability of MON 89034. These
two proteins, Cry2Ab2 and CrylA. 105, therefore, seem to be good candidate proteins for
pyramiding. BPPD agrees with Monsanto that the probability of cross-resistance between
Cry 1 A. 105 and Cry2Ab2 is low and these two proteins have different modes of action.

On the other hand, Roush (1998) cautions that proteins that have already shown significant
levels of cross-resistance in resistant insect strains (e.g., H. virescens, H. armigera, S. exigua,
O. nubilalis, Plutella xylostella), such as between CrylA, CrylFa and CrylJ proteins, should
not be used in pyramiding. This same warning was also given by Hernandez & Ferre (2005).
Cryl A. 105 is a chimera that consists of binding domains of Cryl Ac and CrylFa. There are
commercial Bt crops that express Cryl Ac, CrylAb, CrylFa proteins and these products have
been in the marketplace for nearly a decade. Should there be insect populations resistant to
Cry 1 Ac, CrylAb, and/or CrylFa that are cross-resistant to Cry 1 A. 105 then the durability of
MON 89034 would be significantly reduced and a 5% structured refuge would be insufficient
to maintain high levels of durability.

Given that there is very high amino acid similarity between CrylAb, Cryl Ac, and
CrylA. 105 proteins then the potential for cross-resistance between CrylAb, Cryl Ac and
CrylA. 105 is an important consideration. CrylA. 105 and CrylFa have about 76% amino
acid similarity. However, what is really important is the similarity of the binding domain III
of CrylFa and Cry 1 A. 105 which is presumed to be very high. Cross-resistance is a real

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possibility for these two proteins. There are several lines of evidence that indicate that
CrylFa and Cryl Ab/Cryl Ac share a common binding receptor although each of these
proteins has unique binding receptors as well (Denolf et al. 1993, Hua et al. 2001, Jurat-
Fuentes & Adang 2001; Hernandez & Ferre, 2005). Evidence for a shared binding receptor
would increase the likelihood of cross resistance should resistance evolve through
modification of the shared binding receptor.

Evidence provided by Monsanto indicates that there is little cross-resistance of Cry 1 A. 105
and Cryl Ab. One cannot, however, infer much about the likelihood of cross-resistance of
Cryl A. 105, CrylFa, and Cryl Ac based on the binding patterns of Cryl A. 105 and CrylAb
because binding patterns are unique to each species (e.g., ECB, SWCB, and CEW) and each
protein. Monsanto did not address the likelihood of cross-resistance of Cry 1 A. 105 and
CrylFa, a protein already in existing Bt corn and Bt cotton products, and what impact cross-
resistance would have on the durability of MON 89034. BPPD recommends that Monsanto
provide additional information on cross-resistance of Cryl A. 105 and CrylFa and Cryl Ac
(including binding site models and use of resistant colonies) for the target pests and
determine how such cross-resistance may impact the durability of MON 89034.

Monsanto's second assumption was that resistance will be recessive. Ten years of resistance
monitoring data indicate that the frequency of CrylAb alleles in ECB is very low (<4 x 10"4)
and that this frequency has not changed significantly during this time. The 20% structured
refuge requirement for single-gene Bt corn products has been in place for over a decade and,
as noted earlier in this review, there is no evidence of field resistance to CrylAb and CrylFa
in ECB, SWCB, and CEW during that period in the continental U.S. There is also no
evidence of Cry2Ab2 resistance (CEW) after five years of widespread use of Bollgard II
cotton. The absence of any cases of field resistance to Bt crops after a decade of use
indicates that any relatively common /^/-resistant alleles must be recessive (Tabashnik et al.,
2003). This evidence provides a strong indicator that resistance to the Bt proteins expressed
in MON 89034 would also be recessive. BPPD agrees with this line of reasoning. Pyramids
are considerably more effective when resistance frequencies are low provided that the
susceptible homozygotes are all killed by each of the toxins used separately (Roush 1998;
Figure 4.).

Monsanto's third assumption for its proposed 5% structured refuge depended on whether the
amount of Cryl A. 105 and Cry2Ab2 produced in MON 89034 confers a high level of control
of susceptible target pests (defined as at least 90% and preferably >95% mortality). It is this
third assumption that is the most difficult to prove.

Resistance simulation models predict that the greatest benefits of combining toxins in single
plants by "pyramiding" or "stacking" are achieved when no cross-resistance occurs, when
there are no fitness costs, when resistance to each toxin is rare and recessive, and when a
refuge of plants without toxins are present. Modeling simulations of two-gene products

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predict that the resistance risk associated with a two-gene product will be significantly less
than for a single-gene product (for example, Caprio 1998; Roush 1998). Pyramiding two or
more proteins increases the chance that at least one of the proteins will be especially
favorable to resistance management. Modeling simulations predict that pyramids (without
cross-resistance) can reduce the need for larger refuges (Roush 1998).

Pyramiding relies on the idea that each protein is used individually in a way that would kill
all insects susceptible to that protein, and in so doing, kills insects that are resistant to the
companion protein (Roush 1998). This has been described as "redundant killing" in the sense
that most of the population is susceptible to both proteins and thus is killed twice. The extent
to which the individuals that are resistant to one protein are killed by the other is central to
the effectiveness of the pyramiding strategy.

Monsanto relied on the Roush (1998) model to support the need for a 5% structured refuge
rather than a 20% structured refuge in the Corn Belt. Roush's model (figure 2 in the
publication) indicated that a 5% structured refuge is equal to or greater than a 20% structured
refuge for a highly effective, high dose single-gene product when a two-gene product (MON
89034 in this case) achieves at least 95% control of susceptible homozygotes and 70%
control of heterozygotes assuming there is no cross-resistance. Monsanto's dose studies, as
discussed earlier, presented a mixed picture for MON 89034 (see Section b., "Dose"). Dose
and efficacy data indicated that MON 89034 has a high level of control against the four
major target pests (as described in Head 2006): "(1) the Cry 1 A. 105 and Cry2Ab2 proteins in
MON 89034 each provide essentially 100% control of ECB; (2) the CrylA. 105 protein in
MON 89034 provides approximately 95% control of SWCB, while the Cry2Ab2 protein
provides 80-90% control; (3) the CrylA. 105 and Cry2Ab2 proteins in MON 89034 each
provide >95% control of FAW; and (4) the CrylA. 105 and Cry2Ab2 proteins in MON 89034
each provide 90-95% control of CEW." The actual level of control may be even higher due
to growth inhibition among survivors that would likely preclude developmental completion.
The target pests appear to be somewhat more sensitive to CrylA. 105 than to Cry2Ab2.

Monsanto's dose testing indicated that MON 89034 has a high level of control (greater than
90%) of susceptible homozygotes (ECB, SWCB, CEW, FAW), one of two thresholds needed
to support the durability of a 5% structured refuge for a two-gene pyramided Bt corn product
(as equal to or better than that of a single-gene Bt corn product expressing a high dose of
control against the target pests). However, it was not easily discernable as to whether each
individual toxin kills greater than 95% of susceptible individuals. This is important for
prediction of the durability of MON 89034: Roush's simulations (1998; Figure 3) showed
that the greatest gains of pyramiding two proteins are when the mortality of susceptible
insects is considerably greater than 95%, especially if resistance allele frequencies are quite
low.

On the other hand, Monsanto's dose studies did not directly evaluate whether MON 89034
kills at least 70% of the heterozygotes, the other threshold needed to support a 5% structured
refuge for a two-gene pyramided Bt corn product. Monsanto did not provide enough

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information to determine the mortality of susceptible (SS) homozygotes and heterozygotes
(RS) on MON 89034 plants. It is important to know whether Cry 1 A. 105 and Cry2Ab2 are:
1) both high dose proteins; 2) one high dose and one moderate dose protein (and which one is
high and which one is moderate); or 3) two moderate dose proteins to control ECB (and
SWCB) in the Corn Belt. In other words, one has to establish whether each protein can kill
potentially resistant individuals to the other protein. To evaluate MON 89034 in the context
of Roush's model, it must be determined whether Cryl A. 105 and Cry2Ab2 are produced at
high levels to kill at least 95% of susceptible homozygotes and 70% of the heterozygotes.
Because of this, BPPD recommended that Monsanto further investigate heterozygote
mortality for MON 89034. BPPD recognizes that direct evaluations of heterozygote effects
can be difficult, particularly if resistant colonies for the target pests are unavailable.

However, the 1998 SAP suggested several ways to estimate mortality for less susceptible
larvae (i.e., heterozygotes). These techniques included testing larger, later instar larvae that
may be less susceptible to the either the Cry 1 A. 105 or Cry2Ab2 proteins or with PIPs
expressing less protein (less Cry 1 A. 105 or Cry2Ab2) than MON 89034. As Roush (1998)
cautioned, "... small refuges remain risky..." when mortalities of heterozygotes are lower
than expected. For MON 89034, it has only been assumed (but not verified) that the
heterozygote mortality will be at least 70% for each protein.

Cross-resistance between Cryl A. 105 and Cryl Ac and CrylFa is not known, but published
studies indicate that there is at least some potential for cross-resistance between Cryl A and
CrylFa proteins in a number of insect species (see earlier discussion). The impact of this
potential cross-resistance on the durability of MON 89034 is not known.

Monsanto's use of the Roush (1998) model as a guide to predict the durability of MON
89034 was very useful, but it is only a first step. Roush encouraged researchers to further
investigate the points raised in his 1998 paper with additional modeling and experiments (see
Roush 1998). However, this was not done by Monsanto. Additional modeling using a
species-specific (e.g., ECB and SWCB for the Corn Belt), spatially-explicit, preferably
stochastic, landscape model of available Bt crops expressing many different Cry proteins
(needs to be a multiple gene model, a more complex model) needs to be performed to more
precisely predict the evolution of ECB resistance (or SWCB) to MON 89034. This new
model would need to consider the impact of other Bt proteins in which there may be some
cross-resistance. This is analogous to the species-specific simulation modeling that EPA
required Monsanto do to support the use of natural refuge (instead of a structured refuge) for
management of H. virescens and H. zea to the Cryl Ac and Cry2Ab2 proteins expressed in
Bollgard II cotton. In conclusion, Monsanto's initial data and modeling do not support a 5%
structured refuge for MON 89034 for field corn uses in the Corn Belt.

Given the uncertainties in the dose determination for ECB and SWCB (SS and RS mortality)
(note: CEW and FAW are lesser pests in the Corn Belt), cross-resistance likelihood of
Cryl A. 105, Cryl Ac, and CrylFa, and limitations of the simulation modeling, BPPD
recommended that the current 20% structured refuge requirement for field corn uses of MON
89034 in the Corn Belt be maintained until such time as Monsanto could address these

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uncertainties associated with the durability of a 5% structured refuge. There are many Bt
corn and Bt cotton products in the landscape. Cross-resistance conferred by any of these
proteins may negatively affect the durability of MON 89034. Studies indicate that there is at
least some potential for cross-resistance between Cry 1 A. 105, CrylFa, and Cry 1 Ac proteins
in a number of insect species (see earlier discussion). Monsanto needs to examine the
potential of Cry 1 A. 105, Cry 1 Ac, and CrylFa cross-resistance and what impact it has on the
durability of MON 89034.

ii. 20% Structured Refuge for Field Corn Uses of MON 89034 in Cotton-Growing Areas

Monsanto has proposed that a 20% structured refuge rather than the current 50% structure
refuge requirement for single-gene lepidopteran-control products be used to manage insect
resistance to MON 89034 in cotton-growing areas. The major pest of concern for Bt corn in
cotton-growing areas is CEW (also known as cotton bollworm when it feeds on cotton),
although ECB, FAW, SCB (sugar cane borer) are also sporadic corn pests in cotton-growing
areas. As described earlier in this assessment (Section b "Dose"), Cry 1 A. 105 and CryAb2
proteins have at least 90% control of CEW. Previous studies submitted by Monsanto (Head
& Reding 2001; reviewed in BPPD 2007) demonstrated the low likelihood of cross-
resistance between the Cry2Ab2 and Cry 1 Ac proteins. Both Cry 1 A. 105 and Cry2Ab2 have
a low likelihood of cross-resistance with Cryl Ab (see earlier discussion in Section c,

"Impact of Prior Use of CrylAb-Expressing Bt Corn Products on MON 89034").

Monsanto used its deterministic, non-spatial model (Gustafson & Head 2005) to examine
whether planting a 20% structured non-Bt corn refuge with MON 89034 was sufficient to
manage the risk of resistance evolution to Bt corn and Bt cotton products. In this model, it
was assumed that all cotton planted consisted of Bollgard II cotton, with no non-Bt cotton in
the system, and that 80% of the corn planted in the region consisted of MON 89034 and 20%
non -Bt corn. The modeling was focused on estimation of the likelihood of CEW resistance
in the Mississippi region because of the relatively higher risk of CEW resistance evolution in
this review. Monsanto estimated the effective (all non -Bt hosts of CEW, including current
levels of non -Bt cotton and 20% non-Bt corn refuge associated with MON 89034) and natural
refuge (only non-cotton hosts of CEW, including 20% structured non -Bt corn refuge
associated with MON 89034 and other unmanaged hosts) available for CEW in this region as
described in Gustafson & Head (2005). These estimates were used as parameter values in the
model. One model scenario assumed that MON 89034 is fully cross-resistant with Bollgard
II cotton (i.e., Cryl A. 105 and Cryl Ac are fully cross-resistant). Resistance was assumed to
be complete with no associated fitness costs. Using these assumptions, the simulation
modeling predicted that a 20% non -Bt corn refuge for MON 89034 in the southern cotton-
growing areas would be sufficient to manage the risk of resistance evolution to Bt corn and
Bt cotton products. Resistance to Cry2Ab2 protein evolved first and took >24 modeling
years to evolve (modeling time was 25 years). It is not clear from Monsanto's discussion
whether Cryl A. 105 and CrylFa cross-resistance was included in the modeling. The current
landscape has both CrylFa- and CrylAb-corn and CrylFa- and CrylAc- and Cry2Ab2 +

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Cryl Ac-cotton products. Should there be substantial cross-resistance, then the value of MON
89034 would be dramatically reduced.

A 20% non-Bt corn refuge for MON 89034 in the southern cotton-growing areas would be
sufficient to manage the risk of resistance evolution to Bt corn and Bt cotton products
assuming there is no cross-resistance. Monsanto did not, however, sufficiently address the
cross-resistance of Cryl A. 105, CrylFa, and Cryl Ac in the cotton-growing landscape and
how such cross-resistance may impact the durability of MON 89034. Should cross-
resistance be of concern then the durability of MON 89034 in the southern cotton-growing
areas might be compromised. Monsanto needs to address this potential in subsequent
simulation modeling.

e) Sweet Corn Uses

As stated in Monsanto's submission (Head 2006): "In the U.S., sweet corn is grown on
approximately 500,000 acres, with California, Florida, Georgia, New York, Ohio, and
Pennsylvania accounting for 62% of the acres. The insecticide use per acre on sweet corn is
approximately 35-fold that of field corn (2.7 lb/A versus 0.76 lb/A) (USDA, 2006) and
typically, 12 - 40 applications of insecticides may be applied to a single crop of sweet corn in
the southern U.S (Adams 1996). Therefore, planting of MON 89034 has the ability to
drastically reduce the amount of synthetic insecticides used for sweet corn production."

Monsanto has proposed the use of MON 89034 as a sweet corn product to control certain
lepidopteran insect pests in conjunction with no structured refuge. While sweet corn has a
similar pest spectrum to field corn, agronomic practices differ between sweet corn and field
corn. This makes pest management different between the two crops. As described in
Monsanto's submission (Head 2006): "Sweet corn is harvested approximately 18 to 23 days
after silk emergence, compared to field corn in which the grain is allowed to mature and dry
in the field. For sweet corn, the ears are harvested while still wet and placed in cold storage
for fresh market corn or processed immediately. Shortly after harvest, corn stalks are
typically destroyed in the field by disking, chopping or plowing. Previous work by Lynch et
al. (1999), show that these harvest and post-harvest practices make it unlikely that any
surviving/resistant larvae could survive, complete its development, and contribute any
resistant allele to the next generation in sweet corn. Even if a larva was to survive, sweet
corn farmers, including home gardeners, typically grow sweet corn in small plots along with
many other vegetables that serve as alternative hosts for these polyphagous lepidopteran
pests. Therefore, sufficient non-corn refuge should be present due to the typical practices of
planting multiple host crops."

BPPD requested in a January 17, 2007 letter to Monsanto that the company provide
additional (dose) data to support the sweet corn use. Monsanto responded to BPPD's request
for supplemental data on March 9, 2007. Monsanto provided data that compared the
estimated Cryl A. 105 and Cry2Ab2 protein levels in leaf tissues collected from MON 89034

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sweet corn varieties with field corn varieties (see Table 4 in Bogdanova 2007; MRID#
470794-02). Sweet corn data came from one site and field corn data came from five sites.
The mean levels of the Cry 1 A. 105 and Cry2Ab2 proteins were comparable between field and
sweet corn MON 89034 hybrids.

BPPD agrees with Monsanto that no structured refuge is needed in conjunction with the
sweet corn use based on the destruction of potential resistant larvae through cultivation
practices.

f) Popcorn Use of MON 89034

Monsanto proposed to use the same IRM plan described for field corn with popcorn uses for
MON 89034. Monsanto stated that there are approximately 291,000 acres of popcorn grown
annually in the U.S., with Illinois, Indiana, Iowa, Nebraska, and Ohio accounting for 86% of
the planted acres (Pike 2003). Popcorn, like field corn, is allowed to mature and dry in the
field, and the pest spectra are essentially identical in popcorn and field corn.

Monsanto provided no additional dose and/or efficacy data to what was provided for field
corn to support the use of MON 89034 on popcorn. Without these data, the popcorn use
cannot be supported.

g) Other Elements of IRM for MON 89034

Monsanto proposed to have resistance monitoring, grower education and compliance
monitoring as necessary parts of the IRM program. They proposed to implement a program
similar to what is currently carried out for MON 810 and other single-gene Bt corn products.
In particular, the educational and compliance assurance programs for MON 89034 would
follow the structure established through consultations between EPA and the industry, and
will involve working closely with NCGA and other interested stakeholders.

Similarly, post-commercial resistance monitoring programs would be established as an
extension of existing programs to track the susceptibility of the key lepidopteran corn pests to
the Cry 1 A. 105 and Cry2Ab2 proteins. In the monitoring program, insect populations would
be collected and each protein will be tested separately, rather than a mixture of the two
proteins, because resistance to one protein could be masked by the activity of the other. As
part of this program, baseline susceptibility studies are planned for the Cry 1 A. 105 protein
against ECB (through Dr. Blair Siegfried at the University of Nebraska), and for the
Cry 1 A. 105 and Cry2Ab2 proteins against SWCB (through Dr. Qisheng Song at the
University of Missouri), and CEW (through Bruce Lang of Custom Bio-Products). The
baseline susceptibility of ECB to Cry2Ab2 has already been assessed over a two year period
(see Appendix 4 - Siegfried & Spencer 2001 in Monsanto's submission, MRID# 469514-30).
In the case of CEW, baseline studies and annual monitoring have been conducted for
Cry2Ab2 protein as part of the Bollgard II cotton IRM program, and the resulting data will

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be useful for MON 89034. In addition to the formal monitoring program, any unusual
damage from lepidopteran pests will be monitored by the routine scouting of corn fields and
be reported to Monsanto or local extension agents.

A remedial action plan has been developed and approved by EPA for MON 810 and other
single-gene Bt corn products (EPA 2001). This plan describes a series of actions to
investigate suspected resistance, confirm actual resistance, and mitigate the resistant
population. The basis of this plan also is appropriate for MON 89034. However, because
MON 89034 contains both the Cry 1 A. 105 and Cry2Ab2 proteins, this product has the
advantage of having a "built-in" mitigation program if resistance evolves to one of the Cry
proteins but not the other. Therefore, Monsanto indicated that the remedial action plan should
only be implemented for MON 89034 if a field population evolves resistance to both the
CrylA. 105 and Cry2Ab2 proteins.

Monsanto's proposed program for resistance monitoring, grower education and compliance
monitoring as part of the MON 89034 IRM program was determined to be "acceptable." No
CrylA. 105 baseline susceptibility studies have been conducted at the time of the registration
application, but are planned by Monsanto. Monsanto has indicated that baseline
susceptibility information for ECB to Cry2Ab2 has been collected over a two-year period
(summarized in Monsanto's submission). For each protein, a discriminatory concentration
(diagnostic dose) will have to be determined for use in the annual resistance monitoring
program. Annual reporting to the Agency of the results of the resistance monitoring, grower
education, and compliance monitoring is needed (as is required for all other Bt PIPs). If there
is confirmed resistance to either protein then it must be reported to the Agency (see FIFRA
6(a) incident reporting requirements and the requirements as part of the Remedial Action
plan).

h) Conclusions for Initial Registration12

MON 89034 field corn uses in the Corn Belt

1) Pyramids can reduce the need for large refuges. Monsanto had originally proposed that a
5% structured refuge, rather than the current 20% structured refuge, be used with the field
corn uses of MON 89034. However, Monsanto's initial data and modeling do not
support a 5% structured refuge for MON 89034 for field corn uses in the Corn Belt.

There are uncertainties in the dose determination for ECB, SWCB, CEW, FAW (SS and
RS mortality), cross-resistance likelihood of CrylA. 105, Cry 1 Ac, and CrylFa and its
impact on the durability of MON 89034, and limitations of the simulation modeling. The
current 20% structured refuge requirement for field corn uses of MON 89034 in the Corn
Belt will be maintained until such time as Monsanto can address these uncertainties.

12 The assessment for the amendment to reduce lepidopteran refuge requirements modified these conclusions. (See
section II. E. 2.1

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2) Monsanto relied on the Roush (1998) model to support the need for a 5% structured
refuge rather than a 20% structured refuge in the Corn Belt. Roush's model (1998;

Figure 2) indicated that a 5% structured refuge is equal to or greater than a 20%
structured refuge for a highly effective, high dose single-gene product when a two-gene
product (MON 89034 in this case) achieves at least 95% control of susceptible
homozygotes and 70% control of heterozygotes assuming there is no cross-resistance.
The dose information provided by Monsanto is not sufficient to demonstrate that each
protein will kill 95% of the homozygous susceptible insects and 70% of the
heterozygotes. To support a 5 % refuge Monsanto will have to further investigate
whether MON 89034 consistently has high mortality of susceptible homozygotes (>95%)
and whether the heterozygote mortality is at least 70% for MON 89034 against the target
pests (for the Corn Belt - ECB and SWCB). The 1998 SAP suggested several ways to
estimate mortality for less susceptible larvae (i.e., heterozygotes) (EPA 1998). These
techniques included testing larger, later instar larvae that may be less susceptible

3) Monsanto has demonstrated that Cry 1 A. 105 and Cry2Ab2 have different modes of action
and, therefore, a low likelihood of cross-resistance. Cry 1 A. 105 and Cry2Ab2 would be
suitable partners in a pyramided product. Monsanto has also shown that there is a low
likelihood of cross-resistance between Cry 1 A. 105 and CrylAb. Monsanto has previously
demonstrated that there is a low likelihood of cross-resistance between Cry2Ab2 and
Cry 1 Ac.

4) However, Monsanto did not address the likelihood of cross-resistance of Cry 1 A. 105,
Cry 1 Ac, Cry 1 Fa, proteins already in existing Bt corn and Bt cotton products, and what
impact such cross-resistance would have on the durability of MON 89034. Monsanto
must provide additional information on cross-resistance of Cry 1 A. 105 and CrylFa and
Cryl Ac (including binding site models and use of resistant colonies) for the target pests
and determine how such cross-resistance may impact the durability of MON 89034. The
Cryl A. 105 protein is a chimeric protein consisting of Domains I and II and the C-
terminus of CrylAc and Domain III of CrylFa. It is important to address not only the
likelihood of cross-resistance potential of Cryl A. 105 and CrylAb and, similarly,

Cryl A. 105 and Cry2Ab2 (which was done by Monsanto), but also that of Cryl A. 105 and
CrylAc and CrylFa.

5) Additional species-specific (e.g., ECB and SWCB for the Corn Belt), spatially-explicit,
landscape modeling is recommended to explore the durability of MON 89034 versus
single-protein Bt corn products. Modeling would need to consider the impact of other Bt
proteins in the landscape that may confer some cross-resistance (to Cryl A. 105, in
particular) and how such cross-resistance would impact the durability of MON 89034 in
the Corn Belt (use of simulation modeling). This is analogous to the species-specific
simulation modeling that EPA required Monsanto do to support the use of natural refuge
(instead of a structured refuge) for management of H. virescens and H. zea to the CrylAc
and Cry2Ab2 proteins expressed in Bollgard II cotton.

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6) MON 89034 field corn use in cotton-growing areas. A 20% non-Bt corn refuge for MON
89034 in the southern cotton-growing areas would be sufficient to manage the risk of
resistance evolution to Bt corn and Bt cotton products assuming there is no cross-
resistance. However, Monsanto did not sufficiently address the cross-resistance of

Cry 1 A. 105, CrylFa, and Cry 1 Ac in the cotton-growing landscape and how cross-
resistance may impact the durability of MON 89034. Should cross-resistance be of
concern then the durability of MON 89034 in the southern cotton-growing areas might be
compromised. Monsanto must address this potential in subsequent simulation modeling.
(See item 4 above.)

7) Sweet corn. No structured refuge is needed in conjunction with the MON 89034 sweet
corn use based on the destruction of potential resistant larvae through cultivation
practices. Grower agreements (also known as stewardship agreements) will specify that
growers must adhere to the following refuge requirements or, in the case of sweet corn,
harvest practices, as described in the grower guide/product use guide and/or in
supplements to the grower guide/product use guide:

For MON 89034 sweet corn, growers are required to destroy any MON 89034 sweet corn
stalks that remain in the field following harvest via rotary mowing, discing, or plow-
down within one (1) month of harvest.

8) Poycorn. Monsanto provided no additional dose and/or efficacy data to what was
provided for field corn to support the use of MON 89034 on popcorn. Without these
data, the popcorn use cannot be supported.

9) Other Important Elements of the IRM Plan. Monsanto's proposed program for resistance
monitoring, grower education and compliance monitoring as part of the MON 89034
IRM program is "acceptable." No CrylA.105 baseline susceptibility studies have been
conducted, but are planned by Monsanto. Monsanto has indicated that baseline
susceptibility information for ECB to Cry2Ab2 has been collected over a two-year period
(summarized in Monsanto's submission). For each protein, a discriminatory
concentration (diagnostic dose) will have to be determined for use in the annual
resistance monitoring program. Annual reporting to the Agency of the results of the
resistance monitoring, grower education, and compliance monitoring is needed (as is
required for all other Bt PIPs). If there is confirmed resistance to either protein then it
must be reported to the Agency (see FIFRA 6(a) incident reporting requirements and the
requirements as part of the Remedial Action plan).

i) Insect Resistance Management Plan for MON 89034 X MON 88017 Bt Corn

MON 89034 x MON 88017 expresses the CrylA.105, Cry2Ab2, and Cry3Bbl Bt toxins and

is targeted against lepidopteran corn pests including European corn borer (ECB),

southwestern corn borer (SWCB), corn earworm (CEW), and fall armyworm (FAW) as well

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as the coleopteran corn rootworm sp. pest complex (CRW). MON 89034 (Cry 1 A. 105 and
Cry2Ab2) provides activity against the lepidopteran corn stalk and ear insects while MON
88017 (Cry3Bbl) is active against root-feeding CRW. The product was created by
conventional breeding in which the previously-registered MON 88017 (EPA Reg. No. 524-
551) was crossed with MON 89034 (EPA Reg. No. 524-LTL). The Cry3Bbl toxin in MON
88017 is the same as expressed by MON 863 corn (Yieldgard Rootworm, EPA Reg. No. 525-
528), which was registered by Monsanto for the 2003 growing season.

1) Monsanto has provided information to demonstrate that the dose of MON 89034 x MON
88017 against the major target pests should be comparable to the dose of the MON 89034
and MON 88017 isolines. Therefore, the IRM considerations (dose, refuge, cross
resistance) for MON 89034 and MON 88017 are applicable to the stacked MON 89034 x
MON 88017 product.

2) Monsanto has proposed a 5% lepidopteran refuge as part of the "Separate Refuge" option
for MON 89034 x MON 88017 corn. Due to uncertainties in the review of the MON
89034 IRM plan (see BPPD 2007a), a 5% refuge cannot be supported at the present time.
Instead, BPPD recommends that the separate refuge option include a 20% lepidopteran
refuge (as has been required for other Bt corn products). However, BPPD notes that a
20% refuge can be supported for MON 89034 x MON 88017 in cotton-growing regions
in southeastern U.S. where a 50% refuge has been previously required.

3) Monsanto's proposal for a combined refuge (covering both coleopteran and lepidopteran
pests) is acceptable. This option calls for a 20% refuge throughout the U.S. (as described
in #2 above, a 20% refuge can be supported in southern cotton-growing regions).

4) The other aspects of Monsanto's IRM plan for MON 89034 x MON 88017 including
resistance monitoring, remedial action plans, grower education, compliance, and annual
reporting are acceptable. Resistance monitoring (sampling, bioassays, and data
reporting) and remedial action should be conducted under the terms and conditions of
registration for MON 89034 and MON 88017.

2. Amendment to Reduce Lepidopteran Refuge to 5% (2008).

After the registration of MON 89034 was granted (with a 20% lepidopteran refuge in the Corn
Belt, as described in the preceding section), Monsanto submitted an amendment with supporting
data to request a reduction in refuge to 5% in the Corn Belt. This section contains BPPD's
assessment of this proposal (based on the review contained in BPPD 2008a).

As part of the IRM proposal for MON 89034 corn, Monsanto proposed a 5% lepidopteran
structured refuge for non-cotton growing regions instead of the 20% refuge that has been
required for all other Bt corn registrations. Monsanto reasoned that the combination of two

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toxins targeting lepidopteran corn pests with no cross resistance allowed for a reduced refuge
with little risk of resistance. As described in section 1 above, BPPD's review of the IRM
proposal (BPPD 2007) agreed with much of Monsanto's justification but determined that there
were a number of uncertainties in the request for lower refuge. Specifically, there were three
areas of concern: (1) Cry 1 A. 105 and Cry2Ab2 dose determination for the major target pests
(ECB, CEW, SWCB, and FAW); (2) cross resistance potential between Cry 1 A. 105 and Cry IF
and CrylAc (toxins expressed in previously-registered PIPs); and (3) species-specific (e.g., ECB
and SWCB for the Corn Belt), spatially-explicit, landscape modeling to explore the durability of
MON 89034 versus single-protein Bt corn products. Given the uncertainty of the reduced refuge
request, EPA registered MON 89034 with a 20% structured refuge requirement, similar to other
Bt corn products. Separately, EPA did agree with Monsanto's request to reduce refuge in cotton-
growing areas from 50% to 20% (see discussion in section 1 above and in BPPD 2007). As a
condition of registration, Monsanto was required to address cross resistance in existing Bt corn
and Bt cotton products for Cry 1 A. 105, CrylFa and CrylAc.

Monsanto subsequently provided materials to address these three areas of uncertainty as part of a
new amendment request for a reduced 5% refuge for non-cotton regions. The response,
including a discussion of cross resistance and a new model, is included in a study titled
"Assessment of the Impact of MON 89034 Introduction on Bt Resistance Development in
European and Southwestern Corn Borer" (MRID# 474748-01).

a) Monsanto's Proposed Amendment to Support a 5% Refuge for MON 89034

Monsanto's proposal for a 5% refuge with MON 89034 included two major components: (1) a
discussion of the cross resistance potential between the toxins in MON 89034 and (2) a
deterministic model to simulate a 5% refuge and the risk of resistance for ECB and SWCB.

Each of these sections is described and reviewed individually below.

In lieu of submitting new dose determination data for Cry2Ab2 and Cry 1 A. 105 for the major
target pests, Monsanto has used the existing dose information (submitted for the original
registration) in the new simulation model. Therefore, Monsanto's response to the dose
determination uncertainties (detailed in BPPD 2007 and section 1 above) will be discussed and
reviewed in the modeling portion (section ii.) below.

i) Cross Resistance Potential

MON 89034 contains both Cry 1 A. 105 and Cry2Ab2, which target the same lepidopteran corn
pest complex. The Cry 1 A. 105 toxin is a "chimeric" protein containing domains I and II and the
C-terminal from CrylAc and domain III from CrylFa while the Cry2Ab2 protein is the same as
that currently expressed in Monsanto's Bollgard II cotton. Monsanto has sufficiently
demonstrated that the cross resistance potential between these two proteins should be low,
primarily due to differing modes of action (see discussion in BPPD 2007). In evaluating new
PIP traits, the landscape of previously registered toxins in the same crop must be taken into

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account. In addition, for corn PIPs, cotton must also be considered because one of the key target
pests, corn earworm (also referred to as cotton bollworm, CBW, when a pest on cotton), is a pest
of both crops. As a condition of registration, Monsanto was required to address cross resistance
in existing Bt corn and Bt cotton products for Cry 1 A. 105, Cry lFa and Cry 1 Ac.

Monsanto's amendment submission for MON 89034 contained a discussion of cross resistance
including an analysis of previous studies as well as a summary of recently developed data.
Analysis of existing data was conducted for four toxin combinations: 1) Cryl Ab vs. Cryl Ac; 2)
Cry IF vs. CrylAb and Cryl Ac; 3) Cry2Ab2 vs. Cryl proteins; and 4) Cryl A. 105 vs. CrylAb
and Cryl Ac. New data were presented for comparisons between Cryl A. 105 and Cry2Ab2 vs.
CrylF.

CrylAb vs. CrylAc: Based on a literature review of binding studies with numerous lepidopteran
species, CrylAc is known to have strong cross resistance with CrylAb. Both toxins share a high
affinity binding site in ECB, CEW/CBW, SWCB, FAW, and others (references cited in MRID#
474748-01).

CrylF vs. CrylAb and CrylAc: CrylF also shares a binding site with CrylAb/CrylAc, though
the level of cross resistance between CrylF and Cryl A is not as strong as CrylAb vs. CrylAc.
ECB resistant to CrylAb have been shown to be partially resistant to CrylF although CrylF
resistant ECB were not cross resistant to CrylAb and only slightly resistant to CrylAc. Similar
trends have also been shown with tobacco budworm (Heliothis virescens, TBW) (references
cited in MRID# 474748-01). Overall, CrylF can be considered partially cross resistant to
CrylAb and CrylAc. The availability of binding sites may explain the partial cross resistance:
CrylAb and CrylAc could have more different sites to bind with than CrylF so that resistance to
CrylF still allows for some binding of CrylAb or CrylAc.

Cry2Ab vs. Cryl proteins: A literature review suggests that Cry2Ab has no cross resistance
potential with any of the currently registered Cryl proteins including CrylAb and CrylAc.
Studies have been conducted with numerous cotton pests including CEW, TBW, pink bollworm
(Pectinophora gossypiella, PBW), and Helicoverpa armigera that revealed no shared binding
sites between Cry2A and CrylAb or CrylAc proteins. Additional studies with Cryl Ac-resistant
TBW, CEW/CBW, and PBW found no cross resistance with Cry2Ab (references cited in MRID#
474748-01). Previously submitted data by Monsanto for MON 89034 (Head 2006; reviewed in
BPPD 2007) demonstrated that Cryl Ab-resistant ECB were not found to be cross resistant with
Cry2Ab while Cry2Ab2-resistant H. armigera were not cross resistant with Cryl A. 105 or
CrylAc.

Cryl A. 105 vs. CrylAb and CrylAc: For CrylAb, a previously submitted binding study with
ECB (Head 2006; reviewed in BPPD 2007) showed that the protein has a distinct binding site
from Cry 1 A. 105. This was confirmed by studies with Cry 1 Ab-resistant ECB and sugarcane
borer (Diatraea saccharalis, SCB) that showed no cross resistance with Cry 1 A. 105. Monsanto
argues that due to similar characteristics between CrylAb and CrylAc (i.e., mode of action), it is

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reasonable to assume that Cry 1 Ac should not be cross resistant with Cry 1 A. 105. However, no
binding studies or experiments with resistant colonies were described to verify that assumption.

CrylA.105 and Cry2Ab2 vs. CrylF: New data were cited by Monsanto (Schlenz et al. 2008) to
assess the cross resistance potential between CrylA.105/Cry2Ab2 and CrylF using CrylF-
resistant ECB and FAW colonies. Artificial diet bioassays were used to test Cry 1 A. 105,
Cry2Ab2, and control groups against ECB and FAW colonies previously selected for high-level
CrylF resistance as well as unselected control colonies. A range of five concentrations was used
and the test was conducted over a seven day period to determine growth inhibition (GI50) for
each colony. The results showed that, as expected, CrylF-resistant ECB and FAW were not
cross resistant with Cry2Ab2 — the GI50 resistance ratios (CrylF-resistant: CrylF-susceptible)
were 1.4 for ECB and 0.11 forFAW. With Cryl A. 105, the GI50 resistance ratios were > 3.9 for
ECB and 7.0 for FAW, indicating low level cross resistance.

Table 7: Cross resistance potential of MON 89034 (CrylA.105 and Cry2Ab2) with previously
registered Bt corn toxins.

Bt toxins in MON 89034

Existing Bt toxins

CrylA.105

Cry2Ab2

CrylAb

No cross resistance (ECB,
SCB)

No cross resistance (ECB)

CrylAc

Unlikely cross resistance, but
unverified experimentally

No cross resistance (TBW,
PBW, CEW/CBW)

CrylF

Low level cross resistance
(ECB, FAW)

No cross resistance (ECB, FAW)

BPPD Review - Cross Resistance

BPPD agrees with Monsanto's characterization of the cross resistance potential for the
CrylA.105 and Cry2Ab2 toxins with (1) each other (previously demonstrated in Head 2006), (2)
CrylF, and (3) CrylAb. Binding and resistant colony work conducted by Monsanto and other
researchers clearly show that no cross resistance can be expected between Cry 1 A. 105, Cry2Ab2
and CrylAb (see Table 7 above). New data referenced in Monsanto's amendment request also
experimentally demonstrate the cross resistance potential between CrylF and Cry2Ab2 (no cross
resistance) and CrylA.105 (low cross resistance).

Nonetheless, BPPD still has reservations about CrylAc. While Monsanto has made the case that
Cryl Ac should be expected to behave like CrylAb due to a similar mode of action, no
experimental data (i.e., binding studies or bioassays with resistant insect colonies) were provided
either in the original MON 89034 IRM submission (Head 2006) or the follow-up amendment
request (MRID# 474748-01). BPPD notes that Cry 1 A. 105 (a chimeric protein) contains domains
I and II and the C-terminal from CrylAc. Cross-resistance could result when proteins share key

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structural features, which allows one resistance mechanism to confer resistance to more than one
protein (Tabashnik 1994; Gould et al. 1995).

BPPD recognizes that at the present time there are no registered Bt corn products containing
Cryl Ac. Therefore, exposure to ECB and SWCB to Cryl Ac is unlikely, as neither is known as a
cotton pest. FAW may occasionally feed on cotton, but favors corn and is also unlikely to have
much exposure to Cryl Ac. On the other hand, successive generations of CEW may feed on both
corn and cotton during the same growing season. This could result in a potential "double"
exposure to Bt cotton (including Cry 1 Ab) and Bt corn (including Cry 1 A. 105) and increased
selection pressure for resistance, particularly if there is a risk of cross resistance.

Given that Monsanto has proposed to substantially reduce refuge for MON 89034 from 20% to
5%, cross resistance is an important consideration even for CrylAc. Although improbable,
BPPD cannot rule out that a CEW/CBW population could develop CrylAc resistance in cotton
and then encounter MON 89034 corn. [Tabashnik et al. (2008) have argued that CrylAc
resistance has already evolved in CBW in the south, although this conclusion has been disputed
(Moar et al. 2008).] Should there be a degree of cross resistance between CrylAc and
Cryl A. 105, MON 89034 might functionally have only Cry2Ab2 remaining as an effective toxin
against CEW. With a reduced refuge (5%), selection pressure could be increased for resistance
to MON 89034 and Cry2Ab2 (which also is expressed in Bollgard II cotton). So that BPPD can
fully assess the cross resistance potential of Cryl A. 105 with CrylAc in CEW/CBW, it is
recommended that Monsanto provide additional information either experimentally (e.g., binding
studies or with resistant colonies) or using another analysis. Alternatively, Monsanto could
revise the CEW model submitted with the original MON 89034 IRM plan (Head 2006) to
support 20% refuge in cotton-growing regions. This model simulated CEW resistance to MON
89034 and assumed complete cross resistance between Cryl A. 105 and CrylAc; the model could
be adapted to evaluate a 5% refuge in the Corn Belt with similar assumptions.

ii) Modeling

As part of the review of Monsanto's initial IRM plan for MON 89034, BPPD identified the need
for additional species-specific (e.g., ECB and SWCB for the Corn Belt), spatially-explicit,
landscape modeling to explore the durability of MON 89034 versus single-protein Bt corn
products (BPPD 2007). Previously, Monsanto had cited the modeling work of Roush (1998) to
demonstrate that a 5% refuge was justified with a two toxin pyramided product. Roush's model
made a number of key assumptions, particularly in terms of the toxin expression level in
pyramided product. For homozygote susceptible insects, the model assumed 95% mortality and
70%) mortality for heterozygotes (with one resistance allele) for each toxin. The dose
information provided by Monsanto for MON 89034, however, was not sufficient to demonstrate
that each protein would kill 95%> of the homozygous susceptible insects and 70%> of the
heterozygotes (see BPPD 2007). BPPD recommended that Monsanto further characterize the
dose expression for the MON 89034 toxins for the major target pests of the Corn Belt (ECB and

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SWCB). Given the dose uncertainties, BPPD could not at the time of registration support the use
of Roush's model to justify a lower 5% refuge for MON 89034 (BPPD 2007).

Rather than re-run dose studies for Cry 1 A. 105 or Cry2Ab2, Monsanto created a deterministic
model for ECB and SWCB using dose mortality estimates consistent with the previously
conducted studies. The model (Gustafson & Head 2008; contained in MRID# 474748-01)
included the toxins from other registered Bt corn products (Cryl Ab, CrylF) and had a number of
assumptions and parameters:

•	Dose mortality for ECB: 99.9% for Cryl (CrylAb, CrylF, Cryl A. 105) and Cry2Ab2
toxins (one mortality scenario was modeled);

•	Dose mortality for SWCB: 99 - 99.5% for Cryl and 85 - 95% for Cry2Ab2 (six dose
mortality scenarios were modeled);

•	Complete resistance to Cry2Ab2 and Cry 1 A. 105 (i.e., survival probability of
heterozygote resistant individuals =1) with no fitness costs;

•	Heterozygotes (i.e., with one resistance allele) survival probability is twice that for
homozygote susceptible insects;

•	Three cross resistance scenarios: 1) Cryl A. 105 and CrylAb fully cross resistant (but not
CrylF) (the "base case" scenario); 2) Cryl A. 105 and CrylF fully cross resistant (but not
CrylAb) (alternate "base case" scenario), and 3) Cryl A. 105, CrylAb, and CrylF all fully
cross resistant (worst case scenario);

•	All resistance alleles (Cryl, Cryl A. 105, and Cry2Ab2) have initial frequencies of 0.005.
CrylAb and CrylF are modeled as one output (i.e., estimated time to resistance for

Yi el dgard/Hercul ex);

•	MON 89034 was assumed to have a refuge of 5%; other single gene products (Yieldgard
and Herculex) were assumed to have 20% refuge;

•	ECB and SWCB have no natural refuge (i.e., wild hosts or other cultivated crops that
could serve as a source of susceptible insects) and have two generations per year on corn;

•	A range of market share adoption values for MON 89034 and other products (Herculex
and Yieldgard) were included in the model simulations. MKT 1 = 100% MON 89034;
MKT 2 = 50% MON 89034, 25% MON 810, 25% TCI507; MKT 3 = 0% MON 89034,
50% MON 810, 50% TCI507.

Most of the assumptions above were conservative estimates, with the possible exception of the
dose mortality parameters for SWCB (see discussion in the BPPD review section below).
Simulations were run with both ECB and SWCB to estimate the time to resistance (in years; up
to a maximum of 30 years) and resistance allele frequency for each of the three cross resistance
scenarios described above. Within each cross resistance scenario, model runs were conducted
for three different market adoption contingencies of MON 89034, MON 810 (CrylAb
Yieldgard) and TCI507 (CrylF Herculex).

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ECB Results

For ECB, the results of the model runs were relatively consistent among the different cross
resistance and market adoption scenarios. In almost all cases, the durability of the MON 89034
toxins (Cry 1 A. 105 and Cry2Ab2; assuming a 5% refuge) exceeded the 30 year time frame of the
model. Only in the "worst case" cross resistance scenario (i.e., all three toxins cross resistant)
was the durability of Cry 1 A. 105 less than 30 years (29 years) for ECB — Cry2Ab2 remained
effective in all model simulations (> 30 years). For the other Cryl toxins (Cryl Ab and CrylF)
that are expressed in other Bt corn products, resistance developed in less than 30 years for some
of the cross resistance and market adoption scenarios. In the "base case" (CrylAb and
Cryl A. 105 cross resistant), the durability of CrylAb/CrylF lasted 26 years (0% MON 89034,
50% MON 810, 50% TCI507) and 29 years (50% MON 89034, 25% MON 810, 25% TCI507).
However, for the alternate base case (CrylF and Cryl A. 105 cross resistance), resistance to
CrylAb/CrylF did not evolve within 30 years. In the worst case scenario (all three toxins cross
resistant), resistance to CrylAb/CrylF developed in 29 years.

SWCB Results

For SWCB, more model simulations were run to account for a range of dose mortalities.

Overall, durability of the traits was affected by the dose mortality scenarios — the simulations
with lower dose mortality frequently resulted in fewer years to resistance in Cryl A. 105 and
CrylF than those with higher dose mortalities. As with ECB, Cry2Ab2 remained durable (> 30
years) in all but one of the simulations regardless of the cross resistance or market adoption
scenario.

For the "base case" cross resistance scenario, the time to resistance was lowest in the market
adoption scheme (MKT 3) without MON 89034 (50% MON 810, 50% TCI507) ranging from 17
years (lower dose mortalities for Cryl and Cry2Ab2 toxins) to 20.5 years (higher dose
mortalities). Once MON 89034 was added to the model (MKT 1 and 2), the time to resistance
with the Cryl toxins increased by 2 -2.5 years for all simulations. Cry 1 A. 105 and Cry2Ab2 did
not evolve resistance in any of the model runs for MKT 2, although there were two instances
with MKT 1 (100% MON 89034) in which resistance evolved within 30 years. In both of these
cases, lower dose mortality values for SWCB (85% for Cry2Ab2; 99% for Cryl A. 105) were
included in the model.

Time to resistance in the "alternate base case" (CrylF and Cryl A. 105 cross resistant) was > 30
years in almost all cases. Only in the simulation that incorporated the lowest dose mortality
values (85%) for Cry2Ab2 and 99% for Cryl A. 105) did resistance evolve to one of the toxins
(28.5 years for Cryl A. 105).

In the "worst case" (CrylAb, CrylF and Cryl A. 105 are all cross resistant), resistance developed
in all scenarios for both the Cryl toxins and Cryl A. 105. Conversely, Cry2Ab2 remained
durable (> 30 years) for all of the simulations. Time to resistance in the Cryl and Cryl A. 105

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toxins was lowest (17 years) in the model run using the lower SWCB dose mortality values (85%
for Cry2Ab2 and 99% for Cry 1 A. 105). Resistance also evolved for case with the higher dose
mortality values, ranging up to 22 years for each toxin. A truncated summary of the results for
all of the model simulations is contained in Table 8 below — the complete results of the modeling
are detailed in Tables 5 and 6 in Monsanto's submission (MRID# 474748-01).

Table 8: Results of Monsanto's model simulations of MON 89034 (5% refuge), MON 810,
TC1507 (20%) refuge) expressed in years to resistance (30 year maximum). Derived from data
reported in MRID# 474748-01.





Cross resistance scenario



Pest

Base case1

Alt. base

Worst case3





MKT 1

MKT 2

MKT 3

case2



CrylA.105

>30

>30

N/A

>30

29

ECB

Cry2Ab2

>30

>30

N/A

>30

>30



CrylAb/CrylF

N/A

29

26

>30

29



CrylA.105

22.5 ->30

>30

N/A

28.5 ->30

17-22

SWCB

Cry2Ab2

25 ->30

>30

N/A

>30

>30



CrylAb/CrylF

N/A

19-23

17-20.5

>30

17-22

1	Base case = CrylAb and Cry 1 A. 105 cross resistant; three different marketing scenarios included (Mkt 1 = 100%
MON 89034, 0% MON 810/TC1507; Mkt 2 = 50% MON 89034, 25/25% MON 810/TC1507; Mkt 3 = 0% MON
89034, 50/50% MON 810/TC1507).

2	Alt. base case = Cry IF and CrylA. 105 cross resistant (only Mkt 2 simulated).

3	Worst case = CrylA. 105, CrylAb, and Cry IF all fully cross resistant (only Mkt 2 simulated).

Based on the model work, Monsanto concluded that the durability of the MON 89034 proteins
(CrylA. 105 and Cry2Ab2) will remain strong for both ECB and SWCB. With a 5% refuge,
Monsanto predicted that MON 89034 will have at least 22 years durability even under the "worst
case" model assumptions. The durability of Cry2Ab2 in the model was particularly robust in
almost all simulations for ECB and SWCB (only one simulation predicted less than 30 years
durability). Resistance to Cry 1 A. 105 was also rare in most simulations, although the "worst
case" modeling (assuming complete cross resistance with CrylAb and CrylF) showed resistance
developing in less than 30 years. Monsanto also noted that in the simulations with different
market adoption scenarios, the addition of MON 89034 increased the time to resistance for the
previously registered Cryl toxins (CrylAb and CrylF).

BPPD Review - Modeling

BPPD agrees with Monsanto's overall conclusions that the model simulations demonstrate the
effectiveness in delaying resistance of MON 89034 and provide support for the use of a 5%
refuge in the Corn Belt. However, BPPD notes that some of the parameters and assumptions of
the model could be revised to improve and expand the overall analysis.

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For ECB, the model clearly predicts that resistance is unlikely to evolve to Cry 1 A. 105, Cry2Ab2,
or the previously-registered Cryl toxins. Even under the worst case scenario that assumed
complete cross resistance, the durability of all toxins was at least 29 years. Presumably, a large
reason for this is the high dose mortality of the MON 89034 toxins against ECB. Previous
mortality studies submitted by Monsanto (reviewed in BPPD 2007) showed that the Cryl A. 105
and Cry2Ab2 proteins in MON 89034 each provide essentially 100% control of ECB (Monsanto
assumed 99.9% mortality for each toxin in the model).

For SWCB, the model predictions were more varied, largely due to the different simulations run
with the range of dose mortality assumptions. Not surprisingly, the simulations that were run
with the lower mortality estimates (i.e., 85% for Cry2Ab and/or 99.0% for Cryl) resulted in less
time to resistance than those using the higher dose values. In the worst case simulations with the
lower dose estimates, SWCB resistance evolved in 17 years to both Cryl A. 105 and
CrylAb/CrylF while with the higher doses resistance took 21 or 22 years to develop. As with
ECB, Cry2Ab2 remained durable (>30 years) for almost all of the simulations.

A number of factors appeared to influence the model results. BPPD agrees with Monsanto that
the addition of MON 89034 in the simulations testing various market adoption scenarios delayed
resistance in the other previously-registered Cryl toxins. Likely, these results were due to less
selection pressure on each individual toxin because of a diverse mosaic of toxins in the
landscape. Cross resistance was also an important variable. Monsanto's "base case" for cross
resistance assumed cross resistance between Cry 1 Ab and Cry 1 A. 105. This resulted in resistance
always developing in CrylAb/CrylF (i.e., within 30 years), although Cryl A. 105 and Cry2Ab2
durability remained strong. On the other hand, when cross resistance between Cryl A. 105 and
Cry IF was assumed, resistance rarely developed in either the MON 89034 toxins or the existing
Cryl toxins. In the worst case scenario (all three toxins cross resistant), the durability of
Cryl A. 105 to SWCB was clearly impacted relative to the other cross resistance simulations.
Conversely, Cry2Ab remained durable in almost all cases regardless of the varying assumptions
and scenarios included in the model. Since Cry2Ab is not cross resistant to the Cryl toxins, this
result was not unexpected.

BPPD generally agrees with Monsanto that conservative assumptions were used in the model.
BPPD notes, however, that several of the parameters could have been expanded or have included
an additional degree of conservatism or additional refinement to improve the model analysis.
For example, Monsanto's simulations assumed a 5% refuge for MON 89034 (while maintaining
the 20% refuge for the other Bt toxins). Although MON 89034 is currently registered with a
requirement for a 20% refuge, simulations were not run with the larger refuge size. Separate
simulations with 5% and 20% MON 89034 refuges would have been useful for comparative
purposes. To illustrate using the SWCB "base case" (with the three different marketing adoption
cases), with no MON 89034 adoption resistance to the Cryl toxins occurred in 17 - 20.5 years.
When MON 89034 with a 5% refuge was included, the time to Cryl resistance was 19-23 years
— indicating that the addition of MON 89034 provides some delay in resistance development (2 -

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2.5 years). It would have been interesting to observe the impact of adoption of MON 89034 with
a 20% refuge on Cryl resistance. In all likelihood, the time to resistance would be increased,
although the magnitude of such an increase is unknown. Had the difference been small, it could
be argued that there is little value gained in having a 20% refuge versus a 5% refuge.

The model time frame (maximum 30 years) was another limiting parameter. Many of the
simulations resulted in no resistance within the 30 year time period of the model, so it was
difficult to discern the effects of certain variables (i.e., cross resistance, market adoption, dose
mortality) between model runs. Had the time horizon been extended (e.g. to 50 years),
differences between the various model scenarios may have been apparent.

For the SWCB simulations, Monsanto used dose mortality range of 85-95% for Cry2Ab2 and
99-99.5% for Cryl toxins. Based on the dose data submitted for the registration of MON 89034
(reviewed in BPPD 2007), BPPD believes these estimates to be somewhat high. For example,
dose data for Cry2Ab2 and SWCB suggested a mortality range of 80-90%. The Cry 1 A. 105
protein in MON 89034 provided approximately 95% control in mortality assays, though the other
registered Cryl proteins (Cryl Ab and CrylF) may provide closer to 99% of SWCB. Had the
model simulations been run with these more conservative dose estimates, it is likely the time to
resistance would have been reduced in some scenarios. The extent of this effect is unknown,
although BPPD notes that the differences between the lower Cry2Ab2 dose (85%) and the
highest dose (95%) in the range appeared to be negligible in the model runs (i.e. no differences
in years to resistance).

iii. BPPD Review - Overall Proposal to Reduce Refuge

Taken together, Monsanto's cross resistance and modeling work provide justification for
reducing the MON 89034 structured refuge requirement in the Corn Belt from 20% to 5% non-Bt
corn. Key elements of support include a lack of cross resistance between Cry2Ab2 and Cryl
proteins and model simulations which demonstrate strong durability of Cryl A. 105 and Cry2Ab2
under a variety of dose, market adoption, and cross resistance scenarios. Reducing the refuge to
5%> is unlikely to increase the selection pressure for resistance in either MON 89034 or the other
previously-registered CrylAb or CrylF corn hybrids.

Despite a good case for a refuge reduction, BPPD notes that there are still some limitations and
uncertainties in the analysis that could be addressed to provide additional support for the
proposal. These areas include:

• Cross resistance between Cryl Ac and Cryl A. 105. Cryl Ac is registered in Bt cotton
products and the chimeric protein Cryl A. 105 has two Cryl Ac domains. CEW feed on
both corn and cotton and successive generations may have exposure to both Cryl A. 105
and Cryl Ac during the same growing season;

• No model simulations were conducted to compare 5% vs. 20% refuge for MON 89034;
the model assumed a 5% refuge for MON 89034;

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• The model time horizon was limited to 30 years. Many of the model runs did not evolve
resistance during this time precluding comparisons between some of the scenarios;

• SWCB model simulations included dose mortality estimates somewhat higher than those
suggested by previously-submitted data. For Cry2Ab2, mortality ranged from 80 to 90%
in dose testing submitted for MON 89034 (instead of 85-95% used in the model).

Cry 1 A. 105 caused 95% mortality in submitted dose studies, though a range of 99-99.5%)
was used in the model.

As a condition of registration of MON 89034, Monsanto was required to address cross resistance
in existing Bt corn and Bt cotton products for Cry 1 A. 105, Cry lFa and Cry 1 Ac. Monsanto has
sufficiently addressed cross resistance for Cry 1 A. 105 and CrylFa, but there are lingering
questions regarding Cry 1 Ac and Cry 1 A. 105. The amendment submission included only a
circumstantial discussion of Cryl Ac cross resistance with an assumption that the protein will
behave similarly to CrylAb. But, since Cryl A. 105 contains domains I and II and the C-terminal
from Cryl Ac, BPPD is still concerned about the potential for cross resistance. As such, BPPD
recommends additional work (as described in the cross resistance section above) to satisfy the
condition of registration. Should additional cross resistance work (as previously described)
demonstrate little or no cross resistance potential between Cryl A. 105 and Cryl Ac, further
support could be provided for the use of a 5% refuge in the Corn Belt.

In terms of resistance risk for MON 89034, cross resistance between Cry 1 Ac and Cry 1 A. 105 is
an issue primarily for CEW. This insect is known to feed on both corn and cotton during the
same growing season and could be exposed to Cry 1 A. 105 (in corn) and then Cry 1 Ac (in
Bollgard cotton) later in the growing season. Theoretically, CEW could develop resistance to
Cryl Ac due to exposure in cotton — should there be a degree of cross resistance between Cryl Ac
and Cryl A. 105, MON 89034 could functionally have only Cry2Ab2 remaining as an effective
toxin against CEW. With a reduced refuge (5%), selection pressure could be increased for
resistance to MON 89034 and Cry2Ab2 (which also is expressed in Bollgard II cotton). While
these are legitimate concerns (and reason for additional analysis), BPPD notes that there are
several mitigating factors that reduce the overall resistance risk for CEW and MON 89034.

First, CEW is generally a lesser pest in the Corn Belt than ECB (and in some areas SWCB),
primarily due to poor overwintering capability in much of the Corn Belt (i.e., north of Virginia,
Tennessee, and Missouri). Therefore, selection pressure for resistance will likely be less for
CEW than ECB which does overwinter in the Corn Belt. On the other hand, in cotton-growing
regions south of the Corn Belt where CEW can overwinter, conditions for resistance
development may be more probable. In these areas, a 20% refuge (approved with the initial
registration of MON 89034) will still be required. Along these lines, in Monsanto's original
MON 89034 IRM submission, modeling was conducted to support the use of a 20% refuge for
CEW in southern cotton-growing regions (see discussion in BPPD 2007).

A second mitigating factor is that CEW is a highly polyphagous insect and is known to feed on a
wide variety of plants including weeds, wild hosts, and other cultivated crops (unlike ECB and
SWCB which feed primarily on corn). Analysis conducted for Bollgard II cotton determined

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that a natural refuge is present for CEW (CBW) in cotton growing areas in the southeastern U.S.
(see BPPD 2004b and 2006b). It is likely that in the Corn Belt, there is also at least some degree
of natural refuge that could supplement a 5% structured refuge to help reduce the overall
selection pressure on CEW and MON 89034. BPPD emphasizes that natural refuge for CEW
has been quantified only in cotton-growing regions and that host utilization patterns in the Corn
Belt are speculative.

The other modeling parameter uncertainties detailed above are relatively minor, though a more
expanded model analysis could have provided stronger support for the proposal. Separate model
runs with 5% and 20% MON 89034 refuges would have been useful to compare potential
differences in times to resistance. Although since most of the simulations did not result in
resistance within 30 years, any differences would have been difficult to detect. Expanding the
time horizon of the model (for example, from 30 years to 60 years) possibly could have fleshed
out variation between model scenarios and provided a more thorough basis for comparison.
Finally, BPPD would have preferred if Monsanto had used the more conservative estimates of
SWCB dose mortality (based on the MON 89034 dose data), though the impact on the model
output would likely have been relatively small.

MON 89034 was originally registered as a conditional time-limited registration (with an
expiration date of September 30, 2010) and BPPD recommends reevaluating 5% refuge if
warranted by cross resistance data or other information during this interim period.

3. Conditional IRM Data Submitted for MON 89034 and MON 89034 x MON 88017 (2010)

Monsanto has submitted a number of reports to EPA to satisfy the Insect Resistance
Management conditions of registration for MON 89034 and MON 89034 x MON 88017. These
submissions are summarized in Table 9 below.

Table 9: Conditional IRM data submitted by Monsanto for MON 89034 and MON 89034 x
MON 88017 corn.

Date

Submission

MRU) No.

7/29/08

Copies of grower agreements

None

9/22/08

Grower education: copy of Monsanto's Technology Use Guide
(2009)

None

10/6/08

Compliance Assurance Program (ABSTC plan for lepidoptera
and MON 863 plan for corn rootworm)

None

6/29/09

Cross resistance data: comparative binding of Cry 1 A. 105 and
Cry 1 Ac proteins in tobacco budworm and cotton bollworm

477912-01

6/29/09

Resistance monitoring: baseline susceptibility data for
Cry 1 A. 105 and ECB

477912-02

6/29/09

Resistance monitoring: baseline susceptibility data for
Cry 1 A. 105 and CEW

477912-03

100

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11/3/09

Compliance, grower education, resistance monitoring, remedial
action for MON 89034: updated materials for 5% refuge

479035-01

11/9/09

Compliance, grower education, resistance monitoring, remedial
action for MON 89034 x MON 88017: updated materials for
5% refuge

479083-01

In a letter to EPA dated September 28, 2009, Monsanto requested additional time to fulfill some
of the conditional data requirements. One of these requirements is baseline susceptibility
(resistance monitoring) data for southwestern corn borer. This report was submitted on August
27, 2010.

Each of the submissions detailed in Table 9 was reviewed in BPPD (2010a) and is addressed
below by discipline.

Cross Resistance

For the original registration of MON 89034 corn, Monsanto demonstrated low likelihood of
cross resistance between the expressed toxins Cry 1 A. 105 and Cry2Ab2 (see review in BPPD
2007). BPPD was concerned, however about potential cross resistance between Cry 1 A. 105 in
MON 89034 and Cry IF and Cry 1 Ac toxins that are expressed in other registered Bt corn and
cotton PIPs. A condition of registration was imposed to conduct additional cross resistance
studies on these toxins (documented in the June 10, 2008 MON 89034 registration notice). As
part of the their amendment request for a 5% refuge Monsanto provided sufficient data to
demonstrate that cross resistance is unlikely between Cry 1 A. 105 and Cry IF, though BPPD still
had uncertainties about Cryl Ac (see review in BPPD 2008a). Given this concern, the condition
to conduct cross resistance data with Cryl A. 105 and Cryl Ac was maintained for the 5% refuge
amendment approval (see December 15, 2008 approval letter).

Monsanto addressed this concern by submitting a report: "Comparative Binding of the Bacillus
thuringiensis Cryl A. 105 and Cryl Ac Proteins to Cotton Bollworm (Helicoverpa zea) and
Tobacco Budworm (Heliothis virescens) Brush Border Membranes" (MRID# 477912-01).

Summary of Monsanto's Cross Resistance Submission (MRID# 477912-01)

Monsanto addressed the question of potential cross resistance between Cryl A. 105 and Cryl Ac
by conducting binding assays using brush border membranes (BBM) from two cotton pests,
tobacco budworm (TBW) and cotton bollworm (CBW). To accomplish this objective, BBMs
were obtained from homogenized and filtered whole body third instar larvae (centrifuged into
pellets) using established procedures (English et al. 1991). The BBM proteins were then
suspended onto nitrocellulose membranes and separated using gel electrophoresis (SDS-PAGE)
techniques. Membranes containing the separated BBMs were exposed to incubations containing
either Cry 1 A. 105 or Cry 1 Ac. Bound protein was detected using a mammalian (goat) antibody
capable of recognizing both Cryl A. 105 and Cryl Ac.

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An analysis of the BBMs of TBW and CBW revealed that both insects have similar protein
components (as detected by SDS-PAGE). Because of this, the binding experiments with
Cry 1 A. 105 and Cry 1 Ac showed similar patterns between the insects. The specific binding
characteristics for each insect are summarized in Table 10 below.

Table 10. Binding profiles for Cry 1 A. 105 and Cry 1 Ac in TBW and CBW (preferential binding
sites as analyzed by SDS-PAGE). (Created from data submitted to EPA in MRID# 477912-01)

TBW

CBW

Cryl A. 105 unique binding
sites (kDas)

20, 42, 50, 95

20, 42, 50, 90

Cryl Ac unique binding
sites (kDas)

57, 63

Common binding sites for
both toxins (kDas)

40, 120, 150

Although there are shared binding sites for Cry 1 A. 105 and Cry 1 Ac in both TBW and CBW,
Monsanto concluded that "...these proteins have different insecticidal modes of action." Each
toxin also has a number of unique bands such that the binding profiles were sufficiently different
between the two toxins.

BPPD Review

BPPD agrees with Monsanto that there appear to be distinct binding sites for each toxin in both
TBW and CBW (see Table 10 above). The presence of three common binding sites could
indicate the potential for some cross resistance between Cry 1 A. 105 and Cry 1 Ac. The 120 and
150 kDA bands appeared to be relatively strong (as or more intense than any of the unique
binding bands) in the photographs of the gel plates.

While some shared binding sites between two toxins is not a definitive indicator of cross
resistance, without other supporting data (e.g., assays with toxin-resistant colonies) the
possibility cannot be eliminated. To BPPD's knowledge, no additional studies have been
conducted with Cry 1 A. 105 and Cry 1 Ac. As noted in BPPD (2008a), Cry 1 A. 105 is a chimeric
protein that contains domains I and II and the C-terminal from Cryl Ac. Cross-resistance can
result when proteins share key structural features, which allows one resistance mechanism to
confer resistance to more than one protein (Tabashnik 1994; Gould et al. 1995). Because of the
structural similarities, some shared binding sites may be expected. Since there are a number of
unique binding sites for each toxin, any cross resistance due to common binding sites will likely
be at low levels.

Cryl Ac could be a potential concern due to the presence of Bt cotton PIPs that express the toxin
(Cry 1 Ac is not found in any presently registered Bt corn PIPs). TBW is not likely to be at risk
for cross resistance because it is not known to be a corn pest. Similarly, corn pests such as ECB

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and SWCB are unlikely to be exposed to Cry 1 Ac in cotton. On the other hand, successive
generations of CBW can feed on both corn and cotton during the same growing season and may
be exposed to Bt toxins in both crops. BPPD notes, however that there are several mitigating
factors that could lower the impact of any cross resistance development in CBW to
Cry 1 A. 105/Cry 1 Ac. First, CBW is generally a lesser pest in the Corn Belt than ECB, primarily
due to poor overwintering capability in much of the region (i.e., north of Virginia, Tennessee,
and Missouri). Therefore, the potential for resistance to evolve will likely be less for CBW than
ECB (which does overwinter in the Corn Belt). For cotton-growing regions south of the Corn
Belt, where CBW can overwinter and conditions for resistance development may be more
favorable, a 20% refuge is required for MON 89034.

Resistance Monitoring

As part of the terms of registration, Monsanto was required to implement a resistance monitoring
program for MON 89034 and MON 89034 x MON 88017. For MON 89034, resistance
monitoring was required for the main lepidopteran pests (ECB, CEW, and SWCB) and the two
expressed toxins (Cry 1 A. 105 and Cry2Ab2). In addition to the lepidopteran pests, monitoring
for corn rootworm (CRW) and the Cry3Bbl toxin was also mandated with the MON 89034 x
MON 88017 registration.

Monsanto was directed to utilize existing monitoring strategies for both pest complexes that had
been developed for previous PIP registrations, although a revised monitoring plan for CRW was
requested as a term of registration for MON 89034 x MON 88017. The core components of the
monitoring program include insect sampling in areas of high risk of resistance development,
bioassays to detect resistant individuals, and investigations of report of unexpected pest damage.
To support these objectives, baseline susceptibility data were required to be submitted for
Cry 1 A. 105 and Cry2Ab2 activity against ECB (Cry 1 A. 105 only), CEW, and SWCB.

Resistance monitoring was also required for fall armyworm (FAW) with sweet corn uses of the
MON 89034 and MON 89034 x MON 88017. FAW monitoring is triggered if acreage exceeds
5,000 in any county known to support overwintering of the insect. A proposed monitoring plan
and FAW baseline susceptibility data to Cry 1 A. 105 and Cry2Ab2 were required as terms of
registration.

Monsanto's Responses to Resistance Management Requirements

To address the resistance monitoring terms and conditions of registration, Monsanto submitted
two baseline susceptibility studies for ECB (MRID# 477912-02) and CEW (MRID# 477912-03).
Both studies were conducted with Cry 1 A. 105 only — for Cry2Ab2, Monsanto cited previously
submitted studies for ECB (part of the original MON 89034 IRM submission, MRID# 469514-
30) and CEW (submitted with Bt cotton monitoring data, see review in BPPD 2005a). The
company requested additional time (until August 31, 2010) to conduct baseline studies for
SWCB (letter to EPA dated September 24, 2009). Separately, Monsanto asked for an extension

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(to April 1, 2012) to submit FAW baseline data (letter to EPA dated June 3, 2010). Baseline
studies for SWCB were submitted to the Agency and will be evaluated in the future.

Monsanto also provided a revised lepidopteran monitoring plan (contained in MRID# 479035-
01) that mirrors a previously-submitted plan submitted by the Agricultural Biotechnology
Stewardship Technical Committee (ABSTC) (MRID# 474070-01). For CRW monitoring,
Monsanto cited the plan (MRID# 478836-03) submitted for SmartStax corn (EPA Reg. No. 524-
581), a separate Bt corn product that targets rootworm.

ECB Baseline Susceptibility Data - Cry 1 A. 105 (MRID# 477912-02)

Baseline susceptibility studies for ECB and Cry 1 A. 105 were conducted by Dr. Blair Siegfried
and Dr. Terrence Spencer of the University of Nebraska. Dr. Siegfried's laboratory has
conducted much of the ECB resistance monitoring work since Bt corn PIPs were registered in the
mid 1990's. The methodology used for the Cry 1 A. 105 testing was similar to the procedures
employed for the annual ABSTC corn monitoring program.

ECB were collected from sites consistent with the ABSTC sampling strategy for Bt corn. A total
of 16 populations were collected as either adults or diapausing larvae from five states (Illinois,
Iowa, Minnesota, Nebraska, and South Dakota). Collected ECB were reared in the lab to
produce progeny for testing (F0, Fi, or F2 neonate larvae were tested). A susceptible laboratory
ECB strain was tested as well. Bioassays were conducted with dilutions (range 0.06 to 4.0
ng/cm2) of Cry 1 A. 105 toxin overlaid onto artificial diet. The toxin was microbially-produced
(by E. colt) and supplied by Monsanto. Results were tabulated after seven days by assessing
mortality (non-molting 1st instar larvae were considered dead) and larval weight.

An initial test with a susceptible laboratory colony showed that 96.9% mortality was achieved at
a concentration of 2.0 ng CrylA.105/cm2 and 100% mortality with all concentrations exceeding
6.25 ng/cm2. Of the field collected populations, results were reported for 10 populations; the
remaining six populations collected as diapausing larvae were delayed. The susceptibility assays
(Table 3) for the reported populations resulted in a LC50 range of 0.52 - 1.02 ng CrylA.105/cm2
and a LC90 range of 1.66 - 4.04 ng/cm2. EC50 values (determined from larval weights) ranged
from ~ 0.17 to 0.46 ng/cm2. By comparison, the laboratory colony LC50 was 0.40 ng/cm2, the
LC90 was 1.06 ng/cm2, and the EC50 was 0.19 ng/cm2. The study authors attributed the
differences in susceptibility (only two fold) between field-collected populations to natural
variability similar to that seen for other Bt toxins. A diagnostic concentration for Cry 1 A. 105 was
not reported in the results.

CEWBaseline Susceptibility Data - Cry 1 A. 105 (MRID# 477912-03)

Bioassays for CEW were conducted by Bruce Lang of Custom Bio-Products, who have
conducted the CEW monitoring for Bt corn toxins since 2001. Custom Bio-Products used
similar sampling and testing procedures for assessing CEW susceptibility to Cry 1 A. 105.

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CEW were collected from 24 locations in nine states (Alabama, Arkansas, Georgia, Illinois,
Iowa, Louisiana, Mississippi, North Carolina, and Texas) creating 26 total populations (two sites
had multiple collections). Populations were collected as larvae which were returned to
Monsanto's facility for rearing. Monsanto supplied eggs from subsequent generations (F2 to F5)
to Custom Bio-Products for the bioassays. A susceptible laboratory colony was also included in
the experiments. Susceptibility was assessed with diet bioassays incorporating dilutions of toxin
overlays (concentration range of 0.1 to 6 |ig CrylA.105/cm2). Cry 1 A. 105 toxin used in the
testing was provided by Monsanto. Neonates were exposed to the diet for seven days and then
evaluated for mortality (larvae <10 mg were considered dead) and larval weight.

All of the collected populations were included in the testing. The baseline susceptibility assays
(Table 11) for the field-collected populations resulted in the following ranges: LC50: 0.010 -
0.540 |ig/cm2; LC90: 0.042 - 2.118 |ig/cm2; LC99: 0.114 - 6.457 |ig/cm2; EC50: 0.0016 - 0.0190
|ig/cm2; EC95: 0.0184 - 1.1092 |ig/cm2; EC99: 0.0482 - 10.8520 |ig/cm2. For the laboratory
colony, the susceptibility results were: LC50: 0.256 |ig/cm2; LC90: 1.296 |ig/cm2; LC99: 4.857
|ig/cm2; EC50: 0.0034 |ig/cm2; EC95: 0.2255 |ig/cm2; EC99: 2.3628 |ig/cm2. The study author did
not suggest a potential value for a CEW diagnostic concentration based on these results.

Table 11. ECB and CEW baseline susceptibility to Cry 1 A. 105 (Created from data submitted to
EPA in MRID# 477912-02 and -03)



Susceptibility to CrylA.105

LC501

LC901

EC501

EC951

ECB - Field Collected

0.52- 1.02

1.66-4.04

0.17 to 0.46

—

ECB - Lab Strain

0.40

1.06

0.19

"

CEW - Field Collected

0.010-0.540

0.042-2.118

0.0016-0.0190

0.0184 - 1.1092

CEW - Lab Strain

0.256

1.296

0.0034

0.2255

1 Units are ng CrylA.105/cm2 for ECB and |_ig CrylA.105/cm2 for CEW

BPPD Review

For MON 89034 and MON 89034 x MON 88017, Monsanto has proposed to use existing
programs to monitor for resistance among lepidopteran and corn rootworm pests. Regarding the
lepidopteran pests, the Agricultural Biotechnology Stewardship Technical Committee (ABSTC),
a consortium representing Bt corn registrants, has been responsible for conducting resistance
monitoring activities. ABSTC submitted a unified plan for lepidopteran resistance monitoring
that covered all registered Bt corn PIPs in 2003 (see ABSTC 2003; reviewed in BPPD 2004a).
This plan has formed the basis of all monitoring activities since its submission including insect
sampling, bioassays, procedures for unexpected pest damage, definitions of pest resistance, and
steps to confirm cases of suspected resistance. ABSTC amended the monitoring plan in 2008
(MRID# 474070-01; see review in BPPD 2008b) to adjust the sampling strategy for ECB and

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SWCB and modify the procedures for determining resistance (mainly in CEW). This revised
monitoring program was integrated into the amended registration terms for both products after
the approval of 5% lepidopteran refuge (letters to Monsanto dated December 15, 2008).

Monsanto submitted lepidopteran resistance monitoring program (MRID# 479035-01) largely
follows the revised (2008) ABSTC plan and now required by the terms of registration. In
Monsanto's submission there are several differences from the ABSTC plan and some
components have been adapted to Cry 1 A. 105 and Cry2Ab2. For ECB, Monsanto indicated that
diagnostic concentrations have been developed ("upper 95% confidence limit of the LC99 or
EC99") for the toxins and will be used in the assays, although the specific values were not
reported. Survival of >1% on the diagnostic concentration will trigger follow-up investigations
of the population. For SWCB, Monsanto reported that a diagnostic concentration has not been
developed. Rather, dose-response assays (i.e., LC50 and EC50) will be used to assess field
collected populations relative to historical data for susceptible populations. With CEW,
Monsanto stated that "high diagnostic concentrations are not practical or relevant," but that
diagnostic concentrations will be developed for Cry 1 A. 105 and Cry2Ab2 since they have higher
activity. Dose-response parameters (LC50 and EC50) will also be used for comparisons with
historical data.

BPPD believes that diagnostic concentrations are an integral part of pest monitoring as a means
to distinguish susceptible and potentially resistant individuals. Monsanto submitted acceptable
Cry 1 A. 105 baseline susceptibility studies for ECB and CEW but diagnostic concentrations were
not proposed in the reports and additional work may be needed to develop functional standards.
For ECB, 96.9% mortality was achieved at a concentration of 2.0 ng/cm2 and 100% mortality
with all concentrations exceeding 6.25 ng/cm2, so a diagnostic concentration based on an LC99
should be easy to extrapolate. As is typical for the species, the CEW baseline results revealed
high variability (as much as several orders of magnitude) in LC99 and EC99 ranges. Data are still
pending for SWCB and FAW (Monsanto was granted an extension to fulfill the data needs for
these insects). BPPD recommends that Monsanto continue to work towards developing
susceptibility data and diagnostic concentrations for these pests and report the results with the
annual ABSTC monitoring reports.

Monsanto's submitted monitoring plan for MON 89034 (MRID# 479035-01) also did not
reference definitions of resistance (suspected and confirmed) and the steps to verify resistance
that are detailed in the revised ABSTC program. To confirm resistance, a pest population must
demonstrate: 1) 30% survival and commensurate insect feeding in a bioassay representative of
field exposure to Bt corn (ECB and SWCB only); 2) survival on a laboratory diagnostic
concentration that demonstrates a genetic basis for the tolerance and a resistance allele frequency
> 0.1; 3) a LC50 in a standardized laboratory bioassay that exceeds the upper 95% LC50
confidence interval for a susceptible population. BPPD notes that these criteria are now required
by the amended terms of registration for both MON 89034 and MON 89034 x MON 88017
(approval letters to Monsanto dated December 15, 2008).

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For CRW resistance monitoring with the Cry3Bbl toxin (expressed in MON 89034 x MON
88017), Monsanto cited to the plan developed for the MON 88017 x MON 810 registration
(MRID# 473547-01) and revised for SmartStax corn (MRID# 478875-03). Both of these
documents have been reviewed separately (see BPPD 2009 and 2010b) for their respective
registrations. Specific aspects of CRW resistance monitoring were required as conditions of
registration including development of a diagnostic dose assay and rootworm damage guidelines
(for unexpected pest damage). These conditions were also required for the existing Monsanto
rootworm Bt corn registrations and were addressed with previous submissions.

CRW resistance monitoring remains a work in progress as methodologies are developed to
assess the pest complex. Developing functional detection bioassays have been complicated by
rootworm biology and difficulties rearing and maintaining colonies in laboratory environments.
BPPD recommends that Monsanto continue to work on improvements to the CRW monitoring
program with the goal of implementing a harmonized program for all Bt corn PIPs (similar to the
ABSTC program for lepidoptera).

Remedial Action

Similar to resistance monitoring, Monsanto was required to utilize existing paradigms to address
remedial action plans for lepidoptera and corn rootworm. For lepidoptera, a plan was developed
by ABSTC in 2001 and modified in 2008 (MRID# 474070-01; reviewed in BPPD 2008b). The
modified remedial action strategy was incorporated into the terms of registration for both MON
89034 and MON 89034 x MON 88017 with the approval of 5% lepidopteran refuge (approval
letters to Monsanto dated December 15, 2008). Monsanto submitted a version of this plan with
the lepidopteran monitoring program (MRID# 479035-01).

The lepidopteran remedial action plan submitted by Monsanto contains a description of
procedures to confirm the heritability and field relevancy of resistance, estimate the frequency of
resistance alleles, determine the geographic boundaries of the resistance, and, in cases where
resistance allele frequencies are increasing or proliferating, creation of "an appropriate remedial
action plan based on the knowledge of the genetics and level of resistance it confers in the field."

Monsanto's described remedial action plan for MON 89034 differs significantly from that
required by the amended terms of registration. The terms of registration require that Monsanto
(paraphrased from EPA's December 15, 2008 letter):

•	Notify EPA, affected customers, and extension agents within 30 days of resistance
confirmation;

•	Increase resistance monitoring in the affected area;

•	Utilize alternate control measures in the area including insecticides or other control
measures if appropriate;

•	Stop sales of relevant Bt corn PIPs in the area until an EPA-approved mitigation measure
is in place;

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• Develop a case-specific mitigation plan within 90 days and notify affected parties of the
plan;

• Maintain the sales suspension and alternate control strategy into future growing seasons
until an EPA-approved mitigation plan is implemented.

Although Monsanto's submitted plan does not address many of these elements, they are still
required by the terms of registration. Therefore, the registration requirements for lepidopteran
remedial action essentially supersede the version submitted by Monsanto.

For CRW, Monsanto (as required by the terms of registration for MON 89034 x MON 88017)
referenced the remedial action plan previously developed for Cry3Bbl registrations. This plan
was originally developed for MON 863 and was subsequently carried over to MON 88017 and
SmartStax, both of which also express Cry3Bbl for CRW control.

Conceptually, the CRW remedial action plan (submitted in MRID# 473547-01; reviewed in
BPPD 2009) is similar to the strategy for lepidoptera. Activities are centered on assessing the
genetics (heritability, r-allele frequency) and geographic scope of the resistance event prior to
"design an appropriate remedial action plan." The actual remedial action plan to be deployed is
based on the one originally created for MON 863 corn (see review in BPPD 2004c).

Compliance

For the initial registrations of MON 89034 and MON 89034 x MON 88017 Monsanto was
required to design and submit a compliance assurance program (CAP) to ensure adherence to
refuge requirements by growers. The CAP was to be based on a "phased compliance approach"
to address non-compliant growers, include annual surveys (anonymous and on-farm) to assess
compliance, and provide a means to investigate "tips and complaints" of out-of-compliance
growers. In addition, Monsanto was required to utilize (and provide copies to EPA) a grower
agreement to contractually bind growers to plant refuges.

Compliance Assurance Program (MRID# 479035-01, 479083-01, other submissions with no
MRID#)

Monsanto responded to these terms of registration by submitting copies of the CAP developed by
ABSTC for lepidopteran Bt corn (dated September 23, 2002) and the CAP designed for MON
863 and CRW (dated July 7, 2005) as part of a non-MRID submission dated October 6, 2008.
These documents have been previously reviewed by BPPD — see BPPD 2005b (2002
lepidopteran CAP), BPPD 2006a (2005 CRW CAP), and BPPD 2004c (review of the original
2003 CRW CAP). CAP activities have been conducted by ABSTC since the 2002 growing
season. The core elements of the CAP are the same for lepidopteran and corn rootworm PIPs (as
well as stacked Bt corn PIPs targeting both pest complexes).

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The major components of the ABSTC CAP for Bt corn are as follows (paraphrased from ABSTC
2002):

• Annual IRM survey: The survey (conducted anonymously by an independent research
firm) is intended to provide a statistically representative sample of growers from various
corn-growing regions in the U.S. Results from the survey can assess not only levels of
grower compliances with refuges but also grower motivations, attitudes, and insights into
IRM for Bt corn.

• Tips and complaints: Registrants establish a means for the reporting and investigation of
incidences of refuge non-compliance.

• On-farm assessments: Trained personnel from each company make on-site visits to
farms growing Bt corn. During these visits, compliance with refuge requirements is
assessed and growers out of compliance are identified for corrective action under the
Phased Compliance Approach.

• Phase Compliance Approach (PCA): The PCA provides a stepwise set of procedures to
address non-compliance with the goal of bringing growers back into compliance.

Separate protocols are established for "significant deviations" (or <2/3 refuge fields
within '/2 mile of Bt fields) and "other deviations" (i.e., less than the significant
deviations). Significant deviations include one or more of the following:

• <15% refuge (for 20% requirement) or <40% refuge (for 50% requirement);

• < 2/3 refuge fields planted within V2 of Bt fields (lepidoptera);

• < 2/3 refuge fields planted within adjacent to Bt fields (rootworm);

• < 2/3 in-field strips planted at least 6 rows wide (rootworm)

Responses for both significant and other deviations include warning letters, compliance
assistance visits, educational efforts, and other measures. Growers who have significant
deviations for two years in a row will be denied access to the Bt corn product for at least
one growing season.

• Other measures: Alternate approaches including addressing large scale non-compliance
on a geographic scale and taking action against seed dealers not in adherence with IRM
requirements are detailed in the CAP.

Subsequent to the original registration, MON 89034 was amended to allow for a 5% refuge in
the U.S. Corn Belt (20% refuge is still required in cotton regions). Because of the new refuge
requirements, Monsanto submitted revised CAPs for MON 89034 (MRID# 479035-01) and
MON 89034 x MON 88017 (MRID# 479083-01). The revised CAP follows the framework of
the ABSTC (2002) program with several modifications to address the reduced refuge. Monsanto
removed the distinctions between "significant" and "other" deviations in the phased compliance
approach; instead, all instances of non-compliance are vetted equally. All growers found to be
non-compliant will be issued a warning letter, receive a "compliance assistance" visit, and
provided additional IRM educational materials. As with the ABSTC plan, any grower out of
compliance for two consecutive years will be prevented from purchasing MON 89034 varieties
for at least one growing season.

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BPPD agrees that the modification to eliminate tiered levels of non-compliance for MON 89034
is reasonable given the small (5% refuge). Any deviation from such a refuge will likely be
significant — a 1% refuge deviation for a 5% requirement functionally results in 20% less refuge.
Further, Monsanto's plan to address non-compliant growers in the MON 89034 CAP is the
practical equivalent of the approach for growers with "significant deviations" in the original
ABSTC plan.

Grower Agreements

As required by the terms of registration, Monsanto submitted copies of the "grower agreements"
used with MON 89034 and MON 89034 x MON 88017 customers. These contractual
documents obligate growers to adhere to IRM requirements as well as other conditions imposed
by the registrant (and not under the purview of EPA). Two sets of grower agreements were
submitted: one (for the 2009 growing season) in response to approval of the original registration
(attached with letter to EPA dated July 29, 2008) and the second (2010 growing season) for
approval of the amendment allowing 5% refuge (contained in MRID# 479035-01 and 479083-
01).

The form appears to be updated annually and is generically written to cover all Monsanto
agricultural biotechnology products. Portions of the 2010 contract pertinent to IRM are quoted
below:

In the "Grower Agrees" section:

•	"To implement an Insect Resistance Management ('IRM') program as specified in

the applicable	YieldGard® corn sections of the most recent Technology Use

Guide ('TUG') and the Grower and Insect Resistance Management Guide
('IRM/Grower Guide') and to incorporate and comply with these IRM programs."

•	"To read and follow the applicable sections of the TUG and IRM/Grower Guide,
which are incorporated into and is a part of this Agreement, for specific
requirements relating to the terms of this Agreement, and to abide by and be bound
by the terms of the TUG and the IRM/Grower Guide as it may be amended from
time to time."

In the "Grower Understands" section:

•	"Insect Resistance Management: When planting any YieldGard®	products,

grower must implement an IRM program according to the size and distance
guidelines specified in the TUG and the IRM/Grower Guide, including any
supplemental amendments. Grower may lose grower's limited use license for these
products if grower fails to follow the IRM program required by this Agreement."

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In the "General Terms" section:

• "Grower acknowledges that grower has received a copy of Monsanto's Technology
Use Guide ('TUG') and the Grower and Insect Resistance Management Guide
('IRM/Grower Guide')."

The 2009 and 2010 versions of the contract are functionally the same although the 2010 grower
agreement expands some of the IRM terms.

Grower Education

Monsanto was required by the terms of registration to "design and implement a comprehensive,
ongoing IRM education program" for MON 89034 and MON 89034 x MON 88017. Specific
requirements include at least one communication to growers per year to inform them of current
IRM requirements and the use of multiple media to convey educational messages (e.g., mailings,
bag tags, internet communications, radio/TV ads).

Monsanto Grower Education Submissions (MRID# 479035-01, 479083-01, other submissions
with no MRID#)

To address grower education, Monsanto submitted a copy of its Technology Use Guide (TUG)
after registration (submission dated September 22, 2008; No MRID#). The TUG covers
Monsanto's complete agricultural biotechnology line including MON 89034 and MON 89034 x
MON 88017 (trade name YieldGard VT). A second submission was made in 2009 that includes
a more complete description of the grower education program for MON 89034 (MRID# 479035-
01) and MON 89034 x MON 88017 (MRID# 479083-01).

The initial TUG provided covered the 2009 season when the original refuge requirement of 20%
was in place for all corn-growing areas nationwide. A supplemental section detailing this refuge
requirement (then unique to MON 89034) was included with the TUG. The 2009 TUG was a
comprehensive document (50 pg.) that addressed topics such as biotechnology, stewardship,
IRM, weed resistance, and crop-specific information for corn, cotton, and herbicide-resistant
soybean, alfalfa, canola, and sugarbeet. In terms of IRM, the TUG provided general information
on its importance and emphasized the need to follow refuge requirements. Growers were warned
that non-compliance could lead to loss of access to Monsanto's products and implementation a
monitoring program for refuge planting. Specific information on IRM requirements was
included in the TUG regarding refuge percentage and deployment. Diagrams were provided to
illustrate acceptable refuge configurations such as separate fields, blocks, and strips. Additional
information was supplied on requirements for insecticide treatments of refuges and other aspects
of refuge management. Each Monsanto Bt corn platform (corn borer control only, rootworm
only, and stacked corn borer/rootworm control) was addressed separately in the TUG. For the
stacked YieldGard Triple VT Triple product (i.e., MON 89034 x MON 88017), the TUG
includes descriptions of how to deploy "common" and "separate" refuges for both lepidopteran
(corn borer) and rootworm pest complexes.

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Monsanto's latter submissions (MRID# 479035-01 and 479083-01) contained a more detailed
description of their educational activities for growers. The following components were
described:

• Grower Agreements: Contractual arrangements between Monsanto and growers
(described in the previous section on compliance).

• Annual Affirmation: Monsanto employs a "bag tag" system in which growers
affirm their obligation to comply with refuge requirements when they open the seed
bag.

• Grower Education Program: Monsanto uses a multi-faceted system comprised of
these elements:

o Use of an IRM logo and development of the "Respect the Refuge"
advertising campaign.

o A comprehensive grower guide (TUG).

o Advertising including in or on billboards, seed catalogs, and websites.

o IRM training for sales representatives and communications with seed
dealers.

o Published articles and news releases in farm media to inform growers of
IRM responsibilities.

Subsequent to the registration of MON 89034 and MON 89034 x MON 88017, EPA approved an
amendment to lower the refuge to 5% for lepidopteran pests for the Corn Belt (required refuge
remains 20% in southern cotton-growing regions). Given the new refuge requirement, Monsanto
provided additional information regarding their grower educational efforts to facilitate this
change. To accomplish this, Monsanto will partner with ABSTC and the National Corn Grower
Association to harmonize educational messages. The TUG for the products (2010 version) was
revised to include details on which products and regions are eligible to employ the 5% refuge,
although only an excerpt (one page) was provided in the submission. Seed catalogs and bags
will be clearly marked to display the required refuge size and training will be provided for the
seed distribution network (including an "IRM Quick Guide" for sales representatives). Finally,
Monsanto developed an on-line calculator to help growers make accurate refuge determinations.

BPPD could find no errors or omissions in the education materials submitted for MON 89034
and MON 89034 x MON 88017. But, a complete version of the 2010 grower guide with all of
the IRM information was not provided, so it is not possible to fully verify the revisions made for
the 5% refuge approval. In addition, the grower guide provides little information on IRM
principles (i.e., how Bt corn is at risk for resistance and why refuges can mitigate the threat of
resistance). Such information could be beneficial to growers' understanding of the importance
and need for IRM.

F. BENEFITS AND PUBLIC INTEREST FINDING

To grant a conditional registration under Section 3(c)(7)(C) of the Federal Insecticide, Fungicide,
and Rodenticide Act (FIFRA), EPA must determine that such conditional registration will, inter

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alia, be in the public interest. EPA determines whether conditional registration of a pesticide is
in the public interest in accordance with the criteria set forth at 51 Fed. Reg. 7628 (Conditional
Registration of New Pesticides, March 5, 1986). There is a presumption that registration of a
pesticide is in the public interest if one of the following criteria are met: (i) the use is for a minor
crop; (ii) the use is a replacement for another pesticide that is of continuing concern to EPA; (iii)
the use is one for which an emergency exemption under FIFRA Section 18 has been granted (the
basis for the exemption was lack of a registered alternative product); or (iv) the use is against a
pest of public health significance. Notwithstanding whether a registration of a pesticide may be
presumed to be in the public interest, EPA may determine that such a registration is in the public
interest on the basis of one of the following criteria: (i) there is a need for the new chemical that
is not being met by currently registered pesticides; (ii) the new pesticide is comparatively less
risky to health or the environment than currently registered pesticides; or (iii) the benefits
(including economic benefits) from the use of the new active ingredient exceed those of
alternative registered pesticides and other available non-chemical techniques.

MON 89034 and MON 89034 x MON 88017 do not meet any of the criteria for a presumption of
public interest; however, BPPD has determined that MON 89034 and MON 89034 x MON
88017 are in the public interest based on criteria (ii) and (iii) mentioned above. Specifically,
under criteria (ii), both MON 89034 and MON 89034 x MON 88017 should allow growers the
opportunity to reduce the use of higher risk, and often less effective and more expensive,
conventional pesticides. A reduction in use of conventional pesticides equates to less potential
for adverse effects to human health and the environment. Additionally, MON 89034 and MON
89034 x MON 88017 provide a wider spectrum of protection against primary and secondary corn
pests, which should facilitate greater grain quality, a reduction of mycotoxin contamination,
increased yield and ultimately have positive implications for human health.

1. Agricultural Benefits

MON 89034

BPPD recognizes that MON 89034's unique combination of Cryl A. 105 and Cry2Ab2 proteins
expands the spectrum of protection for corn against lepidopteran pests - past that offered by
already-registered MON 810 (BPPD, 2007a). In addition to providing protection against primary
pests such as European corn borer (Ostrinia nubilalis, ECB), MON 89034 also protects against
secondary corn pests such as corn earworm (Helicoverpa zea, CEW), fall armyworm
(Spodopterafrugiperda, FAW), and black cutworm (Agrotis ipsilon, BCW) (BPPD, 2007a;
BPPD, 2007c; BPPD, 2007d). Use of MON 89034 could reduce or eliminate the need for
conventional pesticide applications on acreage infested with secondary pests, although most
growers do not use conventional pesticides to treat pests that are not part of the soil pest complex
(BPPD, 2001). Finally, yield appears to be comparable to other Bt insect-protected corn. In
situations of increased lepidopteran pressure, yield could be higher than other Bt insect-protected
corn because of the presence of two insecticidal toxins and the effective protection against
particular primary and secondary corn pests.

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MON 89034 x MON 88017

In addition to the agricultural benefits mentioned above for MON 89034, MON 89034 x MON
88017 provides control of corn rootworm complex (Diabrotica spp., CRW) that is functionally
equivalent to already-registered MON 863 and MON 88017. Use of MON 89034 x MON 88017
should encourage replacement and reduction of higher-risk conventional pesticides currently
utilized for CRW control (BPPD, 2003). Additionally, MON 89034 x MON 88017, which has
tolerance for glyphosate, should allow corn growers to utilize a conventional chemical, Roundup,
that is recognized by the Agency as a Category E chemical (i.e., there is evidence of non-
carcinogenicity for humans). Finally, yield appears to be comparable to other Bt insect-protected
corn. In situations of increased lepidopteran and/or coleopteran pressure, yield could be higher
than other Bt insect-protected corn because of the presence of three insecticidal toxins and the
effective protection against particular primary and secondary corn pests.

2.	Economic (Grower) Benefits
MON 89034

MON 89034 will offer protection against a wider spectrum of primary and secondary corn pests
(including FAW and CEW); should create conditions that allow for a reduction in the amount of
mycotoxin contamination; and should facilitate replacement and reduction of the amount of a
small amount of conventional pesticides that may be used against particular non-soil complex
corn pests. It is reasonable to believe that all of these characteristics should result in increased
yield, increased grain quantity, and increased grain quality.

MON 89034 x MON 88017

Because of the presence of MON 88017, which offers protection against CRW, MON 89034 x
MON 88017 should offer the same benefits as MON 89034 with perhaps more reduction in
conventional pesticide use and a slight advantage over the single event for growers that require
protection against lepidopteran pests and CRW.

3.	Human Health and Environmental Benefits

MON 89034
Human Health

The Cry 1 A. 105 and Cry2Ab2 proteins produced by MON 89034 should not present toxicity or
allergenicity problems in humans based on the reviews of the studies submitted in support of
MON 89034's conditional registration. As with other Bt corn products, it is reasonable to
assume that the utilization of MON 89034 should reduce the use of some conventional pesticides

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(BPPD, 2001). Finally, because the Cry 1 A. 105 and Cry2Ab2 proteins target secondary corn
pests - such as FAW and CEW - and protect the ear from damage caused by these pests,
decreased amounts of mycotoxin contamination should be recognized as a substantial benefit.

Environmental

Generally, there should be no risk from the proposed uses for MON 89034 to non-target
organisms, including, mammalian wildlife species, aquatic species, avian species, non-target
insects, and endangered species (BPPD, 2007i; BPPD, 2007j). Finally, use of MON 89034
should encourage a small reduction in the use of conventional pesticides. Fewer chemical
insecticide applications generally result in increased populations of beneficial organisms that
control secondary pests, such as aphids and leafhoppers.

MON 89034 x MON 88017

Human Health

In addition to Cry 1 A. 105 and Cry2Ab2 proteins produced in MON 89034, the introduction of
MON 88017 in the pyramided product results in production of Cry3Bbl protein. Human risk
assessment data has previously been reviewed for MON 88017, and BPPD concluded that there
is reasonable certainty that no harm will result from aggregate exposure to the U.S. population,
including infants and children (BPPD, 2007g). Additionally, an exemption from tolerance was
established for Cry3Bbl protein under 40 CFR 174.518. As with other Bt corn products, it is
reasonable to assume that the utilization of MON 89034 x MON 88017 should reduce the use of
conventional pesticides. In particular, the use of MON 89034 x MON 88017 should result in the
reduction of many conventional pesticides that are currently used, which have significant adverse
effects on human health (BPPD, 2003).

Environment

Cry3Bbl protein, produced in MON 88017, posed no significant risk to test organisms (BPPD,
2003). The only potential concern, brought to BPPD's attention by a recently published study,
relates to MON 89034 and will be dealt with by submission of 7-14-day Daphnia study.
Additionally, use of MON 89034 x MON 88017 should reduce the amount of conventional
pesticides used in the environment. All of the conventional pesticides used for CRW control or
suppression currently cause significant adverse environmental effects under conditions of normal
use (BPPD, 2003). Fewer chemical insecticide applications generally result in increased
populations of beneficial organisms that control secondary pests, such as aphids and leafhoppers.

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4. Insect Resistance Management (IRM) Benefits

MON 89034 (mention of MON 89034 assumes the same conclusions for MON 89034 x MON
88017 also)

BPPD concludes that MON 89034's two modes of action are better than a single mode of action
for mitigating the development of insect resistance. But, because of uncertainties in the data
submitted to support the MON 89034 IRM plan, a 5% refuge cannot be established until
additional data is submitted to support such a reduction. Instead, BPPD recommends that the
separate refuge option include a 20% lepidopteran refuge (as has been required for other Bt
products). A 20% refuge is likely to be supported for MON 89034 in cotton-growing regions of
the southeastern U.S. where a 50% refuge has been previously required. This will likely provide
an economic benefit to certain growers, since they will be required to plant less structured
refuge. In addition, the Cry 1 A. 105 and Cry2Ab2 toxins are new proteins targeting lepidopteran
pests in corn. These additional modes of action will likely provide a benefit to IRM programs
(i.e., a toxin "mosaic" in corn-growing regions may reduce the likelihood of resistance
developing in individual toxins). Also, the use of pyramided Bt corn products (containing 2 or
more toxins targeting the same pest) should further reduce the potential for resistance (BPPD,
2007b; BPPD, 2007h).

BACKGROUND

1. General Information

Corn {Zea mays L.) is the largest cultivated crop grown in the United States in terms of acreage
planted and net value. Monsanto states that 93.6 million of acres of corn were planted in the
U.S. during 2007 and that the net value of the 2006 corn crop was 33.7 billion dollars. The corn
industry suffers substantial economic losses from damage caused by specific lepidopteran and
coleopteran pests.

Two primary corn pests of particular concern to growers are corn rootworm complex (Diabrotica
spp., CRW) and European corn borer (Ostrinia nubilalis, ECB). According to Monsanto, CRW
causes damage to all portions of the plant (i.e., those above and below ground) depending on the
insect's life stage. In 2003, EPA estimated that approximately 28 million acres of corn were
infested with CRW and that untreated corn could result in severe yield loss, which was typically
in the range of 8 -16% reduction, but could be as high as 28% (BPPD, 2003). ECB has been
identified as the second most important insect pest of corn after CRW. ECB causes damage to
the plant based upon the generation: (i) the first generation causes leaf and stalk damage; (ii) the
second generation causes stalk, leaf sheath, collar, and ear damage; and (iii) the third generation
causes leaf sheath, collar, and ear damage. Monsanto estimates that the average annual U.S.
yield loss from ECB infestation is within the range of 3-7%. Deviations from the range are
attributed to level of infestation and region.

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Two secondary corn pests of particular concern to growers are corn earworm (Helicoverpa zea,
CEW) and fall armyworm (Spodoptera frugiperda, FAW). FAW typically has a limited range as
it is primarily found in the Gulf States and overwinters only in extreme southern Texas and
Florida. Monsanto provides an estimate that FAW damage to untreated acreage in Georgia
between 1991 and 1997 resulted in average yield loss of approximately 10%. No average yield
loss for all of the U.S. due to FAW damage was provided. On the other hand, CEW is found
throughout the U.S. corn-growing region, but Monsanto cites its economic damage as being low
and dependent on timing of infestation, region, and number of moth flights per year.

2. MON 89034

Monsanto has developed MON 89034, a corn product that produces Bacillus thuringiensis (Bt)-
derived insecticidal proteins Cry 1 A. 105 and Cry2Ab2. The Cry 1 A. 105 toxin is a "chimeric"
protein containing domains I and II and the C-terminal from Cry 1 Ac and domain III from Cry IF.
The Cry2Ab2 protein is functionally equivalent to that currently expressed in Monsanto's
Bollgard II cotton. MON 89034 is protected from damage caused by larval feeding of ECB,
southwestern corn borer (Diatraea grandiose I la, SCWB), Sugarcane borer (Diatraea
saccharalis, SCB), FAW, and CEW (BPPD, 2007a).

3. MON 89034 x MON 88017

Monsanto has also developed a second generation corn product, MON 89034 x MON 88017.
MON 88017 (EPA Reg. No. 524-551) (plasmid vector ZMIR39) expresses the Cry3Bbl Bt toxin
and is targeted against CRW larvae. The toxin is the same as expressed by MON 863 corn (EPA
Reg. No. 525-528), which was registered by Monsanto for the 2003 growing season. The
Cry3Bbl protein produced in MON 88017 and MON 863 is a variant of the wild-type Cry3Bbl
protein from Bt subsp. kumamotoensis. When compared by amino acid sequencing, the Cry3Bbl
protein expressed in MON 88017 has been reported to be 99.8% similar to the Cry3Bbl protein
expressed in MON 863. The primary difference between the two hybrids is that MON 88017
also expresses a gene for resistance to glyphosate (Roundup)-based herbicides (BPPD, 2005).
By crossing MON 89034 and MON 88017 through conventional breeding, Monsanto has
obtained an insect-protected corn product that expresses the CrylA.105, Cry2Ab2, and Cry3Bbl
Bt toxins, is targeted against lepidopteran corn pests including ECB, SWCB, CEW, and FAW as
well as coleopteran CRW, and is tolerant of glyphosate (BPPD, 2007b).

4. Monsanto's Public Interest Assertions for MON 89034 and MON 89034 x MON 88017

In the introduction of their public interest document (PID), Monsanto outlines the following
reasons why MON 89034 and MON 89034 x MON 88017 are in the public interest according to
some of the criteria set forth in 51 Fed. Reg. 7628:

• Enhanced spectrum of control. MON 89034 provides protection against

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an expanded spectrum of lepidopteran pests when compared to current Bt corn products.
MON 89034 x MON 88017 protects against both particular lepidopteran and coleopteran
pests. The increased protection found in both products improves overall grain quality and
limits yield losses due to root, leaf, stalk, and ear damage.

• Reduced mycotoxin levels. Because MON 89034 and MON 89034 x MON 88017
control the secondary pests, FAW and CEW, the opportunity for fungal infections

to thrive due to plant damage is reduced. This leads to less mycotoxin contamination.

• Improved breeding efficiency. Vector-stacking, which increases the efficiency of
breeding multiple traits into new corn hybrids, was utilized in the creation of
MON 89034 and MON 89034 x MON 88017.

• Compatibility with integrated pest management (IPM) systems. Both MON 89034
and MON 89034 x MON 88017 provide two different modes of action in a single plant
and reduce the probability of lepidopteran pests developing resistance to the Bt proteins.
This allows for a smaller refuge, helps the product maintain efficacy, and guards against
potential insect resistance to Bt crops.

• Reduced use of chemical pesticides. MON 89034 and MON 89034 x MON 88017
reduce the use of conventional chemicals, which saves costs and protects human
health and the environment.

• Easy implementation. No additional labor or machinery is needed to plant, grow,
or harvest MON 89034 and MON 89034 x MON 88017 relative to conventional
corn.

• Presence of glyphosate tolerance. MON 89034 x MON 88017 produces

5-enolpyruvylshikimate-3-phosphate synthase protein from Agrobacterium sp.

Strain CP4, which confers tolerance to glyphosate. Therefore, the agricultural
herbicide, Roundup, can be utilized on MON 89034 x MON 88017 corn to
control weeds and enhance the ability of the corn plants to access soil nutrients. EPA has
classified glyphosate as a Category E Chemical, meaning there is evidence of non-
carcinogenicity for humans.

This review will evaluate most of the assertions presented above in order to determine if MON
89034 and MON 89034 x MON 88017 are in the public interest.

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EPA'S REVIEW OF MONSANTO'S PUBLIC INTEREST DOCUMENT

Monsanto submitted a public interest document in support of the Section 3(c)(7)(C) registrations
of MON 89034 and MON 89034 x MON 88017 (Crawford and Bogdanova 2007, MRID
472797-01). The main portion of this document is divided into the following five sections: (i)
agricultural benefits; (ii) economic (grower) benefits; (iii) human health benefits; (iv)
environmental benefits; and (v) insect resistance management benefits. This document also
includes three appendices: Appendix I provides a summary of reports submitted by Monsanto to
the EPA that support registration of MON 89034 and MON 89034 x MON 88017, Appendix II
contains a study that analyzes the mycotoxin levels in grain of MON 89034 corn exposed to
lepidopteran insect infestation and inoculation with Aspergillus flavus or Fusarium
verticillioides, and Appendix III contains an assessment of efficacy of MON 89034 x MON
88017 corn against corn rootworm complex (Diabrotica spp., CRW) in the US during 2005 and
2006. Information provided by Monsanto will be discussed below, as applicable.

1. Agricultural Benefits

a) Pest Spectrum and Efficacy - Monsanto's Summary (MRID 472797-01)

MON 89034

MON 89034 exhibits the Cry proteins, Cry 1 A. 105 and Cry2Ab2, which specifically target
lepidopteran pests (See Tables 7 and 8). The primary benefit of MON 89034 is that it provides
equal (as compared to MON 810) or improved protection (as compared to MON 810, other Bt
corn products, and non-Bt corn products) from feeding damage caused by particular lepidopteran
pest larvae. The spectrum of protection against lepidopteran insects includes the following:
European corn borer (Ostrinia nubilalis, ECB), southwestern corn borer (Diatraea grandiose/la,
SCWB), Sugarcane borer (Diatraea saccharalis, SCB), fall armyworm {Spodopterafrugiperda,
FAW), black cutworm (Agrotis ipsilon, BCW), and corn earworm (He Iico verpa zea, CEW).

During the 2003 and 2004 growing seasons, Monsanto conducted efficacy field trials in the U.S.,
Puerto Rico, and Argentina. MON 89034's control of ECB, SWCB, and SCB was found to be
comparable to MON 810 (See Table 11). Because of the production of Cryl A. 105 protein by
MON 89034 and the subsequent control of FAW throughout the season and not just the plant's
vegetative growth phase, Monsanto claims a higher level of protection and increased activity
against FAW are shown by MON 89034 as opposed to MON 810 (See Table 11). Of particular
note, under heavy FAW pressure, MON 810 did not provide the significant amount of protection
from leaf damage that MON 89034 exhibited. Finally, the Cry2Ab2 protein produced by MON
89034 provided improved control from CEW, when compared to the activity of MON 810's
CrylAb protein (See Table 11).

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Table 9. Summary of arthropod LC50 values for the CrylA.105 protein exposure in diet
bioassays

In the event that no adverse effect was observed, the LC50 value is considered to be greater than the
maximum concentration tested.



Insect.

Assay

Diet Assay

Maximum

L Cjci

T est Insect

Stage

D mat ion

Type

C 011 rent rat ion

(|ig/mL

(0 rd er /F a mily/Sp ec ies)

T ested

(days)



tested (|xg mL or g

nr ™









d ie t)

.11--1.1

1, <-|i iil up 101 1







Nottuidae











ffehcoverpa zea

larvae

7

In co rp oration

N/A

6

Agrotis ipsilon

larvae

7

Incorporation

N/A

33

Sp 0 dop 12 r a fr u g ip e r da

larvae

7

Incorporation

N/A

6.9

Crninb idae











Diatraea grandiosella

larvae

12

Incorporation

N/A

37

Qstrinia nubilalis

larvae

12

Incorp oration

N/A

0.4 3

Co llv mil 11 l.i











Fohomia Candida

nymphs

28

Overlay

SO2

>80

C 0 lvdl" 1 vi 1









C lire ulino id ae











Anthonomus grandis

larvae

7

Overlay

100

>100

grand is











Clirysomelidae











Diahrotica

larvae

5

Overlay

100

>1 00

undecimpunctata howardi











Cocciiiellidae











ColeomegiUa maculata

larvae

20

Incorporation

240

>240

*Table from page 15 of MRID 472797-01

1 LC50 values with a greater than sign represent the highest dose tested.
2 Assay was performed with lyophilized leaf tissue from MON 89034.

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Table 9. (cont). Summary of arthropod LC50 values for the CrylA.105 protein exposure
diet bioassays

In the event that no adverse effect was observed, the LC50 value is considered to be greater than the
maximum concentration tested.

Test Insect
(0 rd er/F a mily/Sp ecies)

Insect
Stage
Tested

Assay
Duration

(days)

Diet Assay
Type

M axtauim
Co 11c en txation
tested
1 |ij 111L ¦> 1 j '1 iv 11

LCm
(|ig/mL org
diet)1

II *¦" 111 *¦! 1 I"1 1









Ichneumonidae











Ichneumon promissoriu*

adults

21

Incorporation

24 0

>24 0

Ap idae











Apis rnellifera

adults

18

Incorporation

550

>550

Apis mellifera

larvae

18

Overlay

1100 fi,g/mL
as a siflgle dose

>1100 K g/m L
as a single dose

J [ "llllji l-.'L .1











Ap hid id ae









Myzus persiscae
M irid ae

adults/
nymphs

5

Incorporation

80

>80

Lygus hesperus

nymphs

5

Incorporation

80

>80

Antliocoridne











Orius insidiosus

nymphs

14

Incorporation

N/A

240"

*Table from page 16 of MRID 472797-01

1 LC50 values with a greater than sign represent the highest dose tested.
2 The no observed effect concentration was determined to be 120 |ig/g diet.

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Table 10. Summary of arthropod LC50 values for the Cry2Ab2 protein exposure in diet
bioassays

In the event that no adverse effect was observed, the LC50 value is considered to be greater than the
maximum concentration tested.

T est I usee t

( O r d e r/F a m ily /S pec ies )

] »|< ill n|i 11-1 1

Insect.
Stage
T ested

Assay
D ma 11011
¦

Diet Assay
Type

M aximum
Concentration tested
'|i'j in 1. hi l1 iln'ii

L C}(,
(|ig/mL
111 l ¦! i"' •'

Noctuidae

ffelicoverpa zea

larvae

Incorporation

9.9

Agrotis ipstlon

larvae

Overlay

N/A

>100J

Spodoptera frugiperda

larvae

Overlay

N/A

<5 03

Cinmb idae
Ostrinia nubilalis

larvae

Incorporation

N/A

1.5

Diatraea grandioseUa

larvae

Incorporation

N/A

> 1 00*

C 0 llemb 0 l.i

Fohomia Candida

nymphs

Incorporation

703

>7 0

Coleop tei.i

Cuk ulmoid ip

Anthonomus grandis grandis

larvae

Overlay

100

>100

Chrysomelidae

Dibrotica undecimpunctata
h awards

larvae

Overlay

100

>100

*Table from page 17 of MRID 472797-01

1 LC50 values with a greater than sign represent the highest dose tested.

2 42% mortality was observed at the lowest tested dose of 100 |j,g/mL diet.

3 61% mortality was observed at the lowest tested dose of 50 |j,g/mL diet.

4 Significant mortality was not observed at the highest tested dose of 100 ug/mL diet,
however, at the highest tested dose of 100 ug/mL diet >95% growth inhibition was observed
relative to the control treatment in three independent assays

5 Assay was performed with lyophilized leaf tissue derived from MON 89034.

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Table 10. (cont). Summary of Arthropod LC50 Values for the Cry2Ab2 Protein Exposure
in Diet Bioassays

In the event that no adverse effect was observed, the LC50 value is considered to be greater than the
maximum concentration tested.

Test Insect

Insect

Assay

Diet Assay

M aximum

L C50

(01' tl e r/F a in ily / Specie s)

Stage

Duration

Type

Concentration tested

(lig/mL or g



T ested

(days)



({ig/mL or g diet)

diet)1

C 0 f t mellida e











ColeomegiUa maculata

larvae

20

Incorporation

120

>120

H Yitienop tei .1











Ithneuinonidae











JcAme union promissorius

adults

21

Incorporation

100

>100

Nasonia vetripennis

adults

1 0

Incorporation

4500

>4500

Ap idae











Apis meUif&ia

adults

19

Incorporation

68

>68

Apis meHifera

larvae

12

Overlay

100 ^g^inL

>100 ^g/mL









(as a single dose)

(as a single dose)

H <-111 lp I'M 1











_=.p lii ¦! 1*1 j'.1











Myzus persiscae

adults/

5

Overlay

80

>80



nymphs









M iridae











Ijguj hesperus

nymphs

5

Overlay

80

>80

Antliocoi idae











Grins insidiosus

nymphs

14

Incorporation

100

>100

*Table from page 18 of MRID 472797-01

1 LC5o values with a greater than sign represent the highest dose tested.

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Table 11. Summary of field efficacy of MON 89034, MON 810 and control corn against

major lepidopteran pests during t

he 2003-2004 growing season

Field
Location

Infestation method

Damage
measured

Infestation
level

Trait

performance1

Fall Armyworm

Puerto Rico

(I)

Natural

Leaf

High

MON

89034>MON
810>Control

Puerto Rico
(II)

Natural

Leaf

Severe

MON

89034>MON
810=Control

U.S.

Artificial

Leaf

50 larvae /plant

MON

89034>MON
810>Control

Argentina

Natural

Leaf

Low

MON

89034>MON
810>Control

Corn Earworm

Puerto Rico

(I)

Natural

Ear

Moderate

MON

89034>MON
810>Control

U.S.

Artificial

Ear

15 larvae /plant

MON

89034>MON
810>Control

Argentina

Natural

Ear

Low-moderate

MON

89034>MON
810>Control

Southwest Corn Borer

U.S.

Artificial

Stalk tunneling

7 larvae /plant

MON

89034=MON
810>Control

European Corn Borer

U.S.

Artificial

Stalk tunneling

50 larvae /plant

MON

89034=MON
810>Control

Sugarcane Borer

Argentina

Natural

Stalk tunneling

Moderate-high

MON

89034=MON
810>Control

*Table from page 20 of MRID 472797-01

1 Level of protection against lepidopteran pest damage.

> represents statistically significantly improved performance compared to other treatment
= represents no statistically significant difference in performance

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MON 89034 x MON 88017

In addition to producing the Cry 1 A. 105 and Cry2Ab2 proteins, MON 89034 x MON 88017 also
produces the insecticidal protein, Cry3Bbl, that controls damage caused by CRW.

The efficacy of MON 89034 x MON 88017 against CRW was compared in field trials in the
U.S. in 2005 and 2006, and against lepidopteran pests in 2006. The pyramided product showed
protection from feeding damage by lepidopteran pests that was comparable to MON 89034, as
well as protection from damage by CRW that was comparable to MON 88017. The average root
damage rating (RDR) for MON 88017 and MON 89034 x MON 88017 was significantly less
than the RDR for non-CRW protected controls (See Table 12).

Table 12. Field efficacy of MON 88017 and MON 89034 x MON 88017 and non-CRW-
protected control corn against corn rootworm tested in 2005 and 2006

Entry

RDR12

2005

Control

1.399 A

MON 88017

0.165 B

MON 89034 x MON 88017

0.164 B

2006

Control

0.774 A

MON 89034 x MON 88017

0.092 B

*Table from page 21 of MRID 472797-01

1 RDR - Root damage rating calculated as a least-square mean of n=5 plants per plot in 2005 and n = 6
plants per plot in 2006.

2 Values indicated by the same letter in the same column are not statistically different (Fisher's protected
LSD p=0.05 level).

The efficacy of MON 89034 x MON 88017 against ECB was also assessed in 2005 U.S. trials.
Significantly less feeding was observed on MON 89034 and MON 89034 x MON 88017 and
these two insect-protected corn crops also provided a high level of control against leaf damage
and stalk tunneling by ECB.

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a) Pest Spectrum and Efficacy — BPPD's Response
MON 89034

BPPD agrees with Monsanto that MON 89034 targets lepidopteran pests specifically. In two
studies conducted by Monsanto, both the Cry 1 A. 105 protein and Cry2Ab2 protein exhibited
insecticidal activity in the order Lepidoptera but not in the orders Coleoptera and Hemiptera
(BPPD, 2007c; BPPD, 2007d). The Cry 1 A. 105 protein, administered at 50 |ig/mL and 100
[j,g/mL concentrations to insects in the orders Lepidoptera, Coleoptera, and Hemiptera, caused a
range of mortality of 32% to 96% in CEW, ECB, and FAW (BPPD, 2007c). Additionally, all
four lepidopteran insects (CEW, ECB, FAW, and BCW) had a range of 32% to 100% growth
stunting (BPPD, 2007c). On the other hand, the Cry2Ab2 protein, administered at 50 [j,g/mL and
100 |ig/mL concentrations to insects in the orders Lepidoptera, Coleoptera, and Hemiptera,
caused at least 61% mortality (corrected) against CEW, ECB, and FAW (BPPD, 2007d). All
four lepidopteran insects (CEW, ECB, FAW, and BCW) had a range of 97 to 100% growth
stunting (BPPD, 2007d).

BPPD agrees with Monsanto's conclusions from the field trials conducted in the U.S., Puerto
Rico, and Argentina. Across all geographies, the efficacy of MON 89034 against ECB, SWCB,
CEW, FAW, and SCB was equal to or greater than that of YieldGard Corn Borer (MON 810), a
lepidopteran control corn product that expresses the Cryl Ab protein. However, MON 89034 did
offer a broader spectrum of insect protection activity than MON 810 and demonstrated better
control of CEW, FAW, and SCB than MON 810 in these trials (BPPD, 2007a).

MON 89034 x MON 88017

In the past, BPPD has concluded that MON 88017 is functionally equivalent to MON 863 for
CRW control (BPPD, 2005). Therefore, BPPD finds the efficacy benefits of MON 89034 x
MON 88017 are similar to the efficacy benefits of MON 863 (BPPD, 2003) and MON 88017
(BPPD, 2005). A summary of these benefits can also be found in BPPD's Biopesticides
Registration Action Document - Bacillus thuringiensis Plant-incorporated Protectants (BPPD,
2001). BPPD agrees that the efficacy of MON 89034 x MON 88017 should be comparable to
efficacy of the MON 89034 and MON 88017 isolines for FAW and western corn rootworm
(WCRW, Diabrotica virgifera) and to the MON 89034 isoline for ECB, SWCB, CEW, and
FAW. Furthermore, similar to MON 89034, MON 89034 x MON 88017 provides a broader
spectrum of efficacy than MON 810 against lepidopteran pests. Although no SCB, WBCW, or
BCW field trials were conducted with MON 89034 x MON 88017, based on the efficacy against
the other pests, it is reasonable to assume comparable efficacy to MON 89034 for these pests as
well (BPPD, 2007b).

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b) Yield — Monsanto's Summary (MRID 472797-01)

MON 89034 and MON 89034 x MON 88017

In 2006, Monsanto conducted field trials with the objective of comparing yield between MON
89034, MON 89034 x MON 88017, MON 810, MON 810 x MON 88017, and other hybrids not
producing Bt proteins. Results showed comparable yield across several hybrids tested for insect-
protected hybrids and higher yields compared to hybrids that did not produce Bt proteins (See
Table 13). Additionally, the assumption is made that under intense lepidopteran pressure, the
yield benefit from MON 89034 would be significantly higher. Although no economic benefits
can be assessed for MON 89034 until it is actually used by growers, Monsanto predicts that the
economic benefits would be equal or even more advantageous depending on the level and type of
pest infestation that occurs.

Table 13. Yield comparison between MON 89034, MON 89034 x MON 88017, MON 810,
MON 810 x MON 88017, and non-Bt hybrids grown in the U.S. during 2006

Product

Yield (Bu/Acre)

Number of hybrids
tested

MON 89034

180.4

MON 810

180.6

MON 89034 x MON 88017

189.3

MON 810 x MON 88017

185.7

Non-/?/

171.5

*Table from page 13 of MRID 472797-01

c) Yield - BPPD 's Response

BPPD believes that it is reasonable for Monsanto to assume that significant pressure from
lepidopteran pests would cause the yield benefit for MON 89034 to be higher because of the
presence of 2 insecticidal toxins, Cry 1 A. 105 and Cry2Ab2. Furthermore, if the pressure is from
secondary corn pests such as FAW and CEW, then increase of yield is even more logical.
Although Monsanto assumes that economic benefits will be equal or more advantageous for
MON 89034 and is probably correct, BPPD will not assume complete validity of this assumption
until MON 89034 is used over the course of several years and reliable yield data is available.
Although use of MON 89034 x MON 88017 could also result in an overall increase in yield,
BPPD does not expect an increase in yield that exceeds that of the previously-registered single
gene MON 88017 as any increase in yield will result mostly from the characteristics of MON
89034: expanded pest spectrum and the presence of two toxins instead of one.

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2. Economic (Grower) Benefits

a) Monsanto'v Summary (MRU) 472797-01)

MON 89034

For economic benefits, Monsanto cites to National Center for Food and Agricultural Policy
statistics that are based on planted acreage of MON 810 corn in 2005. Monsanto estimated that
MON 810 increased corn production by 103.9 million bushels because of the corn borer resistant
trait. Net returns, decreased costs (fuel, labor, and conventional pesticides purchased), and
premium price of protected seeds were estimated to be 197 million dollars. The decreased use of
conventional pesticides was estimated at 4.85 million pounds. In percentage terms, MON 810
planted in 2005 resulted in a 24% increase in yield, a 27% decrease in pesticide use, and
increased monetary gain of 26% when compared to 2004.

The major economic benefits of MON 89034 are the following: (i) a wider spectrum of pest
protection (to include FAW and CEW), which results in increased grain quality and increased
yield; (ii) reduction in mycotoxin contamination levels which contributes to economic recovery;
(iii) protection that is more effective in controlling corn borers and therefore results in increased
yield, grain quality, and grain quantity; and (iv) reduction in conventional pesticide use that
results in less costs (See Table 14).

Table 14. Summary of economic benefits to growers using corn-borer protected corn

Benefit

Per acre benefit ($)

Total benefit
($ Millions)

Yield increase

13.59 (-3.67-48.76)

217 (-59-780)

Pesticide reduction

1.99 (1.00-2.98)

32 (16-48)

Mycotoxin reduction

1.98 (0.52-7.12)

32 (8.3 - 114)

*Table from page 29 of MRID 472797-01
MON 89034 x MON 88017

Monsanto states that the pyramided product will offer the combined benefits of the individual
parents, MON 89034 and MON 88017. The addition of MON 88017 creates enhanced
protection against CRW, a primary corn pest that can cause total yield losses that exceed $1
billion dollars annually, and adds the glyphosate tolerance trait that limits yield loss from weed
pressure. Overall, MON 89034 x MON 88017 will limit yield losses from corn borer insects,
CRW, and weed pressure, reduce conventional pesticide use, and reduce mycotoxin
contamination while increasing yield, grain quantity, and grain quality.

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b) BPPD's Response

MON 89034

Overall, BPPD agrees that MON 89034 should produce economic benefits for many growers.
Monsanto's numbers are estimates and actual economic benefits may be affected by factors
including pest pressure, climatic fluctuations, and commodity pricing. MON 89034 is effective
against a wider spectrum of corn pests and it is reasonable to assume that this should result in
increased yield. Additionally, BPPD has concluded that a slight decrease in pesticide use should
be realized with the use of Bt corn products similar to MON 89034 (BPPD, 2001); therefore,
BPPD agrees with Monsanto's assertion of possible conventional pesticide use reduction and
associated reduced costs.

BPPD agrees with Monsanto's conclusion that MON 89034 should reduce mycotoxin
contamination. Overall, if primary and secondary corn pest pressure is reduced, then less
mycotoxin contamination will be present, which will in turn lead to increased yield, grain
quantity, and grain quality (BPPD, 2001). Furthermore, field evidence has demonstrated the
ability of Bt corn to reduce the infestation rates of certain mycotoxins (Wu, 2008). This article
specifically associates CEW with aflatoxin accumulation in corn and claims that Bt corn
varieties, perhaps those such as MON 89034, are being developed to combat this insect pest in
order to reduce particular mycotoxin contamination.

MON 89034 x MON 88017

BPPD believes that use of MON 89034 x MON 88017, much like MON 89034, should produce
the same economic benefits mentioned above. Additionally, the combination with MON 88017,
which protects corn against CRW and exhibits glyphosate tolerance, can be expected to create
slightly greater economic benefits than MON 89034 for growers needing to treat both
lepidopteran pests and CRW. The economic benefits of MON 88017 (minus an evaluation of
glyphosate tolerance) have previously been assessed in MON 863's public interest finding
document (BPPD, 2003).

3. Human Health and Environmental Benefits

a) Monsanto'v Summary (MRU) 472797-01)

MON 89034

Human Health

Monsanto states that the Cry 1 A. 105 and Cry2Ab2 proteins produced by MON 89034 are
structurally and functionally related to Cry proteins that have a history of use both as active
ingredients in Bt microbial pesticides and bio-tech derived food and feed. Furthermore, they

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state that Bt has been commercially used in the U.S. since 1958 to produce microbial-derived
pesticides and no adverse effects on humans or animals has been reported during their use on
food or feed crops. Additionally, Monsanto asserts that Cry 1 A. 105 and Cry2Ab2 proteins are
highly unlikely to create any concerns of toxicity or allergenicity to humans. These assertions
are based on acute oral toxicity data, which produced results that agreed with literature showing
that Bt proteins only impact insect species and that no mammalian toxicity or issues have been
reported in nearly 60 years of Bt protein insecticide use, and a comparison to known allergens,
which indicated no allergenicity in the Cry 1 A. 105 and Cry2Ab2 proteins.

Two of the specific human health benefits that Monsanto attributes to use of MON 89034 are
pesticide reduction and mycotoxin reduction. Using numbers from an article from the National
Center for Food and Agricultural Policy, Monsanto demonstrates current usage levels of MON
810, a functional equivalent of MON 89034, results in an estimated decrease in use of
approximately 4.85 million pounds of conventional pesticides per year (equivalent to a 27%
decrease in conventional pesticide use to control corn-boring pests). According to Monsanto,
mycotoxin reduction is also evident with the use of MON 89034 because of its ability to suppress
or control secondary corn pests, such as CEW and FAW, that play a role in damaging corn ears
and facilitating the inoculation and growth of mycotoxin-producing fungi. Monsanto cites to two
types of fungi, Fusarium and Aspergillus that produce fumonisin and aflatoxin, respectively. A
study conducted by Monsanto indicates that MON 89034 is subject to less damage from corn
pests and subsequently, it suffers less mycotoxin contamination, particularly from Aspergillus.

Environmental

Monsanto states that Cry 1 A. 105 and Cry2Ab2 have no toxic effects on non-target organisms (to
include the following: mammalian wildlife species, aquatic species, avian species, non-target
insects, and endangered species) based on studies they submitted to the Agency in conjunction
with the registration application for MON 89034. Additionally, the proteins rapidly degrade in
soil which also minimizes exposure to non-target species.

MON 89034 x MON 88017

Human Health

Since MON 89034 is present in the pyramided product, Monsanto's contentions for the safety of
MON 89034 with regard to human health also apply to MON 89034 x MON 88017. In addition
to the presence of the Cry 1 A. 105 and Cry2Ab2 proteins found in MON 89034, the pyramided
product also produces Cry3Bbl protein. Given that MON 88017 is already a product registered
by the EPA, Monsanto states that the Cry3Bbl protein produced by MON 89034 x MON 88017
already has an exemption from tolerance. Furthermore, human risk assessment data reviewed by
the EPA for registration of MON 88017 has resulted in a conclusion that there is reasonable
certainty that no harm will result from aggregate exposure to the U.S. population, including
infants and children. As with MON 89034, MON 89034 x MON 88017 is expected to result in

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both reduced pesticide use (and perhaps more because of MON 88017' s protection from primary
corn pest, CRW) and mycotoxin contamination.

Environmental

Since MON 89034 is present in the pyramided product, Monsanto's contentions for the safety of
MON 89034 with regard to environmental effects also apply to MON 89034 x MON 88017.
MON 89034 x MON 88017 also produces the Cry3Bbl protein, which is in the previously
registered MON 88017. Upon review of the environmental effects data for MON 8801T s
registration, EPA concluded that no unreasonable adverse effects are expected to the
environment from the cultivation of MON 88017 and MON 88017 x MON 810 corn.

b) BPPD's Response

MON 89034

Human Health

BPPD agrees with Monsanto's conclusions that the Cry 1 A. 105 and Cry2Ab2 proteins produced
by MON 89034 should not cause toxicity or allergenicity problems in humans. The data
submitted and cited regarding potential health effects for the Cry 1 A. 105 and Cry2Ab2 proteins
include the characterization of the expressed proteins in corn, as well as acute oral toxicity
studies, amino acid sequence comparisons to known allergens and toxins, and in vitro
digestibility of the proteins. The results of these studies were used to evaluate human risk
(BPPD, 2007e).

The acute oral toxicity data submitted support the prediction that the Cryl A. 105 and Cry2Ab2
proteins would be non-toxic to humans. When proteins are toxic, they are known to act via acute
mechanisms and at very low dose levels. Since no treatment-related adverse effects were shown
to be caused by the Cryl A. 105 and Cry2Ab2 proteins, even at relatively high dose levels, the
Cry 1 A. 105 and Cry2Ab2 proteins are not considered toxic. Basing this conclusion on acute oral
toxicity data without requiring further toxicity testing or residue data is similar to the Agency
position regarding toxicity and the requirement of residue data for the microbial Bt products from
which this plant-incorporated protectant was derived (See 40 CFR 158.740(b)(2)(i)) (BPPD,
2007e).

Since Cryl A. 105 and Cry2Ab2 are proteins, potential allergenicity was also considered as part
of the toxicity assessment. Considering all of the available information (1) Cry 1 A. 105 and
Cry2Ab2 originate from a non-allergenic sources; (2) Cryl A. 105 and Cry2Ab2 have no
sequence similarities with known allergens; (3) Cryl A. 105 and Cry2Ab2 are not glycosylated;
and (4) Cryl A. 105 and Cry2Ab2 are rapidly digested in simulated gastric fluid; EPA has
concluded that the potential for Cry 1 A. 105 and Cry2Ab2 to be a food allergens is minimal
(BPPD, 2007e).

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The lack of mammalian toxicity at high levels of exposure to the Cry 1 A. 105 and Cry2Ab2
proteins, as well as the minimal potential to be a food allergens, demonstrate the safety of the
product at levels well above possible maximum exposure levels anticipated (BPPD, 2007e).

BPPD agrees with Monsanto's conclusion that MON 89034 should reduce mycotoxin
contamination. Overall, if primary and secondary corn pest pressure is reduced, then less
mycotoxin contamination will be present, which will in turn lead to increased yield, grain
quantity, and grain quality (BPPD, 2001). Further, field evidence has demonstrated the ability of
Bt corn to reduce the infestation rates of certain mycotoxins (Wu, 2008). This article specifically
associates CEW with aflatoxin accumulation in corn and claims that Bt corn varieties, perhaps
such as MON 89034, are being developed to combat this insect pest in order to reduce particular
mycotoxin contamination.

Environmental

BPPD agrees with Monsanto's assessment that there should be no risk from the proposed uses
for MON 89034 to non-target organisms, including mammalian wildlife species, aquatic species,
avian species, non-target insects, and endangered species (BPPD, 2007i; BPPD, 2007j). In
addition to Monsanto's submitted rationale for environmental benefits, Monsanto could have
included a reference to MON 89034 use potentially reducing the amount of conventional
pesticides applied in the environment and the subsequent environmental benefits. BPPD
believes that cultivation of MON 89034 corn may have fewer adverse impacts on non-target
organisms than use of chemical pesticides for corn production, because under normal
circumstances, MON 89034 corn should require substantially fewer applications of chemical
pesticides, compared to production of non-Bt corn. Fewer chemical insecticide applications
generally result in increased populations of beneficial organisms that control secondary pests,
such as aphids and leafhoppers.

MON 89034 x MON 88017
Human Health

BPPD agrees with Monsanto that the use of MON 89034 x MON 88017 should not result in any
unreasonable adverse effects to human health. In addition to Cry 1 A. 105 and Cry2Ab2 proteins
produced in MON 89034, the introduction of MON 88017 results in production of Cry3Bbl
protein. BPPD has already reviewed human risk assessment data for MON 88017 and reached a
conclusion that there is reasonable certainty that no harm will result from aggregate exposure to
the U.S. population, including infants and children (BPPD, 2007g). Additionally, Cry3Bbl
currently has an exemption from tolerance established under 40 CFR 174.518.

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BPPD also agrees with the claim that there should be a reduction of some pesticide use (BPPD,
2001). Additionally, Cry3Bbl protein primarily protects corn plants against CRW. Virtually all
of the registered conventional insecticides used to control CRW are of special concern to the
EPA because of risks to humans. Each year, there are confirmed reports of human illness
associated with these registered conventional chemicals (See BPPD, 2003).

Finally, BPPD agrees with the claim that use of MON 89034 x MON 88017 should combat
mycotoxin contamination because of the production of the Cry 1 A. 105 and Cry2Ab2 proteins (as
mentioned previously).

Environmental

BPPD agrees with Monsanto's assessment that there should be no risk from the proposed uses
for MON 89034 x MON 88017 to non-target organisms, including mammalian wildlife species,
aquatic species, avian species, non-target insects, and endangered species. The only potential
concern will be addressed through a 21-day Daphnia study. For the registration of MON 88017,
a series of studies were completed by Monsanto that exposed non-target organisms to high doses
of leaf tissue, grain, or pollen containing a plant-produced Cry3Bbl variant or to an artificial diet
containing a ifr-produced Cry3Bbl variant. Results indicated that the Cry3Bbl protein posed no
significant risk to test organisms (BPPD, 2003). Additionally, a study was conducted on MON
89034 x MON 88017 to ensure that the interaction between CrylA.105, Cry2Ab2, and Cry3Bbl
proteins in the pyramided product would not change the overall properties of each individual
component. BPPD concluded that the activity of Cry 1 A. 105 and Cry2Ab2 proteins was not
significantly altered by the presence of Cry3Bbl, and the activity of Cry3Bbl was not
significantly altered by the presence of Cry 1 A. 105 and/or Cry2Ab2. The study, along with the
previously reviewed interaction study between Cry 1 A. 105 and Cry2Ab2, indicated that MON
89034 x MON 88017 corn should not result in any unexpected interaction with regards to target
and non-target insects (BPPD, 2007f).

In addition to the submitted rationale for environmental benefits, Monsanto could have included
reference to MON 89034 x MON 88017 use reducing the amount of conventional pesticides
applied in the environment and the subsequent environmental benefits. BPPD believes that
cultivation of MON 89034 x MON 88017 corn may have fewer adverse impacts on non-target
organisms than use of chemical pesticides for corn production, because under normal
circumstances, MON 89034 x MON 88017 corn should require substantially fewer applications
of chemical pesticides, compared to production of non-Bt corn. The reduction in conventional
pesticide use should essentially be the same seen from MON 88017 and MON 863 use (BPPD,
2003). Fewer chemical insecticide applications generally result in increased populations of
beneficial organisms that control secondary pests, such as aphids and leafhoppers. Furthermore,
all of the conventional pesticides used for CRW control or suppression cause significant adverse
environmental effects under conditions of normal use (BPPD, 2003).

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4. Insect Resistance Management (IRM)

a)	Monsanto'v Summary (MRU) 472797-01)

MON 89034 (mention of MON 89034 assumes the same conclusions for MON 89034 x
MON 88017 also)

Monsanto establishes that MON 89034, which produces Cry 1 A. 105 and Cry2Ab2 proteins, has
two different modes of action against lepidopterans, particularly in the way the proteins bind to
the midgut. Therefore, based on the distinct modes of action of the two proteins and reduced
likelihood of insect resistance, Monsanto proposes that a reduced structured refuge is possible:
5% for the corn belt, down from 20% and 20% for cotton-growing regions, down from 50%.

b)	BPPD's Response

MON 89034 (mention of MON 89034 assumes the same conclusions for MON 89034 x
MON 88017 also)

BPPD agrees with Monsanto in that two modes of action are better than one for reducing the risk
of insect resistance to MON 89034. But, due to uncertainties in the data submitted to support the
MON 89034 IRM plan, a 5% refuge cannot be established until additional data is submitted to
support such a reduction. Instead, BPPD recommends that the separate refuge option include a
20% lepidopteran refuge (as has been required for other Bt products). A 20% refuge is likely to
be supported for MON 89034 in cotton-growing regions of the southeastern U.S. where a 50%
refuge has been previously required (BPPD, 2007b; BPPD, 2007h).

Overall, MON 89034 should present two immediate IRM benefits: (i) dual (distinct) modes of
action for Bt corn and (ii) reduced refuge in cotton regions (and the resulting economic benefits
to growers). These benefits can likely be achieved without an unreasonable risk of resistance to
Cry 1 A. 105 and Cry2Ab2. Additional grower benefits may be realized in the long term, if a 5%
refuge can be supported.

5. Efficacy Studies

In addition to the efficacy studies referenced in the preceding sections, the following studies
were submitted and are considered for registration of MON 89034. These studies demonstrate
the efficacy of MON 89034 corn and the individual Bt proteins (Cry 1 A. 105 and Cry2Ab2)
against a range of lepidopteran corn pests including European corn borer (ECB), corn earworm
(CEW), southwestern corn borer (SWCB), fall armyworm (FAW), and sugarcane borer (SCB).

MRU) 46951413

In laboratory bioassays, the insecticidal activity of Cry 1 A. 105 protein was tested against
agronomically important insects from the orders Lepidoptera (four species), Coleoptera (two

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species), and Hemiptera (two species). Neonate larvae, nymphs or adults (eggs for western bean
cutworm prior to hatching) were fed artificial diets containing the appropriate doses of 50 or 100
|ig CrylA.105/mL of insect diet in the diet-overlay bioassays for fall armyworm, black cutworm,
European corn borer, corn earworm, Southern corn rootworm, and boll weevil or 40 or 80 |ig
Cry 1 A. 105/mL of insect diet in diet-incorporation bioassays for western tarnished plant bug and
green peach aphid. Mortality and a reduction in weight or honeydew production over a five or
seven day period depending on the insect were the endpoints used to indicate insecticidal
activity. The 50 |ag/m L and 100 |ag/m L Cry 1 A. 105 concentrations caused a range of 32 to 96%
mortality in three (corn earworm, European corn borer, and fall armyworm) of the four
lepidopterans. All four lepidopteran insects had a range of 32-100% stunting. The Cry 1 A. 105
protein had activity against all four lepidopteran insects, but no activity against the two
coleopteran or two hemipteran insects tested. Classification: Acceptable

MRU) 46951414

In laboratory bioassays, the insecticidal activity of Cry2Ab2 protein was tested against
agronomically important insects from the orders Lepidoptera (four species), Coleoptera (two
species), and Hemiptera (two species). Neonate larvae, nymphs or adults (eggs for western bean
cutworm prior to hatching) were fed artificial diets containing the appropriate doses of 50 or 100
|ig Cry2Ab2/mL of insect diet in the diet-overlay bioassays for fall armyworm, black cutworm,
European corn borer, corn earworm, Southern corn rootworm, and boll weevil or 40 or 80 |ig
Cry2Ab2/mL of insect diet in diet-incorporation bioassays for western tarnished plant bug and
green peach aphid. Mortality and a reduction in weight or honeydew production over a five or
seven day period depending on the insect were the endpoints used to indicate insecticidal
activity. In the diet-overlay bioassays, both the 50 |ag/m L and 100 |ag/m L Cry2Ab2
concentrations caused at least 61% mortality (corrected) against corn earworm, European corn
borer, and fall armyworm; while stunting was at least 97% for all four lepidopteran insects
tested. Only the 28% black cutworm mortality resulting from testing against the 50 |ag/m L
Cry2Ab2 concentration failed to meet the study criterion of >30% mortality. The Cry2Ab2
protein had activity against all four lepidopteran insects, but no activity against the two
coleopteran or two hemipteran insects tested. Classification: Acceptable

MRU) 46951415

Field trials were conducted in 2003-2004 seasons in Puerto Rico, the United States and
Argentina to determine the efficacy of MON 89034 corn (CrylA.105 and Cry2Ab2) and MON
89597 (Cry 1 A. 105 and Cry2Ab2) corn against European corn borer (ECB), corn earworm
(CEW), southwestern corn borer (SWCB), fall armyworm (FAW), and sugarcane borer (SCB).
Across all geographies the efficacy of the MON 89034 and MON 89597 against ECB, SWCB,
CEW, FAW, and SCB was equal to or greater than that of YieldGard® Corn Borer (MON 810),
a lepidopteran control corn product that expresses the Cryl Ab protein. For all geographies
tested, there was no significant difference in efficacy between MON 89034 and MON 89597
with the exception of CEW damage in PR I, (2003 testing), where MON 89034 demonstrated
significantly better control than MON 89597. MON 89034 and MON 89597 offer a broader
spectrum of insect activity than MON 810. MON 89034 and MON 89597 demonstrated

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significantly better control of CEW, FAW, and SCB than MON 810. Details of the field trials
are found below. Data are acceptable for the Puerto Rico and U.S. trials. Data are
supplemental for the Argentina trials due to lack of sufficient rationale as to why certain
locations were excluded from the analysis. No additional data are required.

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III. REGULATORY POSITION FOR CrylA.105, AND Cry2Ab2

A) Original 3(c)(7)(C) Assessment

Pursuant to FIFRA section 3(c)(7)(C), EPA may conditionally register a new pesticide active
ingredient for a period of time reasonably sufficient for the generation and submission of
required data that are lacking because insufficient time has elapsed since the imposition of the
data requirement for those data to be developed. EPA may grant such conditional registration
only if EPA determines that (1) the use of the pesticide product during the period of the
conditional registration will not cause any unreasonable adverse effect on the environment, and
(2) the registration and use of the pesticide during the conditional registration is in the public
interest. EPA determines that all of these criteria have been fulfilled.

The first criterion under FIFRA Section 3(c)(7)(C) mentioned above has been met because
insufficient time has elapsed since the imposition of the data requirements for:

1)	An independent lab validation of the analytical method for the detection of Cry2Ab2
and/or Cry 1 A. 105 to satisfy residue analytical method in plants requirements for event
MON 89034 corn and event MON 89034 x MON 88017 corn.

2)	A 7 to 14 day Daphnia study as per the 885 Series OPPTS Guidelines or alternatively, a
dietary study of the effects on an aquatic invertebrate, representing the functional group of
a leaf shredder in headwater streams.

3)	Additional information on cross-resistance of Cry 1 A. 105 and CrylFa and Cry 1 Ac
(preferably including binding site models and use of resistant colonies) for the target pests
and determine how such cross-resistance may impact the durability of MON 89034.

4)	Baseline susceptibility studies and/or a discriminating concentration assay that are
required for the CrylA.105 protein against ECB, SWCB, and CEW and for the Cry2Ab2
protein against SWCB, CEW.

5)	Baseline susceptibility studies to support sweet corn uses that must be conducted on FAW
populations collected from sweet corn growing areas; Monitoring studies that will be
conducted on FAW populations collected from sweet corn distribution areas in states in
which Monsanto MON 89034 and/or MON 89034 x MON 88017 sweet corn plantings
exceed 1000 acres; and monitoring of the collected populations of FAW for changes in
susceptibility to the CrylA.105 and Cry2Ab2 proteins.

The applicants submitted or cited data sufficient for EPA to determine that conditional
registration of Bacillus thuringiensis Cry2Ab2 and Cry 1 A. 105 proteins and the genetic material
necessary for their production in event MON 89034 field corn and sweet corn under FIFRA
3(c)(7)(C) will not result in unreasonable adverse effects to the environment, as discussed above.

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The applicants submitted and/or cited satisfactory data pertaining to the proposed use. The
human health effects data and nontarget organism effects data are considered sufficient for the
period of the conditional registration. These data demonstrate that no foreseeable human health
hazards or ecological effects are likely to arise from the use of the product and that the risk of
resistance developing to Cry2Ab2 and Cry 1A.105 proteins, during the conditional registrations
are not expected to be significant.

Registration of Bacillus thuringiensis Cry2Ab2 and Cry 1 A. 105 proteins and the genetic material
necessary for their production in event MON 89034 field corn and sweet corn is in the public
interest because:

(1) Registration of MON 89034 is expected to result in the reduction of the use of higher risk,
and often less effective and more expensive, conventional pesticides. A reduction in use of
conventional pesticides equates to less potential for adverse effects to human health and the
environment.

(2) Additionally, MON 89034 provide a wider spectrum of protection against primary and
secondary corn pests, which should facilitate greater grain quality, a reduction of mycotoxin
contamination, increased yield and ultimately have positive implications for human health.

In view of these minimal risks and the clear benefits related to Bacillus thuringiensis Cry2Ab2
and Cry 1 A. 105 proteins and the genetic material necessary for their production in event MON
89034 field corn and sweet corn , EPA believes that the use of the product during the limited
period of the conditional registration will not cause any unreasonable adverse effects.

Although the data with respect to this particular new active ingredient are satisfactory, they are
not sufficient to support an unconditional registration under FIFRA 3(c)(5). Additional data are
necessary to evaluate the risk posed by the continued use of this product. Consequently, EPA is
imposing the data requirements specified earlier in Section III.

EPA has determined, as explained in section II.F., that the third criterion for a FIFRA 3(c)(7)(C)
conditional registration has been fulfilled because the use of Bacillus thuringiensis Cry2Ab2 and
Cry 1A.105 proteins and the genetic material necessary for their production in event MON
89034 field corn and sweet corn under this registration is in the public interest.

The submitted data in support of this registration under section 3(c)(7)(C) of the Federal
Insecticide, Fungicide, and Rodenticide Act (FIFRA) have been reviewed and determined to be
adequate. Studies mentioned above are included in the terms, conditions, and limitations of these
registrations. This registration will not cause unreasonable adverse effects to man or the
environment and is in the public interest.

The expiration date of the registrations has been set to September 30, 2010.

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B) 2010 3(c)(7)(A) Assessment

Section 3(c)(7)(A) of FIFRA provides for the registration or amendment of a pesticide when the
pesticide and proposed use . .are identical or substantially similar to any currently registered
pesticide and use thereof, or differ only in ways that would not significantly increase the risk of
unreasonable adverse effects on the environment, and (ii) approving the registration or
amendment in the manner proposed by the applicant would not significantly increase the risk of
any unreasonable adverse effect on the environment." Unreasonable adverse effects on the
environment are defined under section 2(bb) of FIFRA as "... any unreasonable risk to man or
the environment, taking into account the economic, social, and environmental costs and benefits
of the use of any pesticide..Thus, pursuant to section 3(c)(7)(A), EPA may conditionally
register a pesticide if (1) the pesticide and its proposed use are identical or substantially similar
to a currently registered pesticide; or (2) the pesticide and its proposed use differ only in ways
that would not significantly increase the risk of unreasonable adverse effects; and (3) approving
the registration would not significantly increase the risk of any unreasonable adverse effect.

The Agency concludes that the following Cry 1 A. 105 and Cry2AB2 corn product registrations,
that were set to expire on September 30, 2010 and described in-depth throughout this BRAD,
meet both criteria (1) and (2):

(1)	Event MON 89034 with Cry 1 A. 105 and CryAb2 (EPA Reg. No. 524-575)

(2)	Events MON 89034 with MON 88017, CrylA.105, Cry2Ab2, and Cry3Bbl
(EPA Reg. No. 524-576)

These Cry 1 A. 105 and Cry2AB2 corn products are identical in both composition and use (corn)
to plant-incorporated protectants that are currently registered. Thus, criterion (1) has been
fulfilled.

With regard to criterion (2), the Agency maintains, as was previously determined for the original
registration of these particular products, that cultivation of Cry 1 A. 105 and Cry2AB2-containing
corn will not cause unreasonable adverse effects on the environment. The conditional
environmental effects data, submitted in response to terms and conditions of registration
strengthen the Agency's initial position and also confirm that long-term effects on non-target
organisms are not anticipated. Lastly, the continued use of these products will likely still provide
many of the benefits as were evaluated in section 11(F) of this BRAD to support the 2005
registration of these products.

In conclusion, as the expiring Cry 1 A. 105 and Cry2AB2 products have met the required criteria
under section 3(c)(7)(A) of FIFRA, the Agency is amending these registrations to extend their
respective expiration dates as follows:

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Product Name (EPA Reg. No.)

Expiration Date

Event MON 89034 with Cry 1 A. 105 and Cry2Ab2
(524-575)

September 30, 2022

Events MON 89034 withMON 88017, CrylA.105,
Cry2Ab2, and Cry3Bbl
(524-576)

September 30, 2015

Although data provided were satisfactory to make the determinations required by section
3(c)(7)(A) of FIFRA, they were not sufficient to support an unconditional registration under
FIFRA section 3(c)(5). Additional data, specifically in relation to insect resistance
management,are necessary for a finding of registrability under FIFRA section 3(c)(5) and remain
as terms or conditions for the purposes of the amendments.

C) Period of Registration

In the 2001 Bt Corn reassessment, EPA determined that it was appropriate to amend the then-
existing registrations to extend the period of registration of those products to an expiration date
of October 15, 2008. All of the products being assessed at that time were efficacious against
lepidopteran pests. EPA based this action on the finding that use of Cry 1 A. 105 and Cry2AB2
expressed in corn will not significantly increase the risk of unreasonable adverse effects on the
environment "for the limited time period of 7 additional years (to October 15, 2008)." These
registrations were later amended to extend the period of registration to an expiration date of
September 30, 2010. EPA subsequently granted time-limited registrations to products
efficacious against coleopteran corn rootworm pests. For example, EPA registered Cry3Bbl on
February 24, 2003, to May 1, 2004, and extended that registration twice, to February 24, 2008,
and September 30, 2010.

As set forth elsewhere in this document, EPA's primary concern for the Bt protected transgenic
corn products is the possibility that target pests will develop resistance to one or more of the PIP
toxins. Development of resistance to a Bt toxin would be a grave adverse effect, and, for over 15
years, EPA has imposed stringent requirements intended to countermand the potential
development of resistance. Registrants similarly have been busily developing various products,
product mixes (i.e., so-called "pyramids" and "stacks"), and resistance strategies, to maximize
agronomic benefits and address resistance management issues. The result has been a vast array
of product combinations and, occurring over the past couple of years, a re-emergence of varying
refuge requirements for different products.

As discussed in the 2001 Bt PIP BRAD (at IID13), the earliest Bt corn registrations did not
include mandatory refuge requirements. There was a lack of scientific consensus as to what the
appropriate refuge requirement should be, and, it was assumed that the limited market
penetration of these early crops would be so low as to guarantee that adequate natural refuges
would be available from neighboring non-Bt corn fields. From 1995 to 1997, Bt corn

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registrations included voluntary refuge requirements of 0% to 20% in the corn belt. In 1999, the
ABSTC, in conjunction with the National Corn Growers Association, proposed uniform IRM
requirements for Bt corn registrations. With some modifications, this proposal, put in place for
the 2000 growing season, formed the baseline IRM requirements for almost all Bt corn
registrations for the better part of a decade: farmers were required to plant a 20% refuge that
could be treated for insects, or a 50% treated refuge in cotton-growing areas; all refuges to be
planted within one-half mile of the Bt corn field.

These uniform requirements brought certainty and consistency to the market after the initial
period where many Bt corn products had different refuge requirements. Recently, however, as
product developers have begun to conceive of products with different combinations of
"pyramided" products (i.e., products containing two or more toxins efficacious against the same
pest) and "stacked" products (i.e., products combining toxins efficacious against different pests),
the refuge requirements have begun to vary. For example, certain products require a 20%
external refuge; some products permit a 5% external refuge; one product incorporates a 10%
seed blend refuge; we have applications in process for products that propose to incorporate a 5%
seed blend refuge; and other permutations are possible.

Given the profusion of various toxin combinations and refuge options, we can no longer proceed
on the basis that, as concerns insect resistance management, all products are equal. It was a
relatively simple proposition when the default requirement of a 20% sprayed refuge applied to
almost all of the Bt corn crops in the market. Under those circumstances, the relative durability
of products against the development of resistance was functionally equivalent, and, as a
consequence, imposing functionally equivalent registration periods was appropriate. That is now
no longer the case.

As part of our continually evolving regulatory approach to the continually evolving product mix
wrought by developers, we think it appropriate to revise our regulatory requirements in
scientifically defensible ways to reflect the comparative level of risks posed by the products that
we regulate. Here, for example, where we've determined that a particular product, or category of
products, likely will pose less risk of insect resistance developing to a particular PIP protein, we
think it appropriate to grant that particular product, or category of products, a registration for a
period greater than that granted a corresponding product that poses a greater risk of insect
resistance developing. This approach is reflective of complementary principles: first, to ensure
that we apply our limited resources to the products that pose greater risk of adverse effects to the
environment; and, second, to conserve the resources that registrants and applicants must expend
in amending the registrations of products that pose less risk of adverse effects to the
environment.

The scheme that we are following includes registration periods of five, eight, and twelve years; a
fifteen year registration period will also be available, if adequately supported by our science
assessment. In this scheme, (i) a product with a single PIP toxin, and a 20% external refuge,
qualifies for a five year registration; (ii) a product with pyramided PIP toxins (i.e., two or more

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toxins with distinct, non-cross reacting modes of action), that are non-high dose (the definition
for a high dose product remains unchanged), with either a seed blend or external refuge, qualifies
for an eight year registration; (iii) a product with pyramided PIP toxins (i.e., two or more toxins
with distinct, non-cross reacting modes of action), that are high-dose, with either a seed blend or
external refuge, qualifies for a twelve year registration; (iv) a product with pyramided PIP toxins
(i.e., two or more toxins with distinct non-cross reacting modes of actions), with either a seed
blend or external refuge, that has been determined by EPA's science assessment to be 150% as
durable as the baseline single toxin product with a 20% external refuge, would qualify for a
fifteen year registration. Products determined by EPA's science assessment to be less than 100%
as durable as the baseline single toxin product with a 20% external refuge would not qualify for a
five year registration and the registration period for such products will be determined on a case-
by-case basis consistent with the level of risk they pose. Similarly, instances where other risk
issues may arise, or where novel resistance concerns may be present, would also be determined
on a case-by-case basis, as will novel refuge configurations that may present unique durability
profiles.

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IV. BIBLIOGRAPHY:

Citations Considered as part of the Data Base Supporting the Registration ofMON 89034

MRID: 46471600

Citation: Monsanto Co. (2005) Submission of Product Chemistry and Residue Data in Support
of the Experimental Use Permit of Bacillus thuringiensis in ZMIR245 Corn. Transmittal
of 1 Study.

MRID: 46471601

Citation: Bogdanova, N.; Brown, S.; Huber, S. (2005) Volume 2: Data in Support of an

Application for an Experimental Use Permit to Test ZMTR245 x MON 88017 Combined
Trait Corn Along with ZMIR245 and MON 88017. Project Number: 04/CR/130E.
Unpublished study prepared by Monsanto Company. 15 p.

MRID: 46694603

Citation: Bonnette, K. (2005) Am Acute Oral Toxicity Study in Mice with Cry 1 A. 105 Protein:
Final Report. Project Number: EUF00081, CRO/2005/050. Unpublished study prepared
by Charles River Laboratories, Inc. 119 p.

MRID: 46694605

Citation: McCoy, R.; Silvanovich, A. (2005) Bioinformatics Analysis of the Cry 1 A. 105 Protein
Utilizing the AD5, Toxin5, and Allpeptides Databases: Final Report. Project Number:
19686, 05/01/62/01. Unpublished study prepared by Monsanto Co. 549 p.

MRID: 46694606

Citation: Kapadia, S.; Rice, E. (2005) Assessment of the In Vitro Digestibility of the CryA.105
Protein in Simulated Gastric Fluid: Final Report. Project Number: MSL/19929,
05/01/62/02. Unpublished study prepared by Monsanto Company. 23 p.

MRID: 46694607

Citation: Goley, M.; Thorp, J. (2005) Immunodetection of Cry2Ab2 and Cry 1 A. 105 Proteins in
Corn Grain from MON 89034 Following Heat Treatment: Final Report. Project Number:
MSL/19899, 05/01/39/27. Unpublished study prepared by Monsanto Co. 38 p.

MRID: 46874901

Citation: ABSTC. 2006. Monitoring the Susceptibility of Corn Lepidoptera Pests to CrylAb and
Cry IF Proteins: 2005 Monitoring Results. Report submitted to EPA by the IRM
Technical Subcommittee of the Agricultural Biotechnology Stewardship Technical
Committee.

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MRID: 46951300

Citation: Monsanto Company (2006) Submission of Product Chemistry, Efficacy, Exposure and
Risk Data in Support of the Application for Registration of MON 89034 x 88017.
Transmittal of 6 Studies.

MRID: 46951301

Citation: Bogdanova, N. (2006) Human Health and Environmental Assessment of the Plant-
Incorporated Protected Bacillus thuringiensis CrylA.105, Cry2Ab2 and Cry3Bbl
Proteins Produced in Insect-Protected Corn MON 89034 x MON 88017. Project
Number: MSL/20434. Unpublished study prepared by Monsanto Company. 27 p.

MRID: 46951302

Citation: Groat, J.; Wolff, B.; Rice, J.; et. al. (2006) Confirmation of the Integrity of Corn MON
89034 x MON 88017 by Southern Blot Analysis. Project Number: 06/01/50/03,
MSL/20145. Unpublished study prepared by Monsanto Company. 22 p.

MRID: 46951303

Citation: Hartmann, A.; Niemeyer, K.; Silvanovich, A. (2006) Assessment of the Cry 1 A. 105,
Cry2Ab2, Cry3Bbl, and CP4 EPSPS Protein Levels in Selected Tissues of Insect-
Protected Corn MON 89034 x MON 88017 Produced in 2005 U.S. Field Trials. Project
Number: MSL0020479. Unpublished study prepared by Monsanto Company. 41 p.

MRID: 46951304

Citation: Levine, S. L.; Camp, R.; Uffman, J.; Vaughan, T.; et. al. (2006) An Evaluation of the
Insect Bioefficacy of Combined Trait Products Produced Through Conventional
Breeding: MON 89034 x NK603 and MON 89034 x MON 88017. Project Number:
06/01/50/02, MSL/20336. Unpublished study prepared by Monsanto Company. 24 p.

MRID: 46951305

Citation: MacRae, T.; Brown, C.; Levine, S. (2006) Evaluation of Potential for Interactions

Between the Bacillus thuringensis Proteins CrylA.105, Cry2Ab2, and Cry3Bbl. Project
Number: 05/01/39/19, MSL/20270. Unpublished study prepared by Monsanto Company.
26 p.

MRID: 46951306

Citation: Head, G. (2006) Insect Resistance Management Plan for the Combined Trait Product
MOON 89034 xMON 88017. Project Number: 06/RA/50/03. Unpublished study
prepared by Monsanto Company. 18 p.

MRID: 46951400

Citation: Monsanto Company (2006) Submission of Efficay, Toxicity, Residue, Environmental
Fate, Exposure and Risk Data in Support of the Application for Registration of MON
89034. Transmittal of 30 Studies.

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MRID: 46951401

Citation: Bogdanova, N. (2006) Human Health and Environmental Assessment of the Plant-
Incorporated Protectant Bacillus thuringiensis Cry 1 A. 105 and Cry2Ab2 Proteins
Produced in Corn MON 89034. Project Number: MSL/20405. Unpublished study
prepared by Monsanto Company. 88 p.

MRID: 46951402

Citation: Rice, J.; Wolff, B.; Groat, J.; et. al. (2006) Amended Report for MSL-20072:

Molecular Analysis of Corn MON 89034. Project Number: 05/01/39/12, MSL/20311.
Unpublished study prepared by Monsanto Company. 67 p.

MRID: 46951403

Citation: Hartmann, A.; Niemeyer, K.; Silvanovich, A. (2006) Assessment of the Cry 1 A. 105
and Cry2Ab2 Protein Levels in Tissues of Insect-Protected Corn MON 89034 Produced
in 2005 U.S. Field Trials. Project Number: 05/01/39/32, MSL/20285. Unpublished
study prepared by Monsanto Company. 31 p.

MRID: 46951404

Citation: Karunanandaa, K.; Thorp, J.; Goley, M.; et al. (2006) Characterization of the Cry2Ab2
Protein Purified from the Corn Grain of MON 89034 and Comparison of the
Physicochemical and Functional Properties of the Plant-Produced and E. coli-Produced
Cry2Ab2 Proteins. Project Number: 20071, 60/100075. Unpublished study prepared by
Monsanto Company. 55 p.

MRID: 46951405

Citation: Levine, S.; Uffman, J. (2006) Evaluation of the Functional Equivalence of the

Cry2Ab2 Protein Produced in E. coli and Bt Against a Sensitive Lepidopteran Species.
Project Number: 05/01/39/23, MSL/20132. Unpublished study prepared by Monsanto
Company. 23 p.

MRID: 46951406

Citation: Bonnette, K. (2006) An Acute Oral Toxicity Study in Mice with Cry2Ab2 Protein:
Final Report. Project Number: CRO/2005/049, EUF00080/MSL/19901. Unpublished
study prepared by Charles River Laboratories, Inc. 113 p.

MRID: 46951407

Citation: Kapadia, S.; Rice, E. (2006) Assessment of the in vitro Digestibility of the Cry2Ab2
Protein in Simulated Gastric Fluid. Project Number: MSL/19931, 05/01/62/04.
Unpublished study prepared by Monsanto Company. 23 p.

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MRID: 46951408

Citation: Kapadia, S.; Rice, E. (2005) Assessment of the in vitro Digestibility of the Cry 1 A. 105
Protein in Simulated Intestinal Fluid. Project Number: MSL/19930, 05/01/62/03.
Unpublished study prepared by Monsanto Company. 20 p.

MRID: 46951409

Citation: Thorp, J.; Goley, M. (2006) Assessment of the in vitro Digestibility of the Cry2Ab2
Protein in Simulated Intestinal Fluid. Project Number: MSL/19938, 05/01/62/05.
Unpublished study prepared by Monsanto Company. 20 p.

MRID: 46951410

Citation: McClain, J.; Silvanovich, A. (2006) Bioinformatics Evaluation of the CrylA.105

Protein Utilizing the AD6, TOXIN5, and ALLPEPTIDES Databases. Project Number:
20351, 06/01/62/04. Unpublished study prepared by Monsanto Company. 550 p.

MRID: 46951411

Citation: McClain, J.; Silvanovich, A. (2006) Bioinformatics Analysis of the Cry2Ab2 Protein
Utilizing the AD6, TOXIN5, and ALLPEPTIDES Databases. Project Number: 20307,
06/01/62/01. Unpublished study prepared by Monsanto Company. 361 p.

MRID: 46951412

Citation: Davis, S. (2006) Comparison of Broiler Performance and Carcass Parameters When

Fed Diets Containing MON 89034, Control or Commercial Corn: Amended Final Report.
Project Number: MN/05/2, 05/01/50/13. Unpublished study prepared by Colorado
Quality Research and University of Missouri. 96 p.

MRID: 46951413

Citation: Macrae, T.; Brown, C.; Levine, S. (2006) Spectrum of Insecticidal Activity of Bacillus
thuringiensis Cry 1 A. 105 Protein. Project Number: 04/01/39/27, MSL/20230.
Unpublished study prepared by Monsanto Company. 18 p.

MRID: 46951414

Citation: Macrae, T.; Brown, C.; Levine, S. (2006) Spectrum of Insecticidal Activity of Bacillus
thuringensis Cry2Ab2 Protein. Project Number: 05/01/62/07, MSL/20229. Unpublished
study prepared by Monsanto Company. 17 p.

MRID: 46951415

Citation: Headrick, J.; Heredia, O.; Oyediran, I.; et. al. (2006) Assessment of the Efficacy of

Lepidopteran-protected Corn MON 89034 and MON 89597 Against Major Insect Pest in
the United States, Puerto Rico and Argentina During 2003-2004 Seasons. Project
Number: 05/RA/39/05. Unpublished study prepared by Monsanto Company. 18 p.

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MRID: 46951416

Citation: Teixeira, D. (2006) Evaluation of Dietary Effects of Lyophilized Leaf Tissue from
Corn MON 89034 in a Chronic Exposure Study with Collembola (Folsomia Candida).
Project Number: 252/6197, SE/2005/027, MSL/20169. Unpublished study prepared by
Springborn Smithers Laboratories. 42 p.

MRID: 46951417

Citation: Palmer, S.; Krueger, H. (2006) Evaluation of Exposure to MON 89034 with the
Cladoceran Daphnia magna: An Acute Static-Renewal Test with Corn Pollen: Final
Report. Project Number: 139A/330, WL/2005/011. Unpublished study prepared by
Wildlife International, Ltd. 32 p.

MRID: 46951418

Citation: Sindermann, A.; Porch, J.; Krueger, H. (2006) Evaluation of Potential Effects of

Exposure to Cry 1 A. 105 Protein in an Acute Study with the Earthworm in an Artificial
Soil Substrate: Final Report. Project Number: 139/472, WL/2005/068, MSL/20147.
Unpublished study prepared by Wildlife International, Ltd. 48 p.

MRID: 46951419

Citation: Richards, K. (2006) Evaluation of the Dietary Effect(s) of a CrylA.105 Protein on
Honeybee Larvae (Apis mellifera L.). Project Number: CAR/128/05, CA/2005/071.
Unpublished study prepared by California Agricultural Research Inc. 43 p.

MRID: 46951420

Citation: Richards, K. (2006) Evaluation of the Dietary Effect(s) of a CrylA.105 Protein on
Adult Honeybees (Apis mellifera L.). Project Number: CAR/129/05, CA/2005/072.
Unpublished study prepared by California Agricultural Research Inc. 52 p.

MRID: 46951421

Citation: Paradise, M.; Jiang, C.; Duan, J.; et. al. (2006) Evaluation of Potential Dietary Effects
of Cry 1 A. 105 Protein on the Ladybird Beetle, Coleomegilla maculata (Coleoptera:
Coccinellidae). Project Number: 05/01/39/24, MSL/20150. Unpublished study prepared
by Monsanto Company. 41 p.

MRID: 46951422

Citation: Paradise, M.; Jiang, C.; Duan, J.; et. al. (2006) Evaluation of Potential Dietary Effects
of Cry2Ab2 Protein on the Ladybird Beetle, Coleomegilla maculata (Coleoptera:
Coccinellidae). Project Number: 05/01/39/25, MSL/20151. Unpublished study prepared
by Monsanto Company. 42 p.

MRID: 46951423

Citation: Teixeira, D. (2006) Evaluation of Potential Dietary Effects of CrylA.105 Protein on
Minute Pirate Bugs, Orius insidiosus (Hemiptera: Anthocoridae). Project Number:

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252/6201, SE/2005/074, MSL/20170. Unpublished study prepared by Springborn
Smithers Laboratories. 105 p.

MRID: 46951424

Citation: Teixeira, D. (2006) Evaluation of Potential Dietary Effects of Cry2Ab2 Protein on
Minute Pirate Bugs, Orius insidiosus (Hemiptera: Anthocoridae). Project Number:
252/6200, SE/2005/075, MSL/20171. Unpublished study prepared by Springborn
Smithers Laboratories. 57 p.

MRID: 46951425

Citation: Sindermann, A.; Porch, J.; Krueger, H. (2006) Evaluation of Potential Effects of

Exposure to Cry 1 A. 105 Protein in an Acute Study with the Parasitic Wasp, Ichneumon
promissorius (Hymenoptera: Ichneumonidae). Project Number: 139/475, WL/2005/128,
MSL/20149. Unpublished study prepared by Wildlife International, Ltd. 47 p.

MRID: 46951426

Citation: Sindermann, A.; Porch, J.; Krueger, H. (2006) Evaluation of Potential Effects of
Exposure to Cry2Ab2 Protein in an Acute Study with the Parasitic Wasp, Ichneumon
promissorius (Hymenoptera: Ichneumonidae). Project Number: 139/476, WL/2005/129,
MSL/20148. Unpublished study prepared by Wildlife International, Ltd. 47 p.

MRID: 46951427

Citation: Gallagher, S.; Beavers, J. (2006) Evaluation of Potential Dietary Effects of MON
89034 with the Northern Bobwhite: and Eight-Day Dietary Study with Corn Grain.
Project Number: 139/470, WL/2005/012. Unpublished study prepared by Wildlife
International, Ltd. 60 p.

MRID: 46951428

Citation: Mueth, M.; Curran, T.; Warren, J.; et. al. (2006) Aerobic Soil Degradation of the

Purified Cry2Ab2 and Cry 1 A. 105 Proteins. Project Number: 05/01/39/29, MSL/20174.
Unpublished study prepared by Monsanto Company, Agvise Inc. and University of
Nebraska. 83 p.

MRID: 46951429

Citation: Huesing, J.; Duan, J.; Levine, S. (2006) Endangered Species Risk Assessment for Corn
MON 89034. Project Number: MSL0020394. Unpublished study prepared by Monsanto
Company. 24 p.

MRID: 46951430

Citation: Head, G. (2006) Insect Resistance Management Plan for Second Generation

Lepidopteran-Protected Corn, MON 89034. Project Number: 06/RA/39/06. Unpublished
study prepared by Monsanto Company. 98 p.

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MRID: 47079400

Citation: Monsanto Co. (2007) Submission of Efficacy Data in Support of the Application for
Registration of MON 89034 X 88017. Transmittal of 2 Studies.

MRID: 47079402

Citation: Bogdanova, N. (2007) Supplemental Information to Address EPA Questions

Regarding Applications 524-LTL and 524-LTA to Register Insect-protected Corn MON
89034 and MON 89034 x MON 88017 (MRID 46951400 and 46951300). Project
Number: 04/C4/172/2. Unpublished study prepared by Monsanto Co. 16 p.

MRID: 47079403

Citation: Headrick, J.; Heredia, O.; Oyediran, I. (2006) Assessment of the Efficacy of Insect-
Protected Corn MON 89034, MON 89034 x NK603, and MON 89034 x MON 88017
Against Major Insect Pests in the Field Trials Conducted in U.S. During 2005. Project
Number: 05/RA/50/04. Unpublished study prepared by Monsanto Co. 19 p.

MRID: 47127500

Citation: Monsanto Company (2007) Submission of Product Chemistry and Toxicity Data in
Support of the Application for Registration of MON 89034 and MON 89034 x MON
88017. Transmittal of 5 Studies.

MRID: 47127501

Citation: Bogdanova, N. (2007) Responses to EPA Questions Regarding Applications 524-LTL
and 524-LTA to Register Insect-protected Corn MON 89034 and MON 89034 X MON
88017 (MRID 46951400 and 46951300). Project Number: 04/CR/172/3, 04/CR/172E/3.
Unpublished study prepared by Monsanto Co. 10 p.

MRID: 47127502

Citation: Groat, J.; Wolff, B.; Rice, J.; et al. (2007) Confirmation of the Integrity of Corn MON
89034 X MON 88017 by Southern Blot Analysis. Project Number: 06/CR/172/3,
BR/ME/0878/01, BR/ME/0094/01. Unpublished study prepared by Monsanto Company.
69 p.

MRID: 47127503

Citation: Rice, J.; Wolff, B.; Groat, J.; et al. (2006) Amended Report for MSL-20072: Molecular
Analysis of Corn MON 89034. Project Number: 06/CR/172/3, BR/ME/0878/01,
BR/ME/0094/01. Unpublished study prepared by Monsanto Company. 78 p.

MRID: 47127504

Citation: Hartmann, A.; Niemeyer, K.; Silvanovich, A. (2006) Assessment of Cry 1 A. 105,
Cry2Ab2, Cry3Bbl and CP4 EPSPS protein Levels in Selected Tissues of INsect-
protected Corn MON 89034 x MON 88017 Produced in 2005 U.S. Field Trials. Project
Number: 06/CR/172/3, BR/ME/0197/05, BR/ME/0197. Unpublished study prepared by
Monsanto Company. 93 p.

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MRID: 47127505

Citation: Hartmann, A.; Niemeyer, K.; Silvanovich, A. (2006) Assessment of the Cry 1 A. 105
and Cry2Ab2 Protein Levels in Tissues of Insect-protected Corn MON 89034 Produced
in 2005 U.S. Field Trials. Project Number: 06/CR/172/3, AG/EQ/1023/01,
BR/ME/1026/01. Unpublished study prepared by Monsanto Company. 51 p.

MRID: 47140300

Citation: Monsanto Company (2007) Submission of Exposure and Risk Data in Support of the
Application for Registration of MON 89034. Transmittal of 1 Study.

MRID: 47140301

Citation: Bogdanova, N.; Dubelman, S.; Mueth, M.; et al. (2007) Responses to EPA Questions
Regarding Application 521-LTL to Register Insect-Protected Corn MON 89034 (MRID
46951428). Project Number: 04/CR/172/4. Unpublished study prepared by Monsanto
Agricultural Co. 9 p.

MRID: 47166401

Citation: Ali, I.; Luttrell, R.; Abel, C. (2007) Monitoring Bt Susceptibilities in Helicoverpa zea:
Results of 2006 Studies. Project Number: 04/CT/133E/31, 04/CT/134E/31. Unpublished
study prepared by USD A, ARS Southern Insect Management Laboratory and University
of Arkansas. 23 p.

MRID: 47166403

Citation: Ali, I.; Luttrell, R.; Abel, C. (2007) Monitoring Bt Susceptibilities in Helicoverpa zea
and Heliothis virescens: Results of 2006 Studies. Project Number: 04/CT/133E/31,
04/CT/134E/31. Unpublished study prepared by University of Arkansas and USD A,
ARS Southern Insect Management Laboratory. 16 p.

MRID: 47243700

Citation: Monsanto Company (2007) Submission of Residue Data in Support of the

Experimental Use of Bacillus thuringiensis Cry 1 A. 105 and Cry2Ab2 Proteins and the
Genetic Materials Necessary for its Production in Corn. Transmittal of 4 Studies.

MRID: 47243701

Citation: Murphy, J.; Stillwell, L. (2007) CrylA.105, Cry2Ab2, Cry3Bbl, and CP4 EPSPS
Protein Levels in Selected Tissues of the Combined Trait Corn Product MON 89034 x
TC 1507 x MON 88017 x DAS-59122-7, in Support of an Application for an
Experimental Use Permit. Project Number: 07/CR/185E/2. Unpublished study prepared
by Monsanto Company. 13 p.

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MRID: 47243702

Citation: Phillips, A. (2007) Cry34Abl, Cry35Abl, Cry IF and PAT Protein Levels in Selected
Tissues of the Combined-Trait Corn Product MON 89034 x TC 1507 x MON 88017 x
DAS-59122-7, in Support of an Application for an Experimental Use Permit: Interim
Report. Project Number: 061026/01. Unpublished study prepared by Dow AgroSciences
LLC. 15 p.

MRID: 47243703

Citation: Burns, J. (2007) Petition to Amend 40 CFR 174.502 to Establish a Temporary

Exemption from the Requirement of a Tolerance for Bacillus thuringiensis Cry 1 A. 105
Insecticidal Protein and the Genetic Material Necessary for its Production When Used as
a Plant-Incorporated Protectant in Food and Feed Commodities of Field Corn, Sweet
Corn, and Popcorn, to Support an EUP. Project Number: 07/CR/185E/3. Unpublished
study prepared by Monsanto Company. 19 p.

MRID: 47243704

Citation: Burns, J. (2007) Petition to Amend 40 CFR 174.503 to Establish a Temporary

Exemption from the Requirement of a Tolerance for Bacillus thuringiensis Cry2Ab2
Insecticidal Protein and the Genetic Material Necessary for its Production When Used as
a Plant-Incorporated Protectant in Food and Feed Commodities of Field Corn, Sweet
Corn, and Popcorn, to Support an EUP. Project Number: 07/CR/185E/4. Unpublished
study prepared by Monsanto Company. 18 p.

MRID: 47279700

Citation: Monsanto Company (2007) Submission of Public Interest Data in Support of the
Applications for Registration of MON 89034 and MON 89034 x MON 88017.
Transmittal of 1 Study.

MRID: 47279701

Citation: Crawford, A.; Bogdanova, N. (2007) Public Interest Document for MON 89034 and
MON 89034 x MON 88017. Project Number 07/RA/50/04, 07/RA/39/04. Unpublished
study. 112 p.

MRID: 47444911

Schlenz, M.L., Babcock, J.M., and Storer, N.P., 2008. Response of CrylF-resistant and
susceptible European corn borer and fall armyworm colonies to Cry 1 A. 105 and
Cry2Ab2. Attached as Appendix 1 of the Insect Resistance Management Plan for MON
89034 x TC1507 x MON 88017 x DAS-59122-7.

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MRID: 47474801

Citation: Gustafson, D. I. and Head, G.P., 2008. Modeling the Impact of a Five-Percent
Structured Refuge on the Evolution of European and Southwestern Corn Borer
Resistance to MON 89034 Corn. Report submitted to EPA by Monsanto.

MRID: 47731601

Citation: Murphy, J. (2009) Independent Lab Validation of the EnviroLogix's QuickStix Kit for
Cry2Ab Bulk Grain Test Strip Performance Verification for Corn: MON 89034 and
MON 89034 x MON 88017. Project Number: 06/CR/172E/9. Unpublished study
prepared by EnviroLogix, Inc. 22 p.

MRID: 47791201

Citation: Jin Li, Y.; Hu, C.; Eyrich, M.; et al. (2009) Comparative Binding of the Bacillus

thuringiensis Cry 1 A. 105 and Cry 1 Ac Proteins to Cotton Bollworm (Helicoverpa zea) and
Tobacco Budworm (Heliothis virescens) Brush Border Membranes. Project Number:
06/CR/172E/12. Unpublished study prepared by Monsanto Company. 13 p.

MRID: 47791202

Citation: Siegfried, B.; Spencer, T. (2009) Baseline Susceptibility of the European Corn Borer to
the Cry 1 A. 105 Bt Endotoxin. Project Number: 06/CR/172E/13. Unpublished study
prepared by University of Nebraska. 10 p.

MRID: 47791203

Citation: Lang, B. (2009) Baseline Assessment of Bt Susceptibility of Helicoverpa. Zeta to
CrylA.105: 2008 Collections and Assays. Project Number: 06/CR/172E/11.

Unpublished study prepared by Custom Bio-Products. 12 p.

MRID: 47838801

Citation Gallagher, S.; Krueger, H. (2009) Evaluation of Exposure to Corn Pollen from

MON89034 in a 14-Day Static-Renewal Test with the Cladoceran (Daphnia magna):

Final Report. Project Number: 139A/382, WL/2008/473. Unpublished study prepared by
Wildlife International, Ltd. 44 p.

MRID: 47903501

Citation: Bogdanova, N.; Carden, J.; Head, G.; et al. (2009) Conditions of Registration for MON
89034 Insect-protect Corn: Updated Compliance Assurance Program, Educational
Materials, IRM Monitoring, and a Remedial Action Plan. Project Number:
06/CR/172E/14. Unpublished study prepared by Monsanto Company. 40 p.

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MRID: 47908301

Citation: Bogdanova, N. ; Carden, J. ; Head, G.; et al. (2009) Conditions of Registration for
MON 89034 x MON 88017 Insect-Protected and Herbicide Tolerant Corn: Updated
Compliance Assurance Plan, Educational Materials, IRM Monitoring, and a Remedial
Action Plan. Project Number: 07/CR/192E/15. Unpublished study prepared by
Monsanto Company. 32 p.

Additional Ecological Risk Assessment References:

Andow, David A and Angelika Hilbeck (July 2004) "Science-Based Risk Assessment for
Nontarget Effects of Transgenic Crops." Bioscience. Vol. 54, No. 7.

Andow, D A and Claudia Zwahlen (2006) "Assessing Environmental Risks of Transgenic
Plants." Ecology Letters. 9pl96-214. doi: 10.1111/j. 1461-0248.2005.00846

Beadle G. 1980. The ancestry of corn. Sci. American 242:112-119.

Benz B. 2000. Personal communication. Botanist, Professor, Department of Biology, Texas
Wesleyan University, Fort Worth, Texas.

Blackwood, C.B., and Buyer, J.S. (2004). Soil microbial communities associated with Bt and
non- Bt-com in three soils. Journal of Environmental Quality, 33: 832-836.

Bradley K. 2000. Personal communication. Botanist, Institute for Regional Conservation, Miami,
Florida.

Brusetti ,L., Francia, P., Bertolini, C., Pagliuca, A., Borin, S., Sorlini, C., Abruzzese, A., Sacchi
G., Viti, C, Giovannetti L., Giuntini, E., Bazzicalupo, M., and Daffonchio, D. (2004). Bacterial
communities associated with the rhizosphere of transgenic Bt 176 maize {Zea mays) and its non
transgenic counterpart. Plant and Soil, 266: 11-21.

De Schrijver et al. (2007) Risk Assessment of GM Stacked Events o/^/ained from crosses
between Events. Trends in Food Science and Technology. Vol 18, ppl01-109.

DeWald, Chester. (1999). Personal communication, plant breeder and geneticist, USDA-ARS,
Woodward, OK, December, (580_256_7449).

DeWald, C.L., P. Sims, Y. Li, and V.A. Sokolov. (1999). A novel cytoplasm for maize. Maize
Genetics Conference Abstracts. 41 :P114.

Doebley JF. 1984. Maize introgression into teosinte - A reappraisal. Ann. Missouri Bot. Gard.
71:1100-1113.

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Doebley JF. 1990. Molecular evidence for gene flow among Zea species. Bioscience 40:443-
448.

Doebley JF. 2000. Personal communication. Geneticist/Visiting Professor, Department of
Genetics, University of Wisconsin, Madison, Wisconsin (608-265-5803).

Doebley JF, Goodman MM, Stuber CW. 1987. Patterns of isozyme variation between maize and
Mexican annual teosinte. Econ. Bot. 41(2):234-246.

Duan JJ, Marvier M, Huesing J, Dively G, Huang ZY (2008) A Meta-Analysis of Effects of Bt
Crops on Honey Bees (Hymenoptera: Apidae). PLoS ONE 3(1): el415.
doi:10.1371/journal.pone.0001415

Duvick S. 1999. Personal communication. Geneticist, Department of Plant Genetics, Iowa State
University, Ames, Iowa (515-294-9375).

Edelstein, R. (2007). Review of Human Health and Product Characterization Data for
Registration for B. thuringiensis Cry 1 A. 105 and Cry2Ab2 Proteins and the Genetic Material
Necessary for their Production in MON 89034 Corn. U.S. Environmental Protection Agency.
Washington, D.C.

Edwards JW, Allen JO, Coors JG. 1996. Teosinte cytoplasmic genomes: I. Performance of maize
inbreds with teosinte cytoplasms. Crop Sci. 36:1088-1091.

Galinat WC. 1983. The origin of maize as shown by key morphological traits of its ancestor
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Galinat WC. 1988. The Origin of Corn, pp. 1-31. In: Corn and corn improvement, Third Edition.
Sprague GF, Dudley JW (Eds.). American Society of Agronomy, Crop Science Society of
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Griffiths, B.S., Caul, S., Thompson, J., Birch, A.N.E., Scrimgeour, C., Andersen, M.N., Cortet,
J., Messean, A., Sausse, C., Lacroix, B., and Krogh, P.H. (2005). A comparison of soil microbial
community structure, protozoa and nematodes in field plots of conventional and genetically
modified maize expressing the Bacillus thuringiensis Cryl Ab toxin. Plant and Soil, 275: 135-
146.

Gross, A. (2010). Review of the Evaluation of Exposure to corn Pollen from MON89034 in 14-
Day Static-Renewal Test with the Cladoceran Daphnia magna. U.S. Environmental Protection
Agency. Washington, D.C.

Hall D. 2000. Personal communication. Forensic Botanist and Environmental Consultant,
Gainesville, Florida (352-375-1370).

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Hilbeck et al. (2006) "Methodolgy to Support Nontarget and Biodiversity Risk Assessment."
Environmental Risk Assessment of Genetically Modified Organisms. Vol.2

Hitchcock AS. 1971. In: Manual of the grasses of the United States. Dover Publications,
Mineola, New York. ISBN 0486227170 and 0486227189.

Holm, L, J.V. Pancho, J.P. Herberger, and D.L. Plucknett. (1979). In: A geographical atlas of
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Honemann L, Zurbriigg C, Nentwig W. 2008. Effects of Bt-corn decomposition on the
composition of the soil meso- and macrofauna. Applied Soil Ecology 40:203-209.

Hunter, M. (2006). Review of "Evaluation of the Potential for Interactions Between Bacillus
thuringiensis Proteins Cryl A. 105 and Cry2Ab2" for Monsanto's MON 89034 X MON 88017
Maize Experimental Use Permit 524-EUP-OT. July 6, 2006.

Icoz, I, and G. Stotzky (2007). Cry3Bbl protein from Bacillus thuringiensis in root exudates and
biomass of transgenic corn does not persist in soil. Transgenic Research, September 13, 2007.

litis HH. 1983. From teosinte to maize: The catastrophic sexual transmutation. Science 222:886-
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litis H. 2000. Personal communication. Professor Emeritus of Botany, University of Wisconsin,
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Jemison J, Vayda M. 2000. University of Maine at Orono, Pollen transport from genetically
engineered corn to forage corn hybrids: A case study. Abstract presented to the Maine
Agricultural Trade Show, January 2000.

Kato-Y TA. 1997a. Review of Introgression between maize and teosinte. In: Gene flow among
maize landraces, improved maize varieties, and teosinte: Implications for transgenic maize,
pp. 44-53, Serratos JA, Wilcox MC, Castillo-Gonzalez F (Eds.), Mexico, D.F., CIMMYT.

Kato-Y TA. 1997b. Plenary session: Analysis of workshop reports and discussions. Group I
report. In: Gene flow among maize landraces, improved maize varieties, and teosinte:
Implications for transgenic maize, pp. 94-103, Serratos JA, Wilcox MC, Castillo-Gonzalez F
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Kermicle JL. 1997. Cross incompatibility within the genus Zea. In: Gene flow among maize
landraces, improved maize varieties, and teosinte: Implications for transgenic maize, pp. 40-43,
Serratos JA, Wilcox MC, Castillo-Gonzalez F (Eds.), Mexico, D.F., CIMMYT.

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Kermicle JL, Allen JO. 1990. Cross-incompatibility between maize and teosinte. Maydica
35:399-408.

Lambert J. 1999. Personal communication. Plant Breeder and Geneticist, Department of Crop
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Lawo, Nora C. and Jorg Romeis. (January 2008) "Assessing the utilization of carbohydrate
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Mangel sdorf PC, Reeves RG. 1939. The origin of Indian corn and its relatives, Texas
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Marvier, M., McCreedy, C., Regetz, J. & Kareiva, P. A meta-analysis of effects of Bt cotton and
maize on nontarget invertebrates. Science 316, 1475-1477 (2007).

Milofsky, T. and Z. Vaituzis (2006). Review the soil fate study submitted in support of ABSTC's
CrylAb corn registrations. Biopesticides and Pollution Prevention Division. U.S. Environmental
Protection Agency. Washington, D.C.

Muir WM and RD Howard (2001) Fitness Components and Ecological Risk of Transgenic
Release; A Model Using Japanese Medaka (Oryzias latipes). The American Naturalist. Vol.
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Oliveira AP, Pampulha ME, Bennett JP. 2008. A two-year field study with transgenic Bacillus
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Orzell S. 2000. Personal communication. Botanist/Ecologist, United States Air Force, Avon Park
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Read J. 2000. Personal communication. Professor, Texas Agricultural Experiment Station,

Dallas, Texas (972-231-5362).

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Sanvido,0., Romeis, J., Bigler, F. (2007). Ecological Impacts of Genetically Modified Crops:
Ten Years of Field Research and Commercial Cultivation. Adv Biochem Engin/Biotechnol 107:
235-278

Saxena, D. and Stotzky, G. (2001) Bacillus thuringiensis (Bt) toxin released from root exudates
and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and
fungi in soil. Soil Biol. Biochem., 33, 1225-1230

Schoper, J. (1999). Personal communication. Geneticist, Pioneer Hi-Bred International,

Johnston, IA, (515-270-3544).

Smith JSC, Goodman MM, Stuber CW. 1985. Relationships between maize and teosinte of
Mexico and Guatemala: Numerical analysis of allozyme data. Econ. Bot. 39:12-24.

USDA APHIS. 1997. USD A/APHIS Petition 97-265-01 for Determination of Nonregulated
Status for Bt Cry9C Insect Resistant and Glufosinate Tolerant Corn Transformation Event CBH-
351: Environmental Assessment. USDA, APHIS, Riverdale, Maryland.

USEPA (2000). SAP report No 99-06. Sets of scientific issues being considered by the
Environmental Protection Agency regarding: Section I - Characterization and Nontarget
Organism Data Requirements for Protein Plant Pesticides. Dated February 4, 2000. Available at
the EPA website: http://www.epa.gOv/scipoly/sap/1999/index.htm#december

USEPA. (2001). Biopesticides Registration Action Document - Bacillus thuringiensis Plant-
Incorporated Protectants. Available from:
http: ic ii'ic. eya. gov/ovvbyvdl/biovesticides/viys/bt brad, htm.

USEPA (2001). SAP Report No. 2000-07. Sets of scientific issues being considered by the
Environmental Protection Agency regarding: Bt plant-pesticides risk and benefit assessments.
Dated March 12, 2001. Web site: http://www.epa.gov/scipoly/sap/2000/october/octoberfinal.pdf

USEPA (2002). SAP Report No. 2002-05. A set of scientific issues being considered by the
Environmental Protection Agency regarding: Corn rootworm plant-incorporated protectant
nontarget insect and insect resistance management issues. Dated November 6, 2002. Web site:
http://www.epa.gov/scipolv/sap/2002/august/august20Q2final.pdf

USEPA. (2003). Ecological hazard assessment for Bacillus thuringiensis Cry3Bbl protein,
EPA Reg. No. 524-LEI

Vaituzis, Z. and R.I. Rose. (2000). Reassessment of Bt crop effects on nontarget wildlife.
Biopesticides and Pollution Prevention Division. U.S. Environmental Protection Agency.
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Wilkes GH. 1967. Teosinte: The closest relative of maize. Bussey Institute, Harvard University,
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Wilkes GH. 2000. Personal communication. Professor of Plant Genetics, University of
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Wilson H. 2000. Personal communication. Professor of Biology, Texas A&M University,
College Station, Texas (409-845-3354).

Wolfenbarger LL, Naranjo SE, Lundgren JG, Bitzer RJ, Watrud LS (2008) Bt Crop Effects on
Functional Guilds of Non-Target Arthropods: A Meta-Analysis. PLoS ONE 3(5): e2118.
doi: 10.1371/journal.pone.0002118

Wunderlin R. 2000. Personal communication. Professor of Botany, Institute for Systematic
Botany, University of South Florida, Tampa, Florida (813-974-2359).

Additional Insect Resistance Management Assessment References:

Adams, D.B. 1996. Sweet corn insect management, pp. 22-23. In D.T. Kelly [ed], Proc. 1996
Georgia Vegetable Conference and Trade Show. Georgia Cooperative Extension Service,
University of Georgia, Tifton.

Alves, A.P., Spencer, T.A, Tabashnik, B.E., and Siegfried, B.D. 2006. Inheritance of resistance
to the CrylAb Bacillus thuringiensis toxin in Ostrinia nubilalis (Lepidoptera: Crambidae). J.
Econ. Entomol. 99: 494-501.

Andow, D.A. and Hutchison, W.D., Corn resistance management, in Now or Never: Serious
Plans to Save a Natural Pest Control, Mellon, M. and Rissler, J., Eds., Union of Concerned
Scientists, Washington, D.C., 1998, chap. 2.

Andow, D.A., Olson, D.M., Hellmich, R.L., Alstad, D.N., and Hutchison, W.D. 2000. Frequency
of resistance to Bacillus thuringiensis toxin CrylAb in an Iowa population of European corn
borer (Lepidoptera: Crambidae). J. Econ. Entomol. 93:26-30.

Baskauf, S.J. 2003. Factors influencing population dynamics of the southwestern corn borer
(Lepidoptera: Crambidae): a reassessment. Environ. Entomol. 32:915-928.

Baskauf, S.J., and McCauley, D.E. 2001. Evaluation of low temperature mortality as a range-
limiting factor for the southwestern corn borer (Lepidoptera: Crambidae). Environ. Entomol.
30:18 1-188.

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Baxter, S.W., Zhao, J.Z., Gahan, L.J., Shelton, A.M., Tabashnik, B.E., and Heckel, D.G. 2005.
Novel genetic basis of field-evolved resistance to Bt toxins in Plutella xylostella. Insect Molec.
Biol. 14:327-3 34.

Benedict, J.H. 2004. Biology and Dispersal of the Cotton Bollworm and Tobacco Budworm in
North America. Submitted to EPA as Appendix 1 of MSL-20123.

Bickel, A., Stojsin, R., and Vaughn, T. 2002. Lepidopteran Field Efficacy Results for tIC105
Events. Monsanto Technical Report #MSL-18264.

Bogdanova, N.N. 2007. Supllemental information to address EPA questions regarding
applications 524-LTL and 524-LTA to register insect-protected corn MON 89034 and MON
89034 X MON 88017 (MRIDs# 46951400 and 46951300). Report submitted to EPA by
Monsanto. MRID# 470794-02.

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Additional Benefits Assessment References:

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BPPD, 2007a. Review of Monsanto Company's assessment of the efficacy of lepidopteran-
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