BIOPESTICIDE REGISTRA TION ACTION DOCUMENT

Bacillus thuringiensis Cry1A.105 and Cry2Ab2 Insecticidal Pr oteins and the Genetic Material Necessary for Their Production in Corn [PC Codes 006515 (Cry2Ab2), 006514 (Cry1A.105)]

U.S. Environmental Protection Agency Office of Pesticide Programs Biopesticides and Pollution Prevention DivisionBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

2 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 C RY 1A.105 ....................................................................................19

C. HUMAN HEALTH ASSESSMENT C RY 2A B 2 .............................................................................................27

D. ENVIRONMENTAL ASSESSMENT FOR MON 89034 ................................................................................34

E. INSECT RESISTANCE MANAGEMENT (IRM) ........................................................................................66

F. BENEFITS AND PUBLIC INTEREST FINDING ......................................................................................113

III. REGULATORY POSITION FO R CRY1A.105, AND CRY2AB2 .................................................................... 136

IV. BIBLIOGRAPHY:

............................................................................................................................... ................. 142Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

3 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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

4 I. OVERVIEW

A. BACKGROUND

On June 10, 2008, EPA conditionally registered a pl ant-incorporated protectant product, event MON 89034 corn, containing tw o new active ingredients, Bacillus thuringiensis Cry1A.105 and Cry2Ab2 insecticidal proteins, and the genetic mate rial necessary for their production. EPA also conditionally registered anot her product, MON 89034 x MON 88017 corn, which contains a previously registered Bacillus thuringiensis Cry3Bb1 protein in addi tion to the two new active ingredients. These registrati ons will expire on September 30, 2010. The Agency determined that the use of these pesticide pr oducts is in the public interest and that their use will not cause any unreasonable adverse effects on the environmen t 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 th is product, called Cry1A.105, and Cry2Ab2, have been shown to effectively control highly destru ctive lepidopteran corn pests, including European corn borer (ECB), corn ea rworm (CEW), southwestern corn borer (SWCB), fall armyworm (FAW), and sugarcane borer (S CB), in field trials conducted during the 2003- 2004 growing seasons in Puerto Rico and the Unit ed States. These pests feed on the base of seedlings and on the stalk, leaf, a nd ear tissue of corn plants, ther eby 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 pe st 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 re quired to be at least 20% for the corn belt. On December 15, 2008, EPA am ended these product registrations to allow a reduction in the structured corn borer refuge re quirement (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 Cry1A.105, and Cry2Ab2.”

On October 1, 2009, EPA announced a new policy to provide a more meani ngful opportunity for the public to participate on major registration decisi ons before they occur. According to this new policy, EPA intends to provide a public comment period prior to making a registration decision for, at minimum, the following types of applicati ons: 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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

5 Consistent with the policy of making regist ration actions more transparent, the proposed amendments to the expiring MON89034 corn prod ucts are subject to a 30-day comment period because the Agency believes, given past experiences with PIPs in general, these actions are of significant public interest. While a final deci sion on amending the expiring MON89034 corn products is contingent upon review and consider ation of public comments received, the Agency maintains that, based on all data submitte d in support of the expiring MON89034 corn registrations (both for initial regi strations and as responses to condi tions of registration), it is in the best interest of the pub lic and the environment to amend the currently existing MON89034 registrations by extending the current expirati on dates to September 30, 2015. The basis for this preliminary decision can be found in the ri sk assessment for the expiring MON89034 corn products, which is characterized throughout this Biopesticides Registration Action Document (BRAD). After scientific revi ews of MON89034 conditional data were completed, this BRAD was updated appropriately to reflect the most cu rrent information available to and evaluated by the Agency regarding these registrations.

All data and findings for the expiring 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), as this should be the most familiar format to outside stakeholders interested in providing comments on these actions. In addition to the expiring MON89034 corn products, there are other Bt corn PIPs, expressing different proteins effective in c ontrolling various lepidopteran pest s, that expire in 2010, and for which the associated registrants have formally requested an extension to the current expiration dates. Therefore, within the same docket (EPA-HQ-OPP-2010-0607) as this document, the following information a is also available for public examination:

● Cry1F and Cry1Ab BRAD

• Cry 3Bb1 BRAD

● Cry34/35Ab1 BRAD

● mCry3A BRAD

● Optimum® AcreMaxTM B

. t . Seed Blends BRAD

● 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

EPA has made the preliminary decision to amend the registrations of twenty-two (22) expiring Bt corn PIP registrations to extend the expira tion dates. We have conducted comprehensive assessments of each of these registrations, cons idering all toxicity and environmental effects data, data from insect resistance monitoring, an d insect resistance refuge compliance reports,

a Each of the Biopesticides Registration Ac tion Documents available for public comment in this action are modified from previous versions to account for data/information submitted to fulfill terms and conditions of registration.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

6 received and obtained since the last comprehensive evaluation of these products in 2001. Based upon our comprehensive assessment, we have re ached significant conclusions regarding the positive environmental impact of Bt corn PIPs, and we are proposing 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 signi ficant number of published field studies that, combined with the post-registrati on field studies required to be submitted to the Agency, have demonstrated that non-target i nvertebrates 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 st udies demonstrate th at, not only are the Bt crops not causing any unreasonable adverse effects in the environment, but, arth ropod prevalence and diversity is greater in Bt crop fields. Although no effects have been observed in the environment, the Agency is requiring additional aquatic invert ebrate data in light of published laboratory studies showing reduced growth in shredding caddis flies exposed to Cry1A protein corn litter.

To strengthen insect resistance management of these corn PIPs and to address reports that compliance with the mandated refuge requireme nts has been decreasing, EPA is proposing to require enhanced compliance assurance programs (CAPs), and a phased requirement for seed bag labeling that clearly shows th e refuge requirements. Also, give n the increasing variety of PIP products and combinations, and the differing risk of resistance evolution that the various products represent, we are proposi ng to grant registrations for the corn PIP products for different timeframes, based on assessments of their likelih ood of forestalling the evolution of insect resistance. We are registering di ffering categories of products for differing time periods to reflect

the assessed level of risk of re sistance posed by the va rious corn PIP products. The scheme that we propose to follow will include registration periods generally of five, eight, and twelve years; with the possibility of a fifteen -year registration period for produc ts that are demonstrated to meet specified criteria. We retain, however, the discretion to register pr oducts for time periods differing from these defaults where circumstances warrant.

B. EXECUTIVE SUMMARY

Product Characterization

MON 89034 was developed by Agrobacterium -mediated transformation of corn using the 2TDNA plasmid vector PV-ZMIR245. The transformation produces two Bacillus thuringiensis proteins, Cry1A.105 and Cry2Ab2. Cry1A.105 is a ch imeric protein composed of portions of Cry1Ab, Cry1Ac, and Cry1F proteins.

Mammalian Toxicity and Allergenicity AssessmentBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

7 The acute oral toxicity data submitted by Monsanto demonstrated the lack of mammalian toxicity in rats and mice exposed to pure Cry1A.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 maxi mum exposure levels anticipated for a plantincorporated 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 protecta nts were derived. [See 40 CFR Sec. 158.2130 and 158.2140.] For microbial products, further toxicity testing to veri fy the observed effects an d clarify the source of the effects (Tiers II & III) and residue data ar e triggered by significant acute effects in studies such as the mouse oral toxicity study.

Since no acute effects were observed in the submitte d studies, even at relatively high dose levels, the Cry1A.105 and Cry2Ab2 proteins are not consid ered to be toxic. This conclusion was supported by amino acid sequence comparisons of the Cry1A.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 M onsanto demonstrated that the Cry1A.105 and Cry2Ab2 proteins were substantially degraded by heat when examined by immunoassay. This instability to heat w ould decrease the potential for diet ary exposure to intact Cry1A.105 and Cry2Ab2 proteins in cooked or processed foods. These biochemical features, along with the lack

of adverse results in the acute or al toxicity tests, support the Agen cy’s conclusion that there is a reasonable certainty of no ha rm from dietary exposure to Cry1A.105 and Cry2Ab2 containing

crops.

Since Cry1A.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 “wei ght-of-evidence” approach when considering the allergenic potential for a PIP protein, and base s its conclusions upon the following factors: the source of the trait, the amino acid sequence compar ed with known allergen s, and the biochemical properties of the protein, includi ng in vitro digestibility in si mulated 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 Cry1A.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 Cry1A.105 and Cry2Ab2 with known allergens showed no sequence similarity or identity at the level of eight contiguous amino acid residues, which is considered to be the smallest amino acid sequence necessary to induce an immune response.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

8 3. Digestibility. The Cry1A.105 and Cry2Ab2 proteins were dige sted rapidly in simulated gastric fluid containing pepsi n, the enzyme produced by the st omach that digests proteins so they can be absorbed as nutrients into the body via the small intestine. The rapid degradation of Cry1A.105 and Cry2Ab2 in the simulated gastric environment indicated that the intact protein will not pass from th e stomach into the intestinal lumen, where sensitization of the immune syst em to food allergens occurs.

4. Glycosylation. Cry1A.105 and Cry2Ab2 prot eins expressed in corn are not glycosylated. 1

5. Conclusion. EPA concluded that the poten tial for Cry1A.105 and Cry2Ab2 to be a food allergen is minimal.

The information on the safety of pure Cry1A .105 and Cry2Ab2 proteins provides adequate justification to address possible exposures in all corn crops.

Environmental Hazard Assessment

Maximum hazard dose toxicity testing on representa tive beneficial organisms from several taxa was performed in support of the registrations of Cry1A.105 and Cry2Ab2 pr oteins expressed in corn. The toxicity of the Cr y1A.105 and Cry2Ab2 proteins was ev aluated on several species of invertebrates, including the la dy beetle, minute pirate bug, parasitic hymenoptera, Collembola, Daphnia , honey bee, and earthworm. Developmental obs ervations 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 Cry1A.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 Cr y1A.105 and Cry2Ab2 proteins, and MON 89034 corn leaf tissue, pollen, and grain. The Octobe r 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 subs tance, 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) ar e needed for maximum hazard dose testing. An insect feeding study, which compared the rela tive potency of plant produced Cry1A.105 and Cry2Ab2 proteins to the microbe produced proteins, indicated th at plant produced protein was similar in toxicity to the bacterially produced protein (E delstein Memo, November 7, 2007).

1 Although this was only demonstrat ed 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-Lainé, A-C., Gomord, V., and Faye, L., “N -Glycoprotein biosynthesis in pl ants: recent developments and future trends,” Plant Molecular Biology 38: 31-48, 1998Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

9 The potential interactions between the Cry1A .105 and Cry2Ab2 proteins was addressed in a memorandum for the MON 89034 Experimental Use Permit accompanying the Agency’s review, “Evaluation of the Potent ial for Interactions Between the Bacillus thuringiensis Proteins Cry1A.105 and Cry2Ab2,” (Hunter, M., July 6, 2 006). The study provided evidence that the proteins do not interact in either an antagonistic or synergistic ma nner, and that there will not be any unexpected interactions with regard to ta rget and non-target ins ects. New data on the potential interaction between the combined Cry1A.105, Cry2Ab2 and Cry3Bb1 proteins were submitted. The results from that study demonstrated that the combined Cry1A.105 and Cry2Ab2 activity was not affected by the Cry3B b1 protein, and that the Cry3Bb1 activity was unaffected by combined Cry1A.105 and Cr y2Ab2 activity (MRIDs 469513-05 & 469513-06).

Insect Resistance Management

Monsanto has demonstrated that the Cry1A.105 and Cry2Ab2 toxins have different modes of action, and consequently, a low likelihood of cross-resistance. Therefore, Cry1A.105 and Cry2Ab2 are suitable partners in a pyramided produc t. Monsanto has also shown that there is a

low likelihood of cross-resistance between Cr y1A.105 and Cry1Ab. Monsanto has previously

demonstrated that there is a low likelihood of cross-resistance between Cry2Ab2 and Cry1Ac. Both Cry1Ab and Cry1Ac are expressed in other registered Bt corn and Bt cotton PIPs. Monsanto did not, however, address the likeli hood of cross-resistan ce between Cry1A.105 and Cry1Ac, and Cry1Fa ( 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 Cry1A.105 and Cry1Fa and Cry1Ac (including binding site models and use of resistant co lonies) for the target pests and determine how such cross-resistan ce 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 fi eld corn uses of MON 89034 in the U.S. Corn Belt. But, the data and simulation mode ling 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 a nd heterozygote (i.e., partially resistant) insects (ECB, SWCB, CEW, and FAW), the cross-re sistance potential of Cry1A.105, Cry1Ac and Cry1Fa and any impacts on the durability of MON 89034, and limitations in the simulation modeling. Therefore, the field corn uses of M ON 89034 in the Corn Belt were registered with a 20% refuge requirement until such time as Mons anto 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 th e 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% to 5% in the U.S. Corn Belt. After reviewing th ese data, EPA determined that a 5% refuge in theBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

10 U.S. Corn Belt should not significantly increase the risk of resistance for ECB, CEW, and SWCB. Monsanto sufficiently addressed the require ment to analyze potential cross resistance in existing Bt corn and Bt cotton products for Cry1A.105 and Cry1Fa, but additional analysis and information is still needed to fully assess the cross resistance pot ential for Cry1Ac and Cry1A.105.

C. USE PROFILE

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

Trade and Other Name(s): MON 89034

OPP Chemical Code s: 006515 (Cry2Ab2) and 006514 (Cry1A.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 grandiosella ), Southern cornstalk borer ( Diatraea

crambidoides ), Corn earworm ( Helicoverpa zea ), Fall armyworm ( 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 8 8017. MON 89034 was developed by Agrobacterium -mediated transformation of corn us ing the 2T-DNA plasmid vector PVZMIR245, and produces two Bacillus thuringiensis ( Bt ) proteins, Cry1A.105 and Cry2Ab2. These proteins are intended to provide protection from feedi ng damage caused by a number of lepidopteran pests. Cry1A.105 is a chimeric pr otein composed of porti ons of Cry1Ab, Cry1Ac, and Cry1F proteins. On July 17, 2006, EPA es tablished temporary exemptions from the requirement of a tolerance for both Cry1A.105 (71 FR 40427 and 72 FR 20434; 40 CFR 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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

11 On November 15, 2006, Monsanto submitted pet itions to EPA under the Federal Food, Drug, and Cosmetic Act (FFDCA), as amended by th e Food Quality Protecti on Act of 1996 (FQPA), requesting amendment of the existing te mporary tolerances in 40 CFR 174.503 for the Bacillus thuringiensis Cry2Ab2 insecticidal protein to esta blish 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 1A.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 In secticide, Fungicide and Rodenticide Act (FIFRA).

On March 9, 2007, Monsanto resubmitted petiti ons 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 esta blish a permanent exemption from the requirement of a tolerance for the Bt Cry2Ab2 insecticidal prot ein, and in 40 CFR 174.502 for

the Bt Cry 1A.105 insecticidal protein to establish a permanent exemption from the requirement

of a tolerance for the Bt Cry 1A.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 FR No. 128), the existing perm anent 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 plan t-incorporated protectant in the food and feed commodities: field corn, sweet corn, popcorn, co tton 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 FR No. 137), the Agency es tablished permanent exemptions from the requirement of a tolerance for residues of the Bacillus thuringiensis Cry1A.105 protein in or on the food and feed commodities: field corn, sw eet 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 registra tions were amended for MON 89034 and MON 89034 x MON 88017, to allow for a 5% structured ref uge in the corn belt (in non-cotton growing regions) for corn borers.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

12 II. SCIENCE ASSESSMENT

A. PRODUCT CHARACTERIZATION

MON 89034 was developed by Agrobacterium -mediated transformation of corn using the 2TDNA plasmid vector PV-ZMIR245 and produces two Bacillus thuringiensis proteins, Cry1A.105 and Cry2Ab2. These proteins are intended to provide protection from feeding damage caused by a number of lepidopteran pests. Cry1A.105 is a chimeric prot ein composed of portions of Cry1Ab, Cry1Ac, and Cry1F proteins.

Transformation System : PV-ZMIR245 is a binary vector containing two separate transfer DNAs (2T-DNA). The first TDNA contains the cry1A.105 and the cry2Ab2 expression cassettes. The second T-DNA contains

the nptII (neomycin phosphotransferase II ) expression cassette. The cry1A.105 expression

cassette contains the cry1A.105 coding sequence under the regulation of the e35S promoter,

Ract1 intron, and the Hsp1 7 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- SSU-CTP ), and the nos 3’ end sequence. The nptII expression

cassette contains the nptII coding sequence under the regulation of the CaMV 35S promoter and

the nos 3’ end sequence. During transformation, bot h T-DNAs were inserted into the genome.

The nptII selectable marker gene was used to select for transformed cells. Traditional breeding was then used to isolate pl ants that only contain the cry1A.105 and cry2Ab2 expression cassettes

and not the nptII expression cassette.

Characterization of the DNA Inserted in the Plant and Inheritance and Stability : Characterization of the DNA isolated from ev ent MON 89034 corn using restriction enzyme digests and Southern blot analysis as well as DNA sequencing indi cates that the DNA was inserted in the corn genome at a single locus, and the insert contains one copy each of the cry1A.105 and cry2Ab2 expression cassettes. There were no ot her detectable elements other than

those associated with the respective cassettes. No b ackbone sequences from plasmid PVZMIR245 or nptII coding sequences were detected in the corn genome. Southern blot analysis also demonstrated the stability of the in sert over multiple generations. DNA sequencing indicated that the genetic elements were present in the inserted DNA as e xpected except that the

e35S promoter was modified, and the right bo rder sequence present in PV-ZMIR245 was replaced by a left borde r sequence in MON 89034.

Protein Characterization : Protein characterization data demonstrate that the plant-produced Cry1A.105 and Cry2Ab2 proteins have biochemical and functional ac tivities that are similar to those of the E. coli

produced proteins that were used in several toxi city studies. The following techniques were used to characterize and compare the plant-produced and the E. coli -produced proteins: sodium dodecyl sulfate polyacrylamide gel electrophores is (SDS-PAGE), western blot analysis, densitometry, matrix-assisted laser desorpti on/ionization time-of-fli ght (MALDI-TOF) massBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

13 spectrometry, glycosylation analysis, N-terminal amino acid sequencing, and insecticidal activity assays. Glycoslyation analysis i ndicated that the proteins are not glycoslyated. These analyses

demonstrated the structural and functional si milarity between the plant-produced and the E. coli

produced Cry1A.105 and Cry2Ab2 proteins and justified the use of E. coli -produced proteins in toxicity studies. Monsanto also provided information showing the similarity between Cry1A.105, Cry1Ab, and Cry1Ac.

In addition, Monsanto provided information co mparing 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-te rminus is blocked in both. Therefore, Monsanto was unable to determin e the cleavage site of the chloroplast transit peptide. Because the chloropl ast transit peptides used in MON 89034 and Bollgard II have potential cleavage sites (methi onine) 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 cleavag e site is within the transit peptide. The E. coli

produced Cry2Ab2 protein used in the toxic ity 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 a nd Bollgard II are varian ts of the wild type Cry2Ab2 protein produced in B. thuringiensis . The Bt -produced protein was us ed 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. coli -produced Cry2Ab2

and the Bt -produced Cry2Ab2 have equivalent biologi cal activity (EC50 va lues and rates of growth inhibition) in a larval corn earworm diet-incorporation bioassay.

Analytical Detection Methods : Short descriptions of enzyme- linked immunosorbent assay (ELI SA) methods for detecting and quantifying Cry1A.105 and Cry2Ab2 as well as standa rd 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 Cry1A.105 will distinguish between Cry1A.105, Cry1Ab, Cry1Ac, and Cry1F. Since Cry1A.105 contains por tions of all three proteins, there may be cross-reactivity in the assa y. Monsanto also provided a study demonstrating that a commercially available qualitative immunochromatographic test strip can detect Cry2 Ab2 in MON 89034 corn.

Since event MON 89034 is the on ly product that expresses Cr y1A.105 and the only corn product that expresses Cry2Ab2, the detection method for Cry2Ab2 can be used for detecting both Cry2Ab2 and Cry1A.105. The presence of Cry2Ab2 in corn should also indicate the presence of Cry1A.105.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

14 When Bt Cry1A.105, and Cry2Ab2 Protein in corn (EPA Reg. No. 524-575, and 524-576) were initially registered, the Agency issued regist ration notices to Monsan to that contained the following requirement for further pr oduct characterization information:

“For event MON 89034 corn, an in dependent lab validation of the analytical method for the detec tion of Cry2Ab2 and/or Cry1A.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 mont hs 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 regi stration. When the evaluation of the independent lab validation of an analytical detection method for Cry2Ab2 pr otein 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 base d on the number of blinded samples tested and also confirmed that at the 1.0% LOD the dipstic k reagents show zero fa lse positive and false negative results. This indicates acceptable perf ormance standards for a rapid analytical method but significantly does not addres s several performance criteria re quired by GIPSA for its dipstick

test kit validation. The use of the kit manufacturer for i ndependent validation is also questionable.

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

Protein Expression : Expression level data were provi ded for Cry1A.105 and Cry2Ab2 in di fferent 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 provi des summaries of the product characterization studies and data provided.

Table 1. Mean Expression Levels of Cry1A.105 and Cry2Ab2 from MON 89034 Plant

Tissues Tissue Type Cry1A.105 (μg/g dry weight + standard deviation)* Cry2Ab2 (μg/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 + 0.091Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

15 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

Corn 2 Southern blot analyses indicate that MON 89034 and MON 89597 have the

introduced DNA inserted in the corn genome at a single locus and contain one copy each of the cry1A.105 and cry1A.105 expression cassettes. All expression elements are s hown to be present in each of the inserts, and there

are no other elements detectable ot her than those associated with the respective cassettes. No backbone sequences from plasmid PV-ZMIR245 or nptII coding sequences were detected in the corn genome.

Classification: ACCEPTABLE 46694501

Analytical detection method/Qualitative

Detection Method for the Cry2Ab2 Protein in

Corn Leaf and Seed of

MON 89034 and MON

89597 2 A commercially available qualitative immunochromatographic test strip (QuickStixTM 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 QuickStixTM 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 TM Kit for Cry2Ab2 Bulk Grain ca n 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 Cry1A.105 Protein to Cry1A Class of Bacillus thuringiensis Proteins: Final Report 2 A summary of current information about the structural and functional similarities of the Cry1A.105 protein to other Bt Cry1 proteins is presented in this submission. The Cry1A.105 protein is chimeric, with overall amino acid sequence identity to the Cry1Ac, Cry1Ab and Cry1F proteins of 93.6, 90.0 and 76.7%, respectively. A stru ctural model of the Cry1A.105 protein was developed using the X-ray crystal structure of the Cry1Aa protein. The model demonstrated high overall main chain structural similarity with Cry1Aa. Models of Cry1Ab and Cry1Ac were also prepared using the Cry1A.105 model. Comparison of the aligned folds of all three proteins showed that Cry1Ab and Cry1A.105 have essentially the same main chain structure, and that Cry1Ac differs sligh tly in its main chain structure from the other two in domain III. Thus, comparison of the modeled crystal structures of the Cry1A.105, Cry1Ab, and Cry1Ac with that of the experimental Cry1Aa 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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

16 Study Type/Title Summary MRID #

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

Classification: ACCEPTABLE

Characterization of the

activeingredient/Characterization of the Cry1A.105

Protein Purified from the

Corn Grain of MON 89034 and Comparison of the Physiochemical

and FunctionalProperties of the PlantProduced and E. coli

Produced Cry1A.105

Proteins 2 The physicochemical properties and functional properties of the plantproduced Cry1A.105 were analyzed and compared with the properties of the

E. coli produced Cry1A.105 using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS -PAGE), western blot analysis, densitometry, matrix assisted laser desorption/ioni zation time of flight (MALDI-TOF) mass spectrometry, glycosylation analysis, and a Cry1A.105 insecticidal activity assay. Similar immunoreactive bands migrating between approximately 85 and 130 kDa were observed in the plant-produced Cry1A.105 and E. coli -produced reference samples, and the full-length Cry1A.105 protein (~130 kDa) was observed in both the plant-produced and

E. coli -produced protein samples. MALDI-TOF mass spectrometry analysis of the ~130 kDa band after trypsin digestion yielded peptide masses consistent with peptide masses of the predicted sequence of the Cry1A.105 protein. The identified peptide masses yielded 43.8% overall coverage of the expected peptide se quence (516 of the 1177 amino acids). Immunoreactivity with the N-terminal peptide antibody demonstrated that the N-terminus in the plant-produc ed full-length Cry1A.105 protein was intact. Glycosylation analysis demonstrated that neither the plant-produced

nor the E. coli -produced Cry1A.105 protein is glycosylated. The plantproduced and E. coli -produced proteins gave similar results in the corn earworm diet-incorporation bio activity assay: the mean EC 50 values for the plant-produced Cry1A.105 protein and E. coli -produced reference standard were determined to be 0.0074 and 0.012 μg Cry1A.105 per mL diet, respectively. The results of this study demonstrate the structural and functional similarity between the plant-produced and the E. coli -produced Cry1A.105 proteins.

Classification: ACCEPTABLE 46694604Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

17

Study Type/Title Summary MRID #

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 cry1A.105 and cry2Ab2 expressioncassettes at a single locus. No backbone sequences from plasmid PVZMIR245 or nptII 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 sequen cing; 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 Cry1A.105 and Cry2Ab2 Protein Levels

in Tissues of Insectprotected corn MON

89034 Produced in 2005

U.S. Field Trials The levels of Cry1A.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 Cry1A.105 protein levels across all sites were 5.9 μg/g dry weight (dwt) in grain, 42 μg/g dwt in forage, 12 μg/g dwt in po llen, 520 μg/g dwt in over season leaf collected at growth stage V2-V4 (OSL -1), 120 μg/g dwt in leaves OSL-4 (collected at growth stage pre-VT), 12 μg/g dwt in forage root, and 50 μg/g dwt in stover. In tissues harvested throughout the growing season, mean Cry1A.105 protein levels across all sites ranged from 72-520 μg/g dwt in leaf, 42-79 μg/g dwt in root, and 100-380 μg/g dwt in whole plant. The means for Cry2Ab2 protein levels across all sites were 1.3 μg/g dwt ingrain, 38 μg/g dwt in forage, 0.64 μg/g dwt in pollen, 180 μg/g dwt in OSL1, 160 μg/g dwt in OSL-4, 21 μg/g dwt in forage root, and 62 μg/g dwt in stover. In tissues harvested throughout the growing season, mean Cry2Ab2 protein levels across all sites ranged from 130-180 μg/g dwt in leaf, 26-58 μg/g dwt in root, and 39-130 μg/g dwt in whole plant.

Classification: ACCEPTABLE 46951403Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

18 Study Type/Title Summary MRID #

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 compar ed 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. Simila r 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 migra ting at approximately 50 kDa; Nterminal amino acid analysis of this pr otein indicated that it is a truncatedCry2Ab2 protein with its N-terminus starting at amino acid 145. MALDITOF mass spectrometry analysis of th e ~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 Cry2Ab 2 protein is not glycosylated. The plant-produced

and E. coli -produced proteins gave similar results in the bioactivity assay:

the mean EC 50 values for the plant-produced Cry2Ab2 protein and E. coli

produced reference standard were both determined to be 0.16 μg Cry2Ab2 per mL diet, with standard deviations of 0.04 and 0.01 μg 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 Cry2Ab2 produced from Bt was evaluated using a co rn 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-LTAto Register Insectprotected 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-

47127505Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

19 B. HUMAN HEALTH A SSESSMENT OF Cry1A.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 ce rtainty that no harm will result from aggregate exposure to the pesticide chemi cal residue, including all antici pated 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 o ccupational exposure. Pursuant to section 408(c)(2)(B), in es tablishing or maintaining in effect an exemption from the requirement of a tolerance, EPA must take in to account the factors set forth in section 408(b)(2)(C), which require EPA to give specia l consideration to e xposure of infants and children to the pesticide chemical residue in estab lishing 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 me chanism of toxicity.” EPA perf orms a number of analyses to determine the risks from aggregate exposure to pe sticide 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 re sult of pesticide use in residential settings.

1. Toxicological Profile

Consistent with section 408(b)(2 )(D) of the FFDCA, EPA has review ed the available scientific data and other relevant information in suppor t of this action and c onsidered its validity, completeness and reliability, and the relationship of this inform ation 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 Cry1A.105 protei n. These data demonstrate the safety of the product at a level well above maximum possible e xposure levels that are reasonably anticipated in the crop. Basing this conclusion on acute oral toxicity data w ithout requiring further toxicity testing and residue data is similar to the Ag ency position regarding t oxicity testing and the requirement of residue data for the microbial Bacillus thuringiensis products from which this plant incorporated protectant was derived (S ee 40 CFR Sec. 158.740(b)(2)(i)). For microbial products, further toxicity testi ng and residue data are triggere d by significant adverse acute

effects in studies such as the m ouse oral toxicity study, to verify the observed adverse effects and clarify the source of these effects (Tiers II & III).Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

20 An acute oral toxicity study in mice (MRID 46694603) indicated that Cry1A.105 is non-toxic to humans. Cry1A.105 produced from microbial cu lture was dosed by gavage as two doses separated by 4 hours (±20 minutes) to 10 fema les and 10 males (2072 mg/kg body weight). Two control groups were also incl uded in the study: a bovine serum albumin protein control, and a vehicle control. One male in the test protei n 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 diffe rences in body weight or body weight change among the three groups during the study, and no trea tment-related gross pathological findings

were observed. The oral LD 50 for males, females, and combined mice was greater than 2072 mg/kg.

When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad, Roy D., et al., “Toxicological Consider ations for Protein Components of Biological Pesticide Products,” Regulatory Toxicology and Ph armacology 15, 3-9 (1992)). Therefore, since no acute effects were shown to be caused by Cr y1A.105, even at relatively high dose levels, the Cry1A.105 protein is not considered toxic. Further, amino aci d sequence comparisons showed no similarities between the Cry1A.105 and known toxic proteins in protein databases that would raise a safety concern.

Since Cry1A.105 is a protein, allergenic potential was also considered. Currently, no definitive tests for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight-of-evidence approach where the following factors are considered: source of the trait; amino acid sequence comparison with known alle rgens; and biochemical properties of the protein, including in-vitro digestib ility in simulated gastric fluid (SGF) and glycosylation. This approach is consistent with the approach out lined in the Annex to the Codex Alimentarius “Guideline for the Conduct of Food Safety A ssessment of Foods Derived from RecombinantDNA Plants.” The allergenicity assessment for Cry1A.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 Cry1A.105 with known allergens showed no overall sequence si milarity or identity at the level of eight contiguous amino acid residues.

3. Digestibility. The Cry1A.105 protein was dige sted within 30 seconds in simulated gastric fluid cont aining pepsin.

4. Glycosylation. Cry1A.105 expressed in corn was shown not to be glycosylated.

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

Although Cry1A.105 was only shown not to be glycos ylated 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 site s for the enzymes involved in gl ycosylation, and the mechanismsBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

21 of protein glycosylation are si milar in different plants (Ler ouge, P. Cabanes-Macheteau, M., Rayon, C., Fichette-Lainé, 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

Pursuant to FFDCA section 408( b)(2)(D)(vi), EPA considers avai lable 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 (r esidential and other indoor uses).

The Agency has considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of consumers) to the pesticide chemical residue and to other

related substances. These considerations includ e dietary exposure under the tolerance exemption and all other tolerances or exemptions in effect for residues of the plant-in corporated protectants, and exposure from non-occupational sources. Expos ure 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 inha lation, the potential for Cry1A.105 to be an allergen is low, as discussed previously. Although the allergenic ity assessment focused on the Cry1A.105 protein’s potential to be a food allergen, the data also indicated a low potential for Cry1A.105 to be an inhalation allergen. Exposure to infants and child ren via residential or la wn use is not expected, because the use sites for the Cry1A .105 protein is agricultural. Oral exposure, at very low levels, may occur from ingestion of processed corn products and, theoretical ly, 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 cu mulative 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 plan t-incorporated protecta nt, EPA concluded that there are no cumulative effects for the Cry1A.105 protein.

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

a) Toxicity and Allergenicity Conclusions

The data submitted and cited regarding potenti al health effects for the Cry1A.105 protein included the characterization of the expressed Cry1A.105 protein in corn, as well as the acute oral toxicity study, amino acid se quence comparisons to known alle rgens and toxins, and in vitroBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

22 digestibility of the protein. The results of these st udies were used to evaluate human risk, and the validity, completeness, and reliabil ity of the available data from th e studies were also considered.

Adequate information was submitted to show that the Cry1A.105 test material derived from

microbial culture was biochemica lly and functionally equivalent to the protein produced by the plant-incorporated protec tant ingredient in the plant. Micr obially produced protein was used in

the studies so that sufficient mate rial for testing was available.

The acute oral toxicity data submitted support the prediction that the Cry1A.105 protein would be non-toxic to humans. As mentioned above, when proteins are toxic, they are known to act via acute mechanisms and at very low dose levels. Given that no treatment -related adverse effects were shown to be caused by the Cry1A.105 protei n, even at relatively high dose levels, the Cry1A.105 protein is not considered toxic. Basing this conclusi on on acute oral toxicity data without requiring further toxicity testing or residue data is similar to the Agency position regarding toxicity and th e requirement of residue data for the microbial Bacillus thuringiensis products from which this plant-incorpor ated 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 cl arify 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 Cry1A.105 in corn tissues.

Since Cry1A.105 is a protein, potentia l allergenicity is also consider ed as part of the toxicity assessment. Considering all of the availabl e information (1) Cry1A.105 originates from a nonallergenic source; (2) Cry1A.105 has no sequenc e similarities with known allergens; (3) Cry1A.105 is not glycosylated; and (4) Cry1A.105 is ra pidly digested in simu lated gastric fluid; EPA has concluded that the potential for Cry1A .105 to be a food allergen is minimal.

The Agency did not evaluate information conc erning the dietary cons umption patterns of consumers (and major identifiable subgroups of consumers including infants and children) or apply safety factors that are ge nerally recognized as appropriate when animal experimentation

data are used to assess risks to humans. Th e lack of mammalian toxic ity at high levels of exposure to the Cry1A.105 protein, as well as th e minimal potential to be a food allergen, satisfactorily demonstrated the safety of the products at levels well above the anticipated maximum exposure levels.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

23 The genetic material necessary for the production of the plant-incorporated protectant active ingredient include the nucleic acids (DNA, RNA) that enc ode these proteins and regulatory regions. The genetic material (DNA, RNA), necessary for the production of the Cry1A.105 protein has been exempted from the requireme nt of a tolerance under 40 CFR 174.507 “Nucleic acids that are part of a plan t-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, speci al susceptibility of infants and children to pesticide chemical residues, and the cumulative effects on infants and ch ildren of the residues

and other substances with a common mechanis m 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 ac count 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 in formation, the Agency concluded that there is a finding of no toxicity for the Cr y1A.105 protein. Thus, there are no threshold effects of concern and, as a result, the provision requiring an additiona l margin of safety does not apply. Further, the considerations of consumption patterns, speci al susceptibility, and cumulative effects do not apply.

c) Overall Safety Conclusion

There is a reasonable certainty that no harm w ill result from aggregate exposure to the U.S. population, including infants and ch ildren, to the Cry1A.105 protei n and the genetic material necessary for its production. This includes all anticipated dietary exposures and all other exposures for which there is reliable informa tion. 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), EP A has developed the Endocrine Disruptor Screening Program (EDSP) to determine whether certain substances (inc luding pesticide active and other ingredients) may have an effect in humans or wildlife si milar 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 ap proach to making the statutorily required

determinations. Tier 1 consists of a battery of 11 scr eening assays to identify the potential of a chemical substance to interact with the estrog en, androgen, or thyroid (E, A, or T) hormonalBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

24 systems. Chemicals that go th rough Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal systems will proceed to the next stag e of the EDSP where EPA will determine which, if any, of th e Tier 2 tests are necessary base d on the available data. Tier 2 testing is designed to identify a ny adverse endocrine related eff ects caused by the substance, and establish a quantitative relationship betw een 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 pesticid e active ingredients and 9 inert ingredients.

This list of chemicals was selected based on th e potential for human exposure through pathways such as food and water, residen tial activity, and certain post-applic ation agricultural scenarios.

This list should not be construed as a lis t of known or likely endocrine disruptors.

Bt Cry1A.105, and Cry2Ab2 Protein in corn is not among the group of 58 pesticide active ingredients on the initi al 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 Regi stration Review cases, including t hose for which EPA has already opened a Registration Review docket fo r a pesticide active ingredient.

For further information on the status of the ED SP, the policies and procedures, the list of 67 chemicals, the test guidelines and the Tier 1 screening battery, pleas e 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 Cry1A.105 in co rn tissue has been submitted.

c) Codex Maximum Residue Level

No Codex maximum residue level exists for the plant-incorporated protectant Bacillus thuringiensis Cry1A.105 protein.

The human health studies submitted for Cry1A.105 are summarized in Table 3 below.

Table 3. Summary of Cry1A.105 Human Health Data

Study Type/Title Summary MRID #

Acute oral toxicity (OPPTS 870.1100)/ Acute Oral Toxicity Study in Mice with Cry1A.105 Protein 2 The Cry1A.105 test protein (2072 mg/kg body weight) was dosed by gavage as two doses separated by 4 hours (±20 minutes). The BSA protein control (1998 mg/kg body weight) was dosed using the same procedure as for thetest protein group. The vehicle control group was dosed with carbonatebicarbonate 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. 46694603Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

25 Study Type/Title Summary MRID #

A general health/mortality check was done twice daily. All animals were necropsied. One male in the test prot ein group was moribund and sacrificed on day 1 due to a mechanical dosing error, which resulted in a perforated esophagus. All other mice survived th e study. There were no significant differences in body weight or body weight change among the three groups during the study. The oral LD 50 for males, females, and combined mice was greater than 2072 mg/kg. This pl aces Cry1A.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 Cry1A.105 Protein Utilizing the AD6, Toxin5, and Allpeptides Databases 2 Bioinformatic analyses were used to search for sequence similarities between the Cry1A.105 protein and toxins and allergens. The FASTA alignment tool and the allergen (AD5), toxin (TOXIN5), and public domain (ALLPEPTIDES) database sequences were used to assess structural similarity. No significant similarities were found, other than with the Cry1Ac protein; this alignment is not surprising, since the Cry1A.105 protein contains a significant portion of the Cry1Ac protein. The Cry1A.105 protein sequence was also screened against the AD5 sequence database using a pair-wise comparison algorithm. No matches of 8 amino acids or more were found for the Cry1A.105 protein in the AD5 database. No similarities between Cry1A.105 prot ein 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 Cry1A.105 Protein

in Simulated Gastric

Fluid 2 No bands representative of intact Cry1A.105 protein were identified by

SDS-PAGE after ≥ 30 seconds incubation with simulated gastric fluid containing pepsin. A very faint band of 4.5 kDaltons was observed between the 30 second and 20 minute digestions but was not observed after 20

minutes. The limit of detection for the SDS-PAGE method was determined to be 5 ng for the full-length Cry1A.105 protein. Both the pepsin stability and test material stability controls gave appropriate responses. In the Western Blot assay, Cry1A.105 was not immunologically identifiable within

30 seconds of incubation. The limit of detection for the method was determined to be 1 ng for the full-length Cry1A.105 protein. The pepsin stability and test material stability controls gave appropriate responses.

Classification: ACCEPTABLE 46694606

Heat stability/

Immunodetection of Cry2Ab2 and Cry1A.105 Proteins in

Corn Grain from MON 89034 Following Heat

Treatment 2 The immunodetectability of the Cry1A.105 and Cry2Ab2 proteins in corn grain from MON 89034 following heat treatment was assessed. MON 89034 and conventional grain were ground, mixed with water, and then

heated in an oven at 204 oC for 20 minutes to simulate the heating process used commercially to process grain. Heated and unheated grain was extracted with two buffers: 50 mM CAPS and 50 mM NLS (CAPS containing 2% N-Lauroyl sarcosine). The extracts were analyzed using western blot to detect the presence of the Cry1A.105 and Cry2Ab2 proteins. The amount of immunodetectable Cry2Ab2 protein in either CAPS or NLS buffer extracts of MON 89034 after heating was below the lower LOD. Based on the LOD and the estimated amount of protein in the unheated 46694607Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

26 Study Type/Title Summary MRID #

extract, it was determined that the Cr y2Ab2 protein decreased at least 77% and 70%, respectively following heat treatment, relative to their original values. Likewise, the amount of immunodetectable Cry1A.105 protein in

either CAPS or NLS buffer extracts of MON 89034 after heating was below the limit of detection (LOD) and had decreased by at least 94% and 78%, respectively, relative to their original values. This loss is likely due to protein degradation or aggregation into an insoluble complex as a result of

heat treatment.

Classification: ACCEPTABLE

Amino acid sequence comparison/ Bioinformatics Analysis of the Cry1A.105 Protein Utilizing the AD6, Toxin5, and Allpeptides Databases The amino acid sequence of the Cry1 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 FAST A comparisons, the Cry1A.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 1x10 -5 from the search using the AD6 database, indicating that Cry1A.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 Cry1A.105 protein and sequences in the AD6 database. In the comparisons using the TOXIN5 and ALLPEPTIDES databases, the highes t similarity identified was with Bacillus thuringiensis pesticidal protein Cry1Ac (92% identity over an 1,182 aa window and an E score of 0). This result is not surprising, given that the Cry1A.105 protein contains a significant portion of the Cry1Ac protein. No significant similarities between Cry1A.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 Cry1A.105 Protein in

Simulated Intestinal

Fluid The in vitro digestibility of Cry1A.105 in simulated intestinal fluid containing pancreatin was investigated using western blot analysis. The band for the full-length Cry1A.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 Cry1A.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 trypsinresistant core and appears to be fairly stable throughout the 24-hour digestion experiment.

Classification: ACCEPTABLE 46951408Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

27 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 ce rtainty that no harm will result from aggregate exposure to the pesticide chemi cal residue, including all antici pated 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 o ccupational exposure. Pursuant to section 408(c)(2)(B), in es tablishing or maintaining in effect an exemption from the requirement of a tolerance, EPA must take in to account the factors set forth in section 408(b)(2)(C), which require EPA to give specia l consideration to e xposure of infants and children to the pesticide chemical residue in estab lishing 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 me chanism of toxicity.” EPA perf orms a number of analyses to determine the risks from aggregate exposure to pe sticide 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 re sult of pesticide use in residential settings.

1. Toxicological Profile

Consistent with section 408(b)(2 )(D) of the FFDCA, EPA has review ed the available scientific data and other relevant information in suppor t of this action and c onsidered its validity, completeness and reliability and the relationship of this information to human risk. EPA has also considered available information concerning th e 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 protei n. These data demonstrate the safety of the product at a level well above maximum possible e xposure levels that are reasonably anticipated in the crop. Basing this conclusion on acute oral toxicity data w ithout requiring further toxicity testing and residue data is similar to the position regarding toxicity testi ng and the requirement of residue data for the Agency’s microbial Bacillus thuringiensis products from which this plantincorporated protectant was derived (See 40 CFR Sec. 158.740(b)(2 )(i)). For microbial products, further toxicity testing (Tiers II & III) and resi due data are triggered by significant adverse acute effects in studies such as the acute oral toxicity study, to verify the obs erved adverse effects and clarify the source of these effects.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

28 An acute oral toxicity study in mice (MRID 44966602) indicated that Cry2Ab2 is non-toxic to humans. Three groups of ten male and ten fema le mice were dosed by oral gavage with 30, 300, or 1000 mg/kg bodyweight of microbially produ ced Cry2Ab2 protein. Two negative control groups were also included in the study: bovine serum albumin protein co ntrol, and a vehicle control (purified water). Ther e were no significant differences between the test and control groups; therefore, the Cry2Ab2 prot ein does not appear to cause a ny significant adverse effects at an exposure level of up to 1000 mg/kg bodyweight.

When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels. Therefore, given that no acute effects were shown to be caused by Cry2Ab2, even at relatively high dose levels, the Cr y2Ab2 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 alle rgens; and biochemical properties of the protein, including in vitro digestibility in simula ted gastric fluid (SGF) and glycosylation. This approach is consistent with the approach out lined in the Annex to the Codex Alimentarius “Guideline for the Conduct of Food Safety A ssessment of Foods Derived from RecombinantDNA 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 se quence similarity or identity at the level of eight contiguous amino acid residues.

3. Digestibility. The Cry2Ab2 prot ein was digested within 15 s econds in simulated gastric fluid containing pepsin.

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

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

Although Cry2Ab2 was only shown not to be glycos ylated in corn, it is unlikely to be glycosylated in any other crops because in order fo r a protein to be glycos lyated, it must contain specific recognition sites for the enzymes involve d in glycosylation, and the mechanisms of protein glycosylation are simila r in different plants (Lerouge, P. Cabanes-Macheteau, M., Rayon, C., Fichette-Lainé, A-C., Gomord, V., and Faye, L., “N-Glycopr otein biosynthesis in plants: recent developments and future trends,” Plant Molecular Biology 38 : 31-48, 1998).Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

29 2. Aggregate Exposures

Pursuant to FFDCA section 408( b)(2)(D)(vi), EPA considers avai lable 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 chemi cal residue and to othe r related substances. These considerations include dietary exposure under the tolerance ex emption and all other tolerances or exemptions in effect for the Plan t Incorporated Protectant ’s chemical residue, and exposure from non-occupational sources. Exposure vi a 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 thes e routes to negligible. In addition, even if exposure can occur through inhalation, the potential for Cry2Ab2 prot ein 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 indicate d a low potential for Cry2Ab2 to be an inhalation allergen. Exposure to infants and children via reside ntial or lawn use is also not expected because the use sites fo r the Cry2Ab2 protein is agricultura l. Oral exposure, at very low levels, may occur from ingestion of proces sed corn products and, theoretically, drinking water. However, oral toxicity testing in la boratory 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 cu mulative 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 in corporated 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 protei n in corn, as well as the acute oral toxicity study, amino acid sequence comparisons to known alle rgens 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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

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

was available.

The acute oral toxicity data submitted by M onsanto support the prediction that the Cry2Ab2 protein would be non-toxic to humans. As menti oned 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 th e 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 toxi city testing and residue data is similar to the Agency position regarding toxicity and th e requirement of residue data for the microbial Bacillus thuringiensis products from which this plant-incorpor ated 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, poten tial allergenicity is also consid ered as part of the toxicity assessment. Considering that (1) Cry2Ab2 origin ates from a non-allergenic source, (2) Cry2Ab2 has no sequence similarities with known allerg ens, (3) Cry2Ab2 is not glycosylated, and (4) Cry2Ab2 is rapidly digested in simulated gastri c fluid, EPA concluded th at the potential for Cry2Ab2 to be a food allergen is minimal.

The Agency did not consider information co ncerning the dietary c onsumption patterns of consumers (and major identifiable subgroups of consumers including infants and children) or safety factors that are generall y recognized as appropria te when animal experimentation data are

used to assess risks to humans. The lack of ma mmalian 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 ma ximum 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 enc ode 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-in corporated 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, speci al susceptibility of infants and children to pesticide chemical residues and the cumulative e ffects on infants and children of the residues andBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

31 other substances with a co mmon 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 ac count 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 in formation, the Agency concluded that there is a finding of no toxicity for the Cr y2Ab2 protein. Thus, there are no threshold effects of concern and, as a result, the provision requiring an additiona l margin of safety does not apply. Further, the considerations of consumption patterns, speci al susceptibility, and cumulative effects do not apply.

b) Overall Safety Conclusion

There is a reasonable certainty that no harm w ill result from aggregate exposure to the U.S. population, including infants and ch ildren, to the Cry2Ab2 protei n and the genetic material necessary for its production. This includes all anticipated dietary exposures and all other exposures for which there is reli able information. The Agency has arrived at this conclusion because, as discussed above, no toxicity to ma mmals 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 sour ce 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-incorp orated 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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

32 Table 4. Summary of Cry2Ab2 Human Health Data

Study Type/Title Summary MRID #

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-1 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 seru m 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 LD 50 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 wereobserved in the western blot. The limit of detection (LOD) for the fulllength 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 factthat no band was observed for the Cry2Ab2 protein in the 30 second timepoint 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. Therefor e, 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 46951409Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

33 Study Type/Title Summary MRID #

the 24-hour incubation time-point.

Classification: ACCEPTABLE

Heat stability/

Immunodetection of Cry2Ab2 and Cry1A.105 Proteins in

Corn Grain from MON 89034 Following Heat

Treatment 2 The immunodetectability of the Cry1A.105 and Cry2Ab2 proteins in corn grain from MON 89034 following heat treatment was assessed. MON 89034 and conventional grain were ground, mixed with water, and then

heated in an oven at 204 oC for 20 minutes to simulate the heating process used commercially to process grain. Heated and unheated grain was extracted with two buffers: 50 mM CAPS and 50 mM NLS (CAPS containing 2% N-Lauroyl sarcosine). The extracts were analyzed using western blot to detect the presence of the Cry1A.105 and Cry2Ab2 proteins. The amount of immunodetectable Cry2Ab2 protein in either CAPS or NLS buffer extracts of MON 89034 after heating was below the lower LOD. Based on the LOD and the estimated amount of protein in the unheated extract, it was determined that the Cr y2Ab2 protein decreased at least 77% and 70%, respectively following heat treatment, relative to their original values. Likewise, the amount of immunodetectable Cry1A.105 protein in

either CAPS or NLS buffer extracts of MON 89034 after heating was below the limit of detection (LOD) and had decreased by at least 94% and 78%, respectively, relative to their original values. This loss is likely due to protein degradation or aggregation into an insoluble complex as a result of

heat treatment.

Classification: ACCEPTABLE 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 1x10 -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 pr otein 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. 46951411Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

34 D. ENVIRONMENTAL ASSESSMENT for MON 89034

Background

Monsanto has requested a registration for Bacillus thuringiensis Cry1A.105 and Cry2Ab2 proteins and the genetic material necessary for th eir production in all corn lines and varieties. The nptII selectable marker gene was used in th e transformation process, but was isolated and removed from transformed plants via traditiona l breeding. The result is marker-free MON 89034 corn. The Cry proteins expressed in this even t are intended to contro l the lepidopteran pests European corn borer (ECB, Ostrinia nubilalis ), corn ear worm (CEW, Helicoverpa zea ), fall army worm (FAW, Spodoptera frugiperda ), 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, th ereby 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 organophospha te, carbamate and pyrethroid insecticides.

EPA has conducted an environmental risk assessment of Cry1A.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 su bmitted for registration, Federal Insecticide Fungicide and Rodenticide Act (FIFRA) Scien tific Advisory Panel (SAP) recommendations, consultations with scientific experts, and pub lic 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 e nough confidence that there is no

risk and no further data would be needed. Henc e, 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 mo st conceivable conditions. Wh en screening studies suggest potentially unacceptable risk, additional studies ar e designed to assess risk under more realistic field exposure conditions. These la ter 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 inve stigation. Lower tier, high dose screening studies also allow tighter co ntrol over experimental variables and exposure conditions, resulting in a greater ability to produce statistically reliable re sults at relatively low

cost 3

.

3 Non-target invertebrate ha zard tests often are conducted at exposure concentrations several times higher than theBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

35 Tiered tests are designed to firs t represent unrealistic worst case scenarios and ONLY progress to

real world field scenarios if the earlier tiered te sts fail to indicate adequate certainty of acceptable risk. Screening (Tier I) non-target orga nism hazard tests are conducted at exposure concentrations several times higher than the hi ghest concentrations e xpected 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. Le ss 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) test ing system to assess the toxicity of a PIP to representative nontarget organisms that could be exposed to the t oxin in the field environm ent. Tier I high dose studies reflect a screening appro ach to testing designed to maximi ze any toxic effects of the test substance on the test (non-target ) organism. The screening test s evaluate single species in a laboratory setting with mortality as the end point . Tiers II – IV generally encompass definitive hazard level determinations, longer term greenhous e or field testing, and are implemented when unacceptable effects are seen at the Tier I screening level.

Testing methods that utilize th e tiered approach were last published by EPA as Harmonized OPPTS Testing Guidelines, Series 85 0 and 885 (EPA 712-C-96-280, February 1996) 4 . These guidelines, as defined in 40 CF R 152.20, apply to microbes and micr obial toxins when used as pesticides, including those that ar e naturally occurring, and those th at are strain-improved, either by natural selection or by delib erate 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 (wheneve r possible >10) times the maximum amount of active ingredient expected to be available to te rrestrial and aqua tic non-target organisms in the environment (EEC) 5 . Tier I tests serve to identify poten tial 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 asse ssment, and such tests can be well standardized.

The Guidelines call for initial screening testing of a single gr oup or several groups of test

animals at the maximum hazard dose level. The Gu idelines call for testing of one treatment group of at least 30 animals or th ree groups of 10 test animals at the screening test concentration.

The Guidelines further state that the duration of all Tier I test s 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 te sting. Lower levels of mortality under these conditions of extr eme exposure suggest that population effects are lik ely to be negligible give n 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 appro ach, a sample size of 30 individuals is sufficient to allow a

treatment effect of 30% to be differentiated from a 50% effect with 95% confidence using a one- sided Z test. A one-sided test is appropriate because only effects of less th an 50% indicate that further experiments are not needed to evaluate risk.

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

5 The dose margin can be less than 10x 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 fa ctors. High dose testing also may not be necessary where many species are tested or tests are very sens itive, although the test concentrat ion used must exceed 1X EEC.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

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

Failing the Tier I (10 X EEC) sc reening does not necessarily indicate the presence of an unacceptable risk in the field but it tr iggers the need for additional testing. 6 A less than 50% mortality effect at the MHD is taken to indicat e minimal risk. Greater than 50% mortality does not necessarily indicate the existence of unacceptabl e risk in the field, but it does trigger the need to collect additional dose-response informati on and a refinement of the exposure estimation before deciding if the risk is acc eptable or unacceptable. Where pot ential 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 confir m whether any effect might still be detected at more realistic field [1X EEC] concentrati ons and routes of exposure 7

.

When screening tests indicate a need for additio nal data, the OPPTS Harmonized Guidelines call for testing at incrementally lower doses in order to establish a definitive LD 50 and to quantify the

hazard. In the definitive testi ng, the number of doses and test organisms evaluated must be

sufficient to determine an LD 50 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, we ight 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 environmen tal 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 enha nce statistical power in these

circumstances.

Data that show less than 50 % mortality at the maximum hazard dosage level – (i.e., LC 50 , ED 50 , or LD 50 >10 X EEC) is sufficient to evaluate advers e effects, making lower field exposure dose definitive testing unnecessary. It is also notable that the recomme nded >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 MHD testing approach is not equivalent to what is commonly known as “testing at a 10X SAFETY FACTOR” where any adverse effect is considered signi ficant. 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 assessmen t scenario any adverse effect is viewed as a concern only at 1X the field exposure.

7 The 1X EEC test dose is based on plant tissue content and is considered a high worst case dose (sometimes referred to as HEEC). This 1X 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 (USE PA) (1998). “Guidelines for Ecological Risk Assessment.” EPA 630/R-95- 002F. Washington, DC, USA. [Federal Re gister, May 14, 1998. 63(93): 26846-26924.] Th e 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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

37 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 pr otectants; however, this panel recommended that, for PIPs with insecticidal propert ies, additional testing of benefici al 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 clos ely related to the target ins ect 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-ta rget 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 toxi ns 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 testi ng when performed at field expos ure test doses. Proteins do not bioaccumulate. The biologi cal nature of proteins makes them readily susceptible to metabolic, microbial, and abiotic degradation on ce 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 gr eatest extent possible, that the Agency requires the minimum amount of data n eeded to make scientifically sound regulatory

decisions. EPA believes that maximum hazard dose Tier I screening testing presents a reasonable approach for evaluating hazards relate d 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 effe cts must be ascertained, then higher-tier longerterm 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 requ irements for protein plant-pesticides. SAP report No. 99-06A for FIFRA Scientific Advisory Panel Meeti ng held December 8, 1999, held at the Sheraton Crystal City Hotel, Arlington, VA. EPA-SAP. November 6, 2002. Corn rootwo rm plant-incorporated protectant in sect resistance ma nagement and nontarget insect issues. Transmitt al 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 char acterization, 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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

38 conduct post-registration long term inverteb rate population/commun ity and Cry protein accumulation in soils studies as a condition of registration. As noted above, the October 2000 SAP and the National Academy of Sciences 10 (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 pe rformed during the last 10 years. No unexpected

adverse effects on invertebrate co mmunity structure were reported. 11 The meta analysis of short term and long term field study eff ects 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 specialis t predators and parasi toids of target pests may eventually be considered unnecessary.

2. Environmental Exposure Assessment

The EPA risk assessment is centered only on ad verse effects at the field exposure rates (1X EEC), and not on adverse effects at greater con centrations. The dose margin can be less than 10x where uncertainty in the system is low or wher e 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 sens itive, although the concentration used must exceed 1X EEC. It is important to note that Tier I screen testing is not “safety factor testing.” In a traditiona l “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 1X 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 Cry1A.105 and/or Cry2Ab2 protein expressed in the tissue(s) th at NTO would most likely be exposed to in the environment (see Edelstein, 2 007 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 primar y route of Cry1A.105 and Cry2Ab2 protein exposure for honeybee, ladybird beetle, parasitic wa sp, 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 Plan ts: 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,O., Romeis, J., Bigler, F. (2007) . Ecological Impacts of Genetically Modified Crops: Ten Years of Field

Research and Commercial Cultivation. Adv Biochem Engin/Biotechnol 107: 235–278Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

39 expression in pollen (8.8 ug/g fwt for Cry1A.105 and 0.47 ug/g fwt for Cry2Ab2). The principal route of Cry1A.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 dos e 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 Augus t 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 determ ined 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 invert ebrates, particularly Coleopteran species. Therefore, maximum hazard dose toxicity testing on representative beneficial organisms from several taxa was performed in support of this Sect ion 3 FIFRA registration. The toxicity of the Cry1A.105 and Cry2Ab2 proteins has been evalua ted on several species of invertebrates including the lady bee tle, minute pirate bug, parasitic hymen optera, collembola, daphnia, honey bee, and earthworm. Developmental observation s 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 Cry1A.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 r ecommendations against testing of non-target species not related to susceptible target pests, EPA has done a risk assessment on a range of nontarget wildlife to comply with the Agency’s published non-target data requirements (in the absence of PIP-specific risk a ssessment 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 ascerta in the potential effects of the Cry1A.105 and Cry2Ab2 proteins on benefici al decomposer species.

Test substances used for studies submitted in support of the event MON 89034 registration included bacterially produced purified Cr y1A.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 protei n is also a valid test substance, particularly in scenarios where test animals do not normally consume corn plan t tissue and where large amounts of Cry protein (Cry protein concentrations that exceed levels presen t in plant tissue) are needed for maximum hazard dose testing. An insect feeding study, wh ich compared the relative potency of plant produced Cry1A.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).Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

40 Potential Interaction between the Cry1A.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 Pote ntial for Interactions Between the Bacillus thuringiensis Proteins Cry1A.105 and Cry2Ab2,” (Hunter, M., July 6, 2006). The purpose of this study was to characterize the potential fo r interaction between the lepidopteran-active proteins Cry1A.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 a ny unexpected interaction with regard to target and non-target insects. New data on the pot ential interaction betw een combined Cry1A.105, Cry2Ab2 with the Cry3Bb1 protein was submitted. The results from the study demonstrated that combined Cry1A.105 and Cry2Ab2 activity wa s not affected by the Cry3Bb1 protein and that Cry3Bb1 activity was not affected by combined Cry1A.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 data requirements published in 40 CFR § 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 N/A N/A

Avian acute oral 850.2100 Acceptable MON 89034 corn grain An eight-day dietary study showed

that the LC 50 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 N/A 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 Cry1A.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. 469514-17Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

41 Guideline Classification Test

Substance Results Summary MRID # Data Requirement

Freshwater aquatic

invertebrate Daphnia magna 885.4240

154-20 Acceptable

[The 885 Series

Guidelines call for a 7-14 day study.

The submitted 48 hour acute study is inadequate.] 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 μg 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 Cry1A.105 protein (Lot No. 20-100073) Orius nymphs were fed a diet containing 30 to 240 μg Cry1A.105/g diet for 14 days. In an initial maximum dose test (240 μg) 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 μg group was 55% compared to 91% and 89% in the control groups. No statistically significant effects on survival or development were seen at concentrations less than or equal to 120 μg Cry1A.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 μg Cry2Ab2 protein/mL for 21 days. Mortality in the Cry2Ab2 group was

3% and the LC50 was determined to be >100 μg/L. 469514-26

Non-target insect testing, parasitic wasp

Ichneumon promissorius 885.4340

154-23 Acceptable Cry1A.105 protein (Lot No. 20-100073) Adult female wasps were fed a sucrose solution containing 240 μg Cry1A.105 protein/mL for 21 days. Mortality in the Cry1A.105 group

was 7% and the LC50 was determined to be >240 μg/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 μg 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 469514-22Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

42 Guideline Classification Test

Substance Results Summary MRID # Data Requirement
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 Cry1A.105 protein (Lot No. 20-100073) Ladybird beetle larvae were fed a diet containing 240 μg Cry1A.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 μg Cry1A.105 and 70 μg 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 Cry1A.105 protein (Lot No. 20-100073) Adult honeybees were fed a 30% sucrose solution containing 550 μg Cry1A.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 μg Cry1A.105 protein/mL. 469514-20

Honeybee testing, Honeybee larvae, Apis mellifera 885.4380

154-24 Acceptable Cry1A.105 protein (Lot No. 20-100073) Two-to-three day old honeybee

larvae in brood frames were administered a single 10 μ L dose of Cry1A.105 protein per brood cell (equivalent to 12 μg 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 μg Cry1A.105 protein per brood cell 469514-20

Earthworm

subchronic toxicity, Eisenia fetida 850.620 Acceptable Cry1A.105 protein (Lot No. 20-100073) Adult earthworms were exposed to artificial soil containing 178 mg Cry1A.105 protein/kg dry soil for 14 days. No mortality was observed in the test group. The LC50 was determined to be >178 mg Cry1A.105/kg dry soil and the NOEC was 178 mg Cry1A.105 mg/kg dry soil. 469514-18Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

43 Guideline Classification Test

Substance Results Summary MRID # Data Requirement

Soil fate 885.5200 Acceptable Purified Cry1A.105 (Lot No. 20-100073)

and Cry2Ab2 protein (Lot No. 20-100071) Results of this degradation study indicate that Cry1A.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 s ubmitted and reviewed with prev iously registered products. The interaction study between Cry1A.105 and Cry2Ab2 wa s reviewed for the MON 89034 Experimental Use Permit

* OPPTS Microbial pesticide test guidelines **Microbial pesticide test guidelines identified in the 40 CFR data tables. † Cry1A.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 th e MON 89034 product registration. These studies were GLP compliant and, when considered toge ther, 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. Tr eatments consisted of soybean meal with MON 89034, a similar iso line (negative control), or one of four different commercial hybrid corn varieties. At test end, chickens we re 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 biol ogically significant differences in broiler performance, carcass, or meat quality.

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

In this eight-day dietary st udy, 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 th ree different commercial hybrid corn varieties. At test end, no mort ality was seen in the MON 89034 treatment group, all birds appeared normal for test duration, and feed consumption wa s comparable to that of the control group. The dietary LC 50 of MON 89034 corn grain was determined to be >500,000 ppm

in the diet.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

44 ii. Wild mammalian species

Mammalian wildlife exposure to Cry1A.105 a nd Cry2Ab2 proteins is considered likely; however, mammalian toxicology info rmation gathered to date on Bt Cry proteins does not show a hazard to wild mammals. In addition, acute or al toxicity studies submitted to EPA in support of the MON 89034 registration indica ted that no significant toxicity was seen when rodents were exposed to Cry1A.105 or Cry2Ab2 at the maximum h azard 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-coleopt eran Cry proteins. A published laboratory study with le pidopteran-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 pr evious field study reports by the same authors did

not find adverse effects on head stream invertebra tes. 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 gran ts 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 w ill 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 communitie s. 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 Cr y1A.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 th e potential for acute effects to the aquatic organism, Daphnia magna , during a static renewal exposur e to MON 89034 corn pollen. The test was initially conducted as a limit test using one test concentration. S light effects were noted

at the limit concentration. In response, a dose-response test was conducted. The test substance, MON 89034 pollen expressing Cry1A.105 and Cry2A b2 proteins, was evaluated for potentialBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

45 adverse effects to neonate Daphnia. Test organisms were < 24 hour s 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 M ON 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, immob ility and other clinical signs were made at approximately 3.5, 24 and 48 hours afte r test initiation. At te st termination there was 17% immobility in the 120 mg/L treatment group, with two daphnids exhibiting lethargy. All daphnids in the assay c ontrol 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 wa ter exposed to pollen (120 mg pollen/L) from conventional corn with a genetic background si milar to MON 89034, and an assay control group exposed to well water only. The test and cont rol 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. Ob servations of mortality, immob ility and other clinical signs were made at approximately 5, 24, and 48 hours af ter test initiation. The NOEC was estimated by visual interpretation of the mortality, immob ility 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 EC 50 was estimated to be greater than 120 mg MON 89034 pollen/L. Ba sed 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 duratio n is not sufficient to show mortality for Bt

toxins. The 48 hours test duration is not considered to be suffi cient duration to assess the potential for adverse effect s 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 shredde r in headwater streams, may be performed and

submitted in lieu of the Daphnia study.

(c) Freshwater Aquatic Invertebrates (MRID 478388-01)Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

46 When Bt Cry1A.105, and Cry2Ab2 Protein in corn (EPA Reg. No. 524-575, and 524-576) were initially registered, the Agency issued registra tion 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. Altern atively, a dietary study of the effects on an aquatic invertebrate, representing the func tional 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 si gnificant reduction in survival rate in the pollen control group when compared to the assay control group, the study au thor concluded the exposure to high concentrations of conventional corn pollen resu lted in impacts on the overall health of daphnids. It was ther efore considered most appropriat e 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, eff ects 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 phys ical 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 inte rval when compared to the pollen control group were not statistically sign ificant (p > 0.05). The 14-day medi an effect concentration (EC 50 ) and no observed effect concentration (NOEC) valu es 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 dela y in the onset of repr oduction 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 hi gh pollen concentrations in the test solutions, but were not considered by the study author to be related to toxici ty 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 ac ute toxicity testing. No unreasonable adverse effects to aquatic invertebrates are ex pected 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 system s will be exposed to the Cry1A.105 and Cry2Ab2 proteins produced in MON 89034 corn plant tissues.

iv. Terrestrial and aquatic plant species- Waiver grantedBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

47 Plant toxicity studies were not required for this product because the active i ngredient is an insect toxin ( Bt endotoxin) that has never s hown any toxicity to plants.

v. Terrestrial Invertebrate species

The Cry1A.105 and Cry2Ab2 proteins are meant to target species within the order Lepidoptera (moths and butterflies). Bt toxins are known to have a limite d host range, however, to address any unforeseen change in activity spectrum a nd to fulfill the publishe d registration data requirements EPA requires that test species used for non-target ins ect 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 effect s of the Cry1A.105 protein on the mortality and development of the ladybird beetle, Coleomegilla maculata . The test substance, Cry1A.105 protein, was produced by a recombinant E. coli fermentation system. The test substance was incorporated at 240 μg Cry1A.105 protein/g of di et. The diet consisted of an artificial agar-based diet. Three control treatmen ts were included in the experiment. The buffer control contained 25 mM CAPS buffer, which wa s 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, contai ning potassium arsenate. Ladybird beetle larvae

were less than 48 hours old at test initiation. The larvae were contai ned 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 th an or equal to 20% mortality; the mortality of larvae ranged from 0 to 18.8% in the assay contro l treatment groups. The diet treatments were replaced with fresh diet approxi mately every 48 to 72 hours. Th e 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 dissect ed 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 em ergence. Samples were taken to test the biological activity, homogeneity, an d stability of the Cry1A.105 prot ein. Results showed that there were no differences in the mean survival percentage of C. maculata between the Cry1A.105 protein, buffer control, and assay co ntrol 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 Cry1A.105, buffer control, and assay control treatments (88.5%, 85.4%, 90.6%). None of the larvae developed to adults in the positive control tr eatment. Further, there were no significant differences in the mean weight of C. maculata adults between the Cr y1A.105, buffer control, and assay control. No significant in teraction was found between insect sex and treatment. In the bioactivity confirmation assay, results indica ted equivalent biological activity among theBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

48 Cry1A.105 used in the test diets and the refere nce standard. In addition, the homogeneity and stability study confirmed that Cry1A.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 Cr y1A.105 protein had no adverse effect on the survival, development, a nd growth of the ladybird beetles at a dietary concentration of 240 μg/g of diet.

MRID 469514-22 The objective of this study was to determine th e potential dietary eff ects of Cry2Ab2 protein on the mortality and developmen t 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 thr ough 20 days (some replicates were completed before 20 days if all insects ha d developed to adults). The Cr y2Ab2 protein was incorporated in to an agar-based artificial di et at a concentration of 120 μg Cry2Ab2/g diet. Three control

treatments were included in the study: 1) buffer control, 2) assa y 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. Ea ch 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 st age. Adults were weighed within 30 hours of

eclosion and adults were sexe d. The biological activity, homoge neity, and stability of the Cry2Ab2 protein in the diet were conf irmed 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 a ssay 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 cont rol. 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 th an those of the assay control group, which was a statistically significant difference. However, there was no sign ificant 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 μg Cry2Ab2 protein/g diet. Although the mean adult weight of the Cry2Ab2 protein treatment group was statistically significantly lower than th at of the assay control group, the difference was slight (~5%) and there was no significant differe nce between the weight of the Cry2Ab2 protein group and buffer control groups.

(b) Minute pirate bug

MRID 469514-23Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

49 The purpose of this study was to determine the potential dietary effect s of Cry1A.105 protein on mortality and development of Orius insidiosus , the minute pirate bug or insidious flower bug. The Cry1A.105 protein ( E. coli -produced) was incorporated into a pollen-based diet for treatment of the test group. Both a buffer control diet and an assa y 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 μg Cry1A.105/g diet) for 14 days, resul ting in 47% survival. The a ssay 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 μg Cry1A.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 μL) of the appropriate diet and the capsules were replaced every other day. The test arenas cons isted of 1-ounce plastic cups with plastic covers and each cup contained one Orius . For each dose-response expos ure, 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 re plicate resulted in percent survival in the 30, 60, 120 and 240 μg Cry1A.105 protein/g diet was 88, 84, 88 and 56%, resp ectively. Percent survival in the assay, buffer, and positive control wa s 92, 88, and 36%, respectiv ely. The percent of nymphs developing to adults for the 30, 60, 120 and 240 μg Cry1A.105 protein/g diet, assay control, buffer control and potassium arsena te control organisms was 92, 100, 96, 92, 96, 96 and 40%, respectively. No statistically significant di fferences were detected between the 30, 60, 120 and 240 μg Cry1A.105 protein/g diet. Percent surv ival in the second replicate in the 30, 60, 120 and 240 μg Cry1A.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 positiv e control was 32%. The percent of nymphs developing to adults for the 30, 60, 120 and 240 μg Cry1A.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 μg Cry1A.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 ny mphs developing to adults for the 30, 60, 120 and 240 μg Cry1A.105 protein/g diet, assay cont rol, 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 th e 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 antic ipated levels and was appropriately mixed

in the test diet.

Conclusions: This study is acceptable. Orius insidiosus were exposed for 14 days to a range of dietary concentrations of Cry1A.105. For the thr ee dose-response replicates the mean survival for the 240 μg Cry1A.105/g diet treatments was 55%. Therefore, the LC 50 value was empiricallyBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

50 determined to be greater than 240 μg Cry1A.105/g diet. No adverse effects were observed at concentrations less than or equa l to 120 μg Cry1A.105/g diet.

MRID 469514-24 The purpose of this study was to determine the potential dietary effect s 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 μg 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 stu dy. In addition, a positive control was included which consisted of the pollen-diet tr eated with potassium arse nate. 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 ins ect was contained in its own te st arena. During the exposure period, one capsule of approximately 50 μL of the appropriate test diet wa s provided in each test arena on Day 0 and every other da y 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 Cry2A b2 protein in the test diet we re tested and confirmed in a separate bioassay using Helicoverpa zea . The percent survival of insects exposed to the 100 μg Cry2Ab2 protein/g diet treatment was 91%, which was similar to the percent survival of the insects in the buffer and assay c ontrol groups. The percen t of nymphs developing to adults in the 100 μg 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 μg Cry2Ab2 prot ein/g diet, assay cont rol, 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 μg Cry2A b2 protein/g diet. Therefore, the LC 50 is greater than 100 μg Cry2Ab2 protein/g diet.

(c) Parasitic hymenoptera

MRID 469514-25 This study was conducted to evaluate the poten tial effects of acute exposure of Cry1A.105 protein to the parasitic wasp, Ichneumon promissorius . The Cry1A.105 protein was administered to the wasps at a concentrati on of 240 μg/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 contro l, 2) assay control (suc rose solution only), and 3) positive control (potassium arsenate). Th e 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 wasp s were 3 to 6 days old at the time of test initiation. The test chambers were disposable 64 ounce containers. Observations of mortality and clinical signs were conducted once within the hour of test ini tiation and then continued daily until Day 21 of the test. Samples of the assay control, control substance and protein group dietsBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

51 were collected for analysis by bioassay to te st for bioactivity and stability of the Cry1A.105 protein. Mortality in the assay control, buffer cont rol, and test material treatments was 10%, 8%, and 7% respectively. All surviv ing wasps in those groups appeared normal in appearance and

behavior. There was no statisti cally significant difference in mean mortality in the Cry1A.105

treatment and buffer control treatments. The biological activity and stability of the Cry1A.105 protein was confirmed in a seven-da y bioassay using the corn earworm ( Helicoverpa zea ).

Conclusions: This study is acceptable. The LC 50 for Ichneumon promissorius was greater than 240 μg Cry1A.105 protein/mL and the NOEC was at least 240 μg Cry1A.105 protein/mL.

MRID 469514-26 A laboratory bioassay was conducte d 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 μg/L in a 30% sucrose so lution. Three control groups were utilized, including: 1) buffer control, 2) negative assa y control group (sucrose so lution 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 e qual amounts of solutions containing test and

control substances in deionized water to o Bt ain diets with approximately 30% sucrose. Test diet containing Cry2Ab2 protein was prep ared 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 te st chambers consisted of disposable 64 oz. polypropylene containers. The wasps were approxim ately 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 su bstance and protein group di ets were collected for analysis by bioassay. The biological activity relativ e 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%, respectiv ely. All surviving wasps were normal in appearance and behavior. No stat istically significant differences in mean mortality were found between the Cry2Ab2 treatment group and the negative control group.

Conclusions: This study is acceptable. The LC 50 for Ichneumon promissorius was determined to be >100 μg Cry2Ab2 protein/mL and the NOE C was at least 100 μg Cry2Ab2 protein/mL.

(d) Collembola (MRID 469514-16)

The objective of this study was to determine the potential e ffect 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 ident ity and the concentrations of Cry1A.105 and Cry2Ab2 in the lyophilized leaf material. The concentration of Cry1A.105 was 160 μg/g lyophilized leaf dry weight and the concentration of Cry2Ab2 wa s 140 μg/g lyophilized leaf dry weight. The lyophilized test mate rial was incorporated in to a diet containing 50% Brewer’sBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

52 yeast and 50% test material (0.500g leaf tissue wi th 0.500 g yeast). Therefore, the test diet contained Cry1A.105 at a nominal concentration of 80 μg/g lyophilized leaf dry weight and Cry2Ab2 at a nominal concentration of 70 μg/g l yophilized leaf dry weight. A control diet was prepared by mixing 50% control leaf tissue, by weight, with 50% Br ewer’s yeast. An additional

control treatment consisted of a test diet contai ning only Brewer’s yeast. Three positive control treatments were included. The three treatments included three treatments of thiodicarb, representing nominal concentr ations of 1.0, 10 and 100 mg a.i./kg. Collembola were provided enough food such that an excess was always availabl e. Each treatment cont ained four replicates and each replicate initially contained 10 juvenile Collembola (12 days old). Each replicate was contained in a glass jars contai ning a water saturated substrate c onsisting of plaster of Paris and charcoal at a ration of 8:1 by weight. The biological activit y and concentration of Cry1A.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 refere nce standard. Mortality and observations of sublethal effects of the surviving Collembola were recorded on day 28 (test termination).

Collembola were removed from the test aren as 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 su rvival of Collembola exposed to the control diet (control leaf tissue) a nd MON 89034 diet was 100 and 98%, respectively. The mean number of offspring produced in the control substance di et (control leaf tissu e) and the MON 89034 diet was 260 and 257 offspring per arena, respectiv ely. Mean survival in the positive control substance treatments 1.0, 10 and 100 mg thiodica rb/kg diet was 95, 63 and 35% respectively. The mean number of offspring produced in the 1.0, 10 and 100 mg thiodica rb/kg diet treatments was 136, 93, and 16 offspring per arena, respectiv ely. Statistical analysis demonstrated no significant reduction in survival or reproduc tion 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 μg Cry1A.105 and 70 μg Cry2Ab2 per gram of diet.

(e) Honeybee

MRID 469514-19 The objective of this study was to evaluate potential dietary e ffects of Cry1A.105 protein when administered to honeybee larvae ( Apis mellifera ). Honeybees were approximately 2 to 3 days old during the experiment. The test substance was Cry1A.105 protein produced by an E. coli fermentation system. The protein was used to pr epare a test solution at a concentration of 1200

μ g/mL. This concentration is equivalent to 12 μ g total protein per cell. Additional treatment groups included an assay control, buffer contro l, and two reference s ubstance concentrations (potassium arsenate at low and high doses). Each treatment include d four replications of 20 bees for a total of 80 bees per treatm ent. The larvae were exposed to a single dose (10 mL) of the appropriate dosing solution at in itiation and observed during larv al and pupal development. Survival of larvae was assessed at study comple tion (Day 18) by observing adult emergence.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

53 The Cry1A.105 treatment group resulted in 95% surv ival. 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 veri fy test concentration and bioactivity of the Cry1A.105 protein, samples of the te st material were taken at te st initiation. A bioassay using Helicoverpa zea was conducted to test for bioactivity and no significant difference was observed

between the test substance and th e Cry1A.105 reference standard.

Conclusions: This study is acceptable. The NOEC for Cry1A.105 protein to honey bee larvae

was determined to be at least 12 μ g Cry1A.105 protein/cell.

MRID 469514-20 An acute bioassay was conducted to determin e the effects of Cry1A.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 cont rol mortality). Adult bees were approximately five days old at the start of the bioassay. The test material was E. coli -produced Cry1A.105 protein supplied by the study sponsor . The protein was used to prepare a 30% sucrose solution containing 550 μg Cry1A.105 protei n/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 pr epared and consisted of only the 30% sucrose solution. A positive control diet was prepared an d contained 100 μg/mL potassium arsenate in a solution of 30% sucrose. Honeybees were mainta ined 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 provid ed via an inverted 12 mL glass vial fitted with a plastic screw cap containing two ~1.0 mm hol es. 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 acce ptance criteria stipulat ed that the assay be terminated at 30 days or when the adult cont rol 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 Cry1A.105 treatments (20.37%). Mo rtality in the Cry1A.105 treatment was not statistically different than the sucrose tr eatment on Day 18. On Day 19, no significant differences were detected among the three trea tment groups (buffer, sucrose, and Cry1A.105 protein) with mortalities of 52.22%, 51.48%, and 47.04%, respectively. The potassium arsenate

treatment resulted in 100% morta lity by Day 2 of the study. A diet incorporation assay using the Cry1A.105 test diet was conducted to confirm the bioactivity of the protein. The biological activity of the test subs tance was evaluated using Helicoverpa zea and compared with the biological activity of a Cry1A.105 reference standar d. In addition, the control substance and the assay control substance were evaluated in the di et incorporation assay. The bioassay confirmed the test diet contained the exp ected level of Cry1A.105 activity.

Conclusions: This study is acceptable. The NOEC for Cry1A.105 protein fed to adult honey bees is at least 550 μg/mL.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

54 (f) Earthworm (MRID 46954-18)

The objective of this study was to evaluate the potential eff ects of acute exposure of Cry1A.105 protein administered to earthworms ( Eisenia fetida ) during a 14-day exposure period. In the test, earthworms were exposed to a single concentrat ion of Cry1A.105 protein that was incorporated

into an artificial soil substrate. Th e Cry1A.105 protein used in this study was E. coli produced.

The concentration of the test substance was 178 mg Cry1A.105 protein/kg soil dry weight. A

total of four control tr eatments were included in the study, in cluding: 1) buffer solution control, 2) assay control group containing neither test su bstance 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 ch ambers consisted of one-liter glass beakers covered with plastic wrap what was perforated for air exchange. The artifi cial soil was prepared in bulk by blending 70% sand, 20% kaolin clay an d 10% sphagnum peat. Each test container contained 750 grams of prepared soil. The worm s were not provided food during the test period. At test initiation (Day 0), the worms were plac ed 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 surviv ing 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 Cry1A.105 treated soil was also compared against a reference standard of Cry1A.105 provided by the study sponsor. There were no mortalities in the assay control group, buffer control group, or Cry1A.105 pr otein 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 terminati on. A slight loss in average individual body weight from test

initiation to test termination was noted in all te st groups and was expected since the worms were not fed during the 14-day test. Losses in body weight in the Cry1A.105 pr otein test substance group were not statistically significant when compared to the control substance group. Analysis

of the test soil showed that Cr y1A.105 was present in the soil and was biologically active against Helicoverpa zea

.

Conclusions: This study is acceptable. The 14-day LC 50 for earthworms was determined to be greater than 178 mg Cry1A.105 protein/kg dry soil. The NOEC was determined to be greater than 178 mg Cry1A.105 protein/kg dry soil.

vi. Soil Fate (MRID 469514-28)

Soil organisms may be exposed to Cry1A.105 a nd Cry2Ab2 protein through contact with corn plant roots (by direct feeding), corn plant root exudates, inco rporation of above-ground plant tissues into soil following harvest, or by soil-de posited pollen. Some eviden ce suggests that soilsBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

55 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 co rn production sites, tend to have high microbial activity and microbes contribute to Cry protein de gradation. Despite evidence that soils high in clay and humic acids may bind Cr y 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 degradati on in soil evaluated clay, silt loam, and loamy sand soils that were spiked with Cry2Ab2 (0.60 μg/g) or Cr y1A.105 (0.062 μg/g) protein and incubated under

controlled conditions for four m onths. The soils were dosed with an approximately 500-fold excess of the maximum calculated pr otein concentrations in the fiel d. Samples of the treated soils were collected eleven times during the incubatio n period and analyzed for protein content using western blot analysis (Cry2Ab2 only) and a co rn 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 Cry1A .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 spik ed with purified insec ticidal protein. This approach is useful because dose responses can be eas ily quantified. But, the degradation and accumulation of Cry proteins found within decaying plant tissue may beha ve 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 a ll plant tissue is ‘mineralized’. The data reviewed here do, however, show that Cry protei ns will be quickly degraded upon release from decaying plant tissue. More specifically, a study that evalua ted Cry1Ab protein accumulation in a field with three years of continuous Cry1Ab field corn production showed that the protein had not

accumulated in soil to a level that would elicit a t oxic response from ECB larvae, a species that is highly susceptible to Cry1Ab 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. Th erefore no additional long term soil degradation studies are re quired for Cry2Ab2 or Cry1A.105 proteins.

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 et al . 2007; Oliveira et al . 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 Bt -related risks have evolve d from the decomposition of Bt -cornBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

56 leaves for the meso- and macr ofauna soil community (Hönemann et al

. 2008). Although a

minimal transient increase and shift in microbial populations ma y result from the presence of transgenic plant tissue in soil, no adverse effect s 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 resear ch projects evaluati ng 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 engineer ed 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 seas ons and weather, plant gr owth stage, and plant varieties, independent of being genetically engineered, are also re sponsible for sign ificant shifts in soil microbial communities. To date, most st udies with genetically engineered crops have shown minor or no effects on soil microbes beyo nd 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 implica tions if such a transfer were to occur. Genes that have been engineered into Bt crops are mostly found in, or have their origin in, soilinhabiting bacteria. Soil is al so the habitat of anthrax, teta nus, and botulinum toxin-producing bacteria. Transfer of these genes and/or toxins to other microorganisms or plants has not been detected. Furthermore, several experiments (publ ished 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 la boratory conditions designed to favor transfer.

As a result of these findings, whic h suggest that HGT is at most an artificial event, and the fact

that the Bt toxins engineered into Bt Cry1A.105, and Cry2Ab2 Protein in corn are derived from soil-inhabiting bacteria, EPA has c oncluded that there is a low probability of risk from HGT of transgenes found in Bt Cry1A.105, and Cry2Ab2 Protein in corn.

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 Cry34/35Ab1 protein by wild or weedy relatives of corn in th e U.S., its possessions, and/ or its territories. In addition, the Animal and Plant Health Inspect ion Service (APHIS) of the United StatesBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

57 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 pot ential for gene capture and expression of Bt endotoxins by wild or weedy relati ves 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 speci es that, for the most part, do not have a reasonable possibility of passing their traits to wild na tive plants. Feral species related to these crops, as found within the United States, cannot be pollinated by thes e 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 ha bitat. 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 consider ation 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 recipien ts 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 planti ng 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 spec ies with imperfect flowers. This means that spatially separate tassels (male flowers) and silks (femal e flowers) are found on the same plant, a feature that limits inbreeding. A large vari ety of types are known to exist (e.g., dent, flint, flour, pop, sweet) and have been selected for sp ecific seed characteris tics through standard breeding techniques. Maize cultivars and landr aces 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; Me xican maize landraces x Chalco teosinte). Zea mays has been domesticated for its current use by sel ection 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 arch aeological 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 declinedBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

58 exponentially to 0.1% at 130 feet and further de clined to 0.03% at 160 feet. At 1,000 feet, the farthest distance measured, no cross-pollinati on was detected (Jemison and Vayda 2000). For production of Foundation Seed, a distance of 660 f eet has been generally required to mitigate outcrossing between different genot ypes. 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 te mperature or low humidity, corn pollen may only survive for a matte r of minutes. Under more favorable conditions in the field or with controlled handling in the la boratory, pollen life may be extended to several

hours.

b. Tripsacum species - Gama Gr ass - 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 ag rostologists; 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 im portance. It is commonly grown as a forage grass and has been the subject of some agrono mic improvement (i.e., se lection 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 repres ent 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 respec ts, 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, pers onal communication, 1999; Galinat 1988; Wilkes 1967). Tripsacum /maize hybrids have not been observed in the field but have been accomplished in the laboratory using sp ecial techniques under highl y controlled conditions.

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; Iltis 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 tetr aploid 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 (Mangelsdo rf and Reeves 1939; DeWald, personalBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

59 communication,1999). In most cases, th ese 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 mai ze under natural conditions is highly unlikely, if not impossible (Beadle 1980). Hybrids of Tripsacum species with Z

. mays are difficult to o Bt ain outside of the

controlled conditions of laborator y and greenhouse. Seed obtained from such crosses are often sterile or progeny have greatly reduce d fertility. Approximately 10–20% of maize- 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 prope rties that indicate it has weedy potential and, other than o ccasional volunteer plants in th e 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 (Agr icultural Research Service of the USDA; Woodward, Oklahoma), a geneticist working on improveme nt 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 ev en in those that do. In controlled crosses, if the female parent is maize, there is a greater likelihood of obtaining viable see d. 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 dir ected between these two species, the resultant genome is lacking in most or all of the chromoso mal 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 accessi ons were tested and crosses

made before this came to fruition. The Tripsacum -derived mitochondrial chondrome and chloroplast plastome in these hybrid s contribute to the seed qualitie s 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 agricultu ral or natural situations is extremely remote based upon his experience trying to create hybrids under the best of conditions. He also felt that no other known grass species present in the con tinental U.S. would interbreed with commercial maize populations (i.e., be recipien ts of pollen-directed gene flow ). This is in agreement with

Holm et al . (1979), who determined that none of the sexua lly compatible relatives of corn in the U.S. are considered to be serious, pr incipal, or common weeds in the U.S.

c. Zea species - Teosintes - General BiologyBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

60 Teosintes—specifically Zea mays ssp. mexicana (Schrader) Iltis, Zea mays ssp. parviglumis Iltis and Doebley , Zea mays ssp. huehuetenangensis (Iltis and Doebley) Doebley, Zea luxurians (Durieu and Ascherson) Bird , Zea perennis (Hitchc.) Reeves and Mangelsdorf, and Zea diploperennis Iltis, 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/in florescence 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. Te osintes and teosinte-maize hybrids do not survive even mild winters and could not propagate in th e U.S. Corn Belt. Additionally, some types have strict day length requirements th at preclude flowering within a nor mal season (i.e., they would be induced to flower in November or December) and, hence, seed production under our temperate climate (Beadle 1980; Iltis, personal communication, 2000; W ilkes, 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 transfor med 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 wi nd pollinated, and tend to outcross but are highly variable species that are often genetically compatib le and interfertile with corn, especially when maize acts as the female parent. Z

. perennis (perennial teosinte) ha s 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 capab le of hybridization when in proximity to each other. In Mexico and Guatemala, teosintes exist as weeds around the margins of corn fields. The

F 1 hybrids have been found to vary in their fertil ity and vigor. Those that are fertile are capable of backcrossing to corn. A few is olated populations of annual and pe rennial teosinte were said to exist in Florida and Texas, respectively (USDA APHIS 1997). The Florida populations were presumably an escape from previous use of Z

. mays ssp. mexicana as a forage grass, but local botanists have not documented any natural populati ons of this species for approximately twentyfive years (Bradley, personal communicati on, 2000; Hall, personal communication, 2000; Wunderlin, personal communication, 2000).Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

61 Consultation with botanists and ag ronomists familiar with Texas flora suggested that no teosinte populations exist in the state (Benz, personal communication, 2000; Read, personal communication, 2000; Orzell, personal comm unication, 2000; Wilson, personal communication, 2000). Further, given the day length characteristics of Z

. diploperennis , it is highly unlikely a sustaining population would result fr om 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). N one of these teosinte species have, however, been shown to be aggressive weeds in their na tive or introduced habi tats (Schoper, personal communication, 1999). Except for special plantings as noted above, teosinte is not present in the

U.S. or its territories. Its na tural distribution is limited to Mexico, Honduras, Nicaragua, El Salvador, and Guatemala.

Given the cultural and biological re lationships 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 thes e two groups of plan ts, and that no weedy types have successfully evolved as a result. More recent cytogenetic, biochemical, and molecular analyses have indicated that th e degree of gene exchange is far less than previously thought (Doebley 1984; Doebley et al . 1987; Kato 1997a; Kato 1997b; Smith et al . 1985). Partial and complete gametophytic incompatibility has been documented among cultivated maize, landraces, and teosinte (Kermicle 1997). The former is dem onstrated by differential pollen growth and a skewed recovery of alleles linked to incomp atibility genes. Complete incompatibility mechanisms serve to isolate a species or subspe cies 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 cultivar s (W22, W23) yielded no or fe w 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 cen turies, and their genera l 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 fr om 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. Additi onally, the genus does not represent any species considered as serious or pernici ous weeds in the United States or its territories. Any introgression 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 speci es, the resultant genome is lacking in

most or all of the maize chromosomal complement in subsequent generations.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

62 Many of the Zea species loosely referred to as “teosi ntes” 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, subspeci es, 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 consid ered to be incapable of surviv al 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 Cry1A.105 and Cry2Ab2 proteins in corn is through ingestion of corn tissue. There are no reports of threaten ed or endangered species feeding on corn plants, therefore such species would not be exposed to corn tissue containing Cry protein. Since Cry1A.105 and Cry2Ab2 proteins have not been show n to have toxic effects on mammals, birds, plants, aquatic species, insects outside the order Lepidoptera and other invert ebrate species at the Estimated Environmental Concentration (EEC), a "may affect" situation for endangered land and aquatic species is not anticipa ted. In addition, EPA does not e xpect that any threatened or endangered plant species will be affected by outcro ssing to wild relatives or by competition with such entities. Hybrid corn does not exist in the w ild, nor are there wild plants that can interbreed

with corn in the United States.

Because of the selectivity of Cry1A.105 and Cry2Ab2 proteins for lepidopteran species, endangered species concerns are ma inly 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 rega rding range overlap with corn production was mainly restricted to the Karner blue butterfly ( Lycaeides melissa samuelis ). Research demonstrates that the Cry1A.105 a nd Cry2Ab2 proteins are selectively toxic to lepidopteran larvae at field con centrations 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 LC 50 value for larvae of the most sensitive
known lepidopteran species (ECB) and the maximum estimated level of Cry protein in pollencontaminated food (9.27 μg/g fresh weight), the margin of safety was calculated to be >10X maximum estimated exposure of Karner blue larv ae to corn pollen. These results indicate that cultivation of MON 89034 is not likely to pose a risk to endangered species.

After careful review of available data, EPA determined that exposur e of the Karner blue butterfly to harmful levels of MON 89034 corn plant tissues is not expected. Like wise, a review of the preferred habitats of other lepidopteran speci es listed as endangered by the U.S. Fish and Wildlife Service indicated that exposure to harmful levels of Cry1A.105 or Cry2Ab2 protein would not take place. The main reasons for th e lack of exposure are geographical and habitatBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

63 limitations. These species are lo cated in non-corn production areas and/or their habitat does not encompass agricultural areas.

Likewise, other insect species in the orders Diptera, Hemipt era, Lepidoptera, Odonata and Orthoptera that are listed as e ndangered/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 plan ts. The reviewed toxico logical data shows the relative insensitivity of a range of insects in non-lepidopteran orders to the Cry1A.105 and Cry2Ab2 proteins, indicating that MON 89034 maize hybrids are not li kely to have detrimental effects on non-lepidopteran insect s included on the endangered/th reatened 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 li sted by the United States Fish and Wildlife Service (USFWS) and the National Ma rine Fisheries Servic es (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 Cry1A.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 toxici ty and/or exposure, no effects from Bt Cry1A.105, and Cry2Ab2 Protein in corn are antic ipated for any nontarget specie s, including federally-listed threatened and endangered (“listed”) lepidoptera n 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 lepi dopteran pollinators that are important and/or essential to an endangered or threatened pl ant. The Agency is therefore upholding its determination that the registered uses of Bt Cry1A.105, and Cry2Ab2 Protein in corn will have “No Effect,” direct or indirect, on endangered or th reatened 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 effect s at the maximum hazard screening dose are observed on representative non-ta rget species, the Agency concludes that there are no unreasonable adverse effects on non-target popula tions from the use of the pesticide.

a) Direct Effects

At present, the Agency is aware of no identified significant adverse effect s of Cry protein on the abundance of non-target organisms in any popula tion in the aquatic or terrestrial field environment, whether they are animals, plants, pe st parasites, pest pred ators, or pollinators.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

64 Further, EPA believes that cultivation of M ON 89034 corn may have fewer adverse impacts on non-target organisms than use of chemical pes ticides for corn production, because under normal circumstances, MON 89034 corn requires substa ntially fewer applications of chemical pesticides, compared to production of non- Bt corn. Fewer chemical insecticide applications generally result in increased populations of benefi cial 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 pr oposed lepidopteran-resi stant corn registration. Further, EPA has determined that there is no si gnificant risk of gene cap ture and expression of Cry1A.105 or Cry2Ab2 proteins by wild or weedy rela tives of corn in the U.S., its possessions, or territories (see Section 5. “Gene Flow and Weediness Potentia l” 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 bact eria 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 timelimited 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 economi c 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 potenti al for these indirect ecological effects on biological c ontrol organisms should not be re garded as a unique ecological risk associated with the PIP crop. Some reduc tions, however, should be expected if the pest management strategy is effective. Since PIP crop s are often grown in vici nity with conventional crops to prevent resistance build- up by the target pest(s), speciali st antagonists can persist in these ‘refuges’, in other crops and in non-crop ha bitats and retain the pot ential for recolonization of the PIP crop area. Based on these considerati ons, regulatory testing of the specialist predators and parasitoids of target pests may even tually be consider ed unnecessary.

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

The Agency has sufficient information to believe th at there is no risk from the proposed uses of MON 89034 corn to non-target terres trial wildlife, aquatic, and so il organisms. The Agency has been frequently asking the registrants to c onduct post-registration l ong term invertebrate population/community and Cry protein accumula tion in soils studies as a condition of 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 perfor med during the last 10 years (Mar vier, et al. 2007; Sanvido, et al. 2007). No unexpected adverse effects on invertebra te community structure were reported. The Agency is in agreement with these conclusi ons. Similarly, no unexpected accumulation of CryBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

65 proteins in agricultural soils was seen in publ ished 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 recently published laboratory studies showing redu ced growth in shredding caddis flies exposed to anti-lepidopteran Cry1A protei n corn litter (Rosi-Marshall, et al. 2007), additional aquatic invertebrate data are required. The submitted Daphnia magna study is unacceptable because it is an 850 Series OPPTS Guideline study. The 48 hour duration of this study is not sufficient to detect mortality due to Bt proteins. It takes more than 48 hours for the target pests to succumb to the Cry proteins, th erefore 48 hours is also not expected to show mortality or reproductive effects on Daphnia. A 7 to 14 day Daphnia study as per the 885 Series OPPTS Guidelines needs to be performed. The study may be submitted as a condition of registration. Alternatively, a diet ary study of the effects on an a quatic invertebrate, representing the functional group of a leaf shredder in headwa ter streams, can be performed and submitted in

lieu of the Daphnia study.

Table 6. Supplemental non-target data re quirements for MON 89034 corn.

Testing Category Type of Data

Aquatic invertebrate A 7 to14 day Daphnia study as per the 885 Series OPPTS Guidelines has to 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, can be performed and submitted in lieu of the Daphnia study.

8. Potential Interaction Between Cry1A.105, Cry2Ab2 and Cry3Bb1 Proteins (MRID 469513-05 & 469513-06)

The purpose of these studies was to characte rize the potential for interaction between the lepidopteran-active proteins Cry1A.105 and Cr y2Ab2 and the coleopteran-active protein Cry3Bb1. The Cry1A.105 and Cry2Ab2 proteins were tested alone and in combination with either the Cry3Bb1 protein against European corn borer (ECB, Ostrinia nubilalis ) and corn ear worm (CEW, Helicoverpa zea ) in diet incorporation studies. Also, the Cry3Bb1 protein was tested alone and with the Cry1A.105 and/or the Cry2Ab2 proteins, against the Colorado potato beetle (CPB, Leptinotarsa decemlineata ). The activity of Cry1A.105 and Cry2Ab2 proteins was not significantly altered by the presence of Cry3Bb1, and the activ ity of Cry3Bb1 was not significantly altered by the pres ence of Cry1A.105 and/or Cry2A b2. Collectively these data provide evidence that the proteins do not interact in an antagonistic or synergistic manner. This study, along with the interac tion study between Cry1A.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 anta gonistic, or synergistic manner with regards toBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

66 target insects. It is therefore extremel y unlikely that the Cry1A.105, Cry2Ab2 and Cry3Bb1 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)

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

88017

This section presents the orig inal assessment for MON 89034 (b ased on BPPD’s review -- see BPPD, 2007). Subsequent to registration, M onsanto 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 Resi stance 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 ( Bt )-derived insecticidal prot eins Cry1A.105 and Cry2Ab2. The Cry1A.105 toxin is a “chimeric ” protein containing domains I and II and the C-terminal from Cry1Ac and domain III from Cry1Fa (domain III). The Cry2Ab2 protein is exactly the same as that currently expr essed 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), Spodoptera frugiperda (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 Cry1A.105 and Cry2Ab2 proteins in MON 89034 is expected to be at least partially recessive; (2) the probability of cross-resistance between Cry1A.105 and Cry2Ab2 is low; and (3) the level of both Cry1A.105 and Cry2Ab2 produced in MON 89034 confer high level of control of su sceptible 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 re fuge in cotton growing regions (currently, a 50% structured refuge) to mitigate insect re sistance to the Cry1A.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 Cro ss-Resistance to the Cry1A.105 and Cry2Ab2

Proteins

The Cry1A.105 toxin is a “chimeric ” protein containing domains I and II and the C-terminal from Cry1Ac and domain III from Cry1Fa (domain III). The Cry2Ab2 protein is exactly the same as that currently expre ssed in Monsanto’s Bollgard II cotton. There are a number of Bt corn products on the market that produce th e insecticidal proteins, Cry1Ab and Cry1FaBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

67 (potential cross resistance w ith Cry1A.105/Cry2Ab2 and Cry1Ab is discussed in section c, “Impact of Prior Use of Cry1Ab-Expressing Bt Corn Products on MON 89034”). There are

also Bt cotton products that produce the Cry1Ac, Cry1F, 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 greates t if there is a low probability of crossresistance (See Roush 1998). Cross-resistance is most likely when proteins share key structural features, which allows one resistance mechanism to c onfer 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 Cry1A toxin mode of action (see discussion in Piggott and Ellar, 2007). The most accepted Bravo model proposes that both the cadherin and aminopep tidase (APN) receptors are required for full Cry1A toxicity. This model s uggests that receptor binding is se quential: 1) ingestion of the protein inclusions by a susceptibl e insect larva, 2) solubilizati on of the protein in the insect midgut, 3) cleavage of the protoxin by host pr oteases 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 st ructures that inject into the membrane, 6) passage of ions and water through the pores, re sulting in swelling, lysis, and the eventual death of the host. Differences in any of th ese steps will reduce the probability of crossresistance between any two Cry proteins. The more controversial Zha ng 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 th e binding site receptor has been the primary Bt resistance mechanism reported to date (reviewed in Ferré & 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 ac tive 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 (Fer ré and Van Rie 2002). Ferré 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 auto somal gene, and cross-resistance extends only to Cry proteins sharing binding si tes. Cry proteins that do not share high levels of sequence similarity tend to have differe nt binding sites and different modes of action. Analyses of

resistance to Bt Cry proteins indicate that cross-resi stance occurs most often with proteins that are similar in structure (T abashnik, 1994; Gould et al., 1995).

With this information in mind, Monsanto has assessed the probability of cross-resistance between Cry1A.105 and Cry2Ab2 on three levels: 1) structural similarity between the proteins, which is indicative of mode of action; 2) characteri zation of elements of the mode of action, such as the biophysic al nature of binding of the Bt proteins to the target insectBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

68 midgut; and 3) demonstration that the indivi dual 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 Cry1A.105 protein has high sequence similarity with the Cry2Ab2 protein. Monsan to provided BPPD with a summary of current

information about the structural and functiona l similarities of the Cry1A.105 protein to other

Bt Cry1 proteins. The Cry1A.105 protein is a chimeric protein with overall amino acid sequence identity to the Cry1Ac, Cry1Ab and Cry1Fa proteins of 93.6, 90.0 and 76.7%, respectively. The Cry1A.105 protein expresse d in MON 89034 corn plants results in increased activity against FAW, SCB, and CEW compared to Cry1A b expressed in MON 810 corn plants (see BPPD review of effi cacy data, Matten, 2007; Monsanto study MRID# 46951415). A structural model of the Cry1A .105 protein was developed using the X-ray crystal structure of the Cry1Aa protein. This model demonstrated high overall main chain structural similarity with Cry1Aa. Models of Cry1Ab and Cry1Ac were also prepared using the Cry1A.105 model. Comparison of the aligned folds of all three proteins showed that Cry1Ab and Cry1A.105 have essentially the same main chain structure (i.e., similar three domain structures) and that Cry1A c differs slightly in its main chain structure from the other two in domain III. Thus, comparison of the modeled crystal structures of the Cry1A.105, Cry1Ab, and Cry1Ac with the experimental Cr y1Aa X-ray crystal structure demonstrated high three-dimensional structural similarity between the four pr oteins (i.e., Cry1A.105, Cry1Ab, Cry1Ac, and Cry1Fa).

In the case of Cry1A.105 and Cry2Ab2 proteins , however, there is on ly a 14% amino acid sequence similarity. Based on the available da ta, Monsanto has sufficiently demonstrated that there is low sequence similarity between the Cry1A.105 protein and the Cry2Ab2 protein. Lack of sequence similarity would suggest that cross-resistance between the Cry1A.105 and Cry2Ab2 proteins would be unlik ely. On the other hand, high sequence similarity between the Cry1A.105 protein and Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Fa proteins is one indicator that cross-resistance may be a concern for these proteins. This is important because Cry1A.105 is composed of domains I and II and the C-terminus of Cry1Ac and domain III of Cry1Fa. This subjec t will be discussed further in the review.

Previous studies have shown that Cry1A 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-termi nus of domain I and the C-terminal part of

domain III. Different activation mechanisms would tend to decrease the likelihood of crossresistance between the Cry1A and Cry2A proteins.

Assessment of binding characteristics is one way of determining the potential for crossresistance between the two prot eins. As noted above, changes in the nature of protein binding to the insect midgut is th e mode of action step that ha s most often been associated

with insect resistance to Bt Cry proteins (for reviews, see Tabashnik, 1994; Baxter et al.,Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

69 2005). Biacore is used to quantif y 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, su ch as ligand blotting, may be used. Ligand blotting is a qualitative tool us ed to identify protein bands that have the specific secondary

modification to bind Bt proteins. Monsanto used bot h Biacore and ligand blotting to characterize Cry1A.105 and Cry2Ab2 binding to ECB brush border membranes (studies by Li and Guzov, 2006 were provided in Appe ndix 1 of Monsanto submission, MRID# 469514- 30, Head, 2006 and are discussed below).

Binding constants for the interaction of Cr y1A.105 and Cry2Ab2 with immobilized BBMV (ECB) differed by more than an order of ma gnitude with essentially no BBMV-specific binding being observable for Cry2Ab2. The Biac ore system could not distinguish unique aspects of non-competitive binding for Cry1A.105 and Cry2Ab2 on BBMV. This result suggests that there are very different bi nding sites for Cry1A.105 and Cry2Ab2 in the ECB midgut. Additional Biacore analyses i ndicated that Cry1A.105 and Cry2Ab2 bound to different glycosyl moieties lin ked to bovine serum albumin (B SA). Cry1A.105 preferentially bound to galactosamine (K D =1.5x10 -8 M and R

max =2419 RU). Cry2Ab2 preferentially bound to N-acetyl glucosamine (K D =7.0x10 -11 M and R

max =32 RU), but also bound galactosamine

with a K D =2.0 x10 -8 M and R

max =625 RU. Furthermore, Cry1A.105 binding to galactosamine filled a two-binding-site model as evidenced by the reduction in the Chi 2 value from 2583 to 53, but the fit of Cry2Ab2 binding wa s similar for both models suggesting that the Cry2Ab2 and Cry1A.105 proteins not only bind to different sugars but also differ in their binding kinetics.

The ligand blotting analysis demonstrated that the Cry1A.105 and Cry2Ab2 proteins bound to different components on ECB brush border membrane filaments (BBMF) separated by SDS-Page and immobilized on a nitrocellu lose membrane. Trypsin-treated Cry1A.105 protein was shown to bind to a ~150 kDa ba nd while the trypsin-tr eated Cry2Ab2 protein was shown to a ~130 kDa band, but weakly to a ~150 kDa band. The trypsin-treated Cry2Ab2 protein had a greater ra te of binding than the Cry1 A.105 protein. Overall these results support the conclusion, as Monsanto has described, that the Cry2Ab2 and Cry1A.105 proteins displayed different binding components and different kinetics in binding to ECB

BBMF. These results are consistent with th e differences in binding affinity for Cry1A.105 and Cry2Ab2 proteins observed with Biacore. In addition, Monsanto noted that Cry2Aa did not bind to a specific, high affinity Cry1Ac r eceptor in work performed by English et al. (1994).

In conclusion, Biacore and ligand blotting analyses demonstrate that Cry1A.105 and Cry2Ab2 proteins bind to some unique compone nts on ECB brush border membranes. They also share many common binding sites. Screen ing a limited number of glycosylated BSAs, indicated that galactosamine is recognized by Cry1A.105 only, while Cry2Ab2 demonstrated a high affinity for both N-acetylglucosamine a nd galactosamine. These data support theBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

70 conclusion that Bt protein binding to carbohydrate moietie s is the principal basis of the specific interactions betw een the Cry1A.105 and Cry2Ab2 proteins and the ECB brush border membrane. Specific binding of Bt proteins to the target in sect gut membrane is a key step in their mode of action. Differences in the Cry1A.105 and Cry2Ab2 protein interactions with the BBM suggest that these two proteins have differences in mode of action. BPPD agrees with Monsanto that these differences should minimize the development of crossresistance by the target insect pests to these two proteins.

Monsanto also provided evidence to show that there is a lack of cross-reactivity between Cry1A.105 and Cry2Ab2 antibodies. The hom ologous primary-secondary antibody pairs recognized only their correspond ing antigens (i.e., trypsin-t reated Cry1A.105 or Cry2Ab2) with no cross-reactivity. Similarly, Monsanto previously demonstrat ed that anti-Cry2Ab antibodies do not cross-react with the Cry1Ac proteins, nor do the anti-Cry1Ac antibodies cross-react with the Cry2A b2 protein (Head and Reding 2001, MRID# 455457-01). The lack of cross-reactivity shows that the epitope bi nding sites for antibody recognition are different and therefore the tertiary structur e is different. Lack of similar tertiary structur e supports the conclusion that there will be a very low likeli hood of high levels of cross-resistance in the target insect pests for the Cry1A.105 (and all Cry1A proteins) and Cry2Ab proteins. Monsanto provided indirect information (i.e., th ere are no colonies of lepidopteran corn pests resistant to either Cry1A.105 or Cry2Ab2 proteins) to indicate that insect s resistant to one of the two insecticidal proteins, Cry1A.105 or Cry2Ab2, will be controlled by the other insecticidal protein. First, M onsanto cited to studi es provided in suppor t of the Bollgard II cotton registration (i .e, Cry2Ab2 and Cry1Ac Bt plant-incorporated protectants as expressed in cotton) that indicated that Cry1Ac-resistan ce did not confer Cry2Ab2 resistance to tobacco budworm, cotton bollworm, and pink bollwor m (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 crossresistance to Cry1Ac and the microbial ins ecticide, DiPel®, that contains the Cry1Ab, Cry1Ac, and Cry2Aa proteins. Monsanto te sted this Cry2Ab2-(SP15) resistant colony against purified Cry1A.105, Cry1Ac, and Cry2A b2 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 cr oss-resistance to th e Cry1Ac and Cry1A.105 proteins. Other published resear ch indicates that there is ev idence for broad cross-resistance (low levels of resistan ce) to Cry1A 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 crossresistance, but that high levels of cross-re sistance to Cry1A.105 and Cry2Ab2 is unlikely. In the field, this would translate to the effi cacy of MON 89034 being maintained even though resistance might occur to one of the proteins.

b) DoseBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

71 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 eff ective strategy for delaying resistance in Bt crops. The high dose/refuge strategy assumes that resistance to Bt is recessive and is conferred by a single locus with two alleles resulting in three genotypes: susceptible homo zygotes (SS), heterozygotes (RS), and resistant homozygotes (RR). It also assu mes that there will be a low initial resistance allele frequency and that there will be extensive random mating between resistant and susceptible adu lts. In practice, a high dos e PIP should express sufficient quantities of toxin to kill all susceptible insects (SS) as well as heterozy gous insects with one resistance allele (RS). Lower dose PIPs might al low 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 a ll susceptible insects. The SAP also outlined five techniques to determine high dose: 1) Serial dilution bioassa y 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 ma ke sure that the cu ltivar is at the LD 99.9 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 laborator y strain of the pest

that had an LD 50 value similar to field strains; and 5) De termine if a later larval instar of the targeted pest could be found with an LD 50 that was about 25-fold higher than that of the neonate larvae. If so, the late r 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 verifi cation techniques were developed in 1998 when all of the registered Bt crops were single t oxin 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. Thes e “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 t oxins with unrelated mode s 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 s hould 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 sing le toxin lines may not be available (orBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

72 commercialized) for comparisons. The dual toxi ns 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 contro l (>95% of heterozygotes ) is provided to be considered high dose.

MON 89034 was created with recombinant DNA technology (and not conventional breeding) to express the Cry1A.105 and Cry2Ab2 toxins. Bo th 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 Cry1A.105 or Cr y2Ab2 only) for dose comparisons, although single gene events were separa tely engineered. The Cry1A.105 toxin is a “chimeric” protein containing domains I and II and the C-termin al from Cry1Ac and domain III from Cry1Fa (domain III). By creating this chimera, Monsanto hoped to improve efficacy against several target pests including fall armyworm and corn earworm. The Cry2Ab2 pr otein is exactly the same as that currently expresse d in Monsanto’s Bollgard II cotton.

To evaluate dose, Monsanto conducted a number of laboratory and fiel d studies with diet bioassays and MON 89034 plant ma terial. Three sets of expe riments were conducted: 1) bioassays with purified toxin incorp orated 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 co rn borer (ECB), southwestern corn borer (SWCB), fall armyworm (FAW), and corn earw orm (CEW). A description of the test procedures is included in Monsanto’s submission (Head 2006; MRID# 469514-30). Toxin expression data was also obtai ned 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 suscepti bility (molting inhibito ry concentration, MIC 90 ) to the MON 89034 toxins. Molting inhibition is of ten used instead of straight mortality (i.e.

an LC 50 or LC 90 ) because it can be assumed that insects th at fail to develop as larvae will be functionally dead in the field. A MIC 90 bioassay can also reduce the amount of purified toxin needed for the testing relative to an LC 90 determination, though it is unclear whether Monsanto had insufficient purified protein to determine LC 90 values. The MIC 90 tests showed that all four target species were more susceptible to Cry1A.105 than Cry2Ab2 (as measured in ppm). ECB was more sensitive to both Cry1A.105 and Cry2Ab2 than the other tested lepidoptera by at least an order of magnitude. For Cry1A.105, BPPD agrees with Monsanto that the amount expres sed 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 MIC 90 for all four species (both measured in ppm). On the other hand, the amount of Cry2A2b expressed in MON 89034 exceeded the MIC 90 value only for ECB. For the other three pests, the level of Cry2Ab2 was at (for SWCB) or below (CEW and FAW) the MIC 90 level. These data suggest th at the Cry1A.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’sBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

73 contention that the results of la boratory 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 performa nce of MON 89034 against the target pests. Since MON 89034 expresses both Cry1A.105 and Cry2Ab2 si multaneously, 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 Cry1A.105 or Cry2Ab2, though none of these were ultimately commercialized or us ed to create MON 89034. To relate the single gene isolines to MON 89034, Monsanto supplied so me 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 Cry1A.105 isolines were used; one (LAJ138) with toxin expression equivalent to that of MON 89034 and another (LAJ129) with le ss than half the expression. Both of the Cry2Ab2 lines that were used ( 70774 and 67620) had less t oxin 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 pe sts). Two-toxin MON 89034 was highly effective against all four targ et pests with at l east 90% mortality among exposed larvae and significant growth inhibition in the surv ivors. On the other hand, mortality was more variable for the single gene is olines that were also tested. For ECB, both MON 89034 and the single gene (Cry1A.105 and Cr y2Ab2) 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 fo r SWCB with some survival (up to 41% of the control gro up) observed on both the isolin es 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 CE W 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 fo r CEW. This test revealed relatively high survival (up to 35%) on the lower expressing isolines (LAJ129 and 67620) and 9% survival on MON 89034 (growt h 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 te sts 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 M ON 89034 was not included in the trials. The trials showed that Cry1A.105 and Cry2Ab2 isol ines 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 forBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

74 ECB and SWCB. While these studies demons trated field efficacy of the Cry1A.105 and Cry2Ab2 isolines, they provide limited informa tion 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 we re not included in the tria ls. Monsanto recognized the limitations of the field work, but indicat ed that they should be considered in toto with the laboratory bioassays and leaf disk tests.

Overall, the dose studies presented a mixed pi cture of the dose profile for MON 89034. Dose and efficacy data indicated that: (1) the Cry1A.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% c ontrol of SWCB, while the Cr y2Ab2 protein provides 80-90% control; (3) the Cry1A.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 ag ainst the four major target pests including greater than 95% cont rol 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 bioassa ys, the target pests appear to be somewhat more sensitive to Cry1A.105 than to Cry2Ab2. However, much of the dose information is circumstantial; the leaf disk assays were the only trial phase th at directly evaluated MON 89034. The other data were obtained from susceptibility assays with purified protein (that we re compared to MON 89034 expression data) and tests with (non- commercialized) single gene isolines.

BPPD agrees with Monsanto that M ON 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 defi nition put forth by the 1998 SAP (a level of toxin 25 times greater than needed to kill sus ceptible larvae; i.e. a dose greater than the LC 99 of the pest). Some survival of MON 89034 plant tissue was noted for ECB, SWCB, and

CEW. Monsanto assumed that the survivor s would not reach adulthood due to growth inhibition (and therefore are func tionally dead), but that assumption was not tested due to the short time frame of the experiment.

Monsanto’s dose studies did not directly eval uate the effect of MON 89034 on potentially heterozygous larvae (i.e. with one copy of a re sistance 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 he terozygotes 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), bu t 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 invest igate whether MON 89034 consistently has high mortality of susceptible homozygotes (>95%) an d further investigate heterozygote mortalityBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

75 for MON 89034. BPPD recognizes that direct evaluations of hetero zygote effects can be difficult, particularly if resistant colonies fo r the target pests are una vailable and given that there is no field resistance to either protei n. Monsanto has not pr ovided enough information to determine the “killing power” of each indivi dual protein -- it would be useful to assess whether Cry1A.105 and Cry2Ab2, individually, will kill greater than 95% of the susceptible homozygotes. The 1998 SAP suggested severa l ways to estimate mortality for less susceptible larvae (i.e. hetero zygotes) (EPA 1998). These tec hniques included testing larger, later instar larvae that may be less susceptible to the toxins or with PIPs expressing lower

levels of toxin than the commer cial event (see the discussion of the SAP recommendations at the beginning of this section).

c) Impact of Prior Use of Cry1Ab-Expressing Bt Corn Products on MON 89034

Monsanto examined the impact of prior use of Cry1Ab-expressing Bt corn products on MON

89034 Bt corn. Cry1Ab-expressing Bt corn products have been on the U.S. market since 1997 and planted on millions of acres. This se lection pressure could result in increased Cry1Ab-resistant allele frequenc ies in lepidopteran corn pests, particularly those that are more dependent on corn as a primary host such as ECB and SWCB. Should there be Cry1Ab-resistant insects that are cross-resi stant to either the Cry1A.105 protein and/or Cry2Ab2 protein then IRM value of MON 89034 w ould be significantly reduced. Given that there is very high amino acid similarity between the Cry1Ab and Cry1A.105 proteins, the potential for cross-resistance between Cry1Ab and Cry1A.105 is an important consideration.

In an earlier section of this assessment, BPPD concluded th at there is a low likelihood of cross-resistance between Cry1A.105 (Cry1A proteins) and Cry2Ab2 proteins.

European corn borer (ECB) populations have be en monitored for susceptibility to Cry1Ab since the 1995 growing season (d iagnostic concentration info rmation has been collected since 1999). Since 1998, monitoring has also been required for corn earworm (CEW), southwestern corn borer (SWCB), and fall army worm (FAW, sweet corn only) susceptibility to Cry1Ab. All of the Cry1Ab monitoring da ta through the 2000 growing season were reviewed by the Ag ency during the 2001 Bt crops reassessment (EPA 2001). Data for the 2001 through 2005 growing seasons were indepe ndently reviewed by BPPD (see Reynolds 2004a, 2004b, 2006; Milofsky 2007). Estimates of the frequency of Cry1Ab resistance in ECB indicate that Cry1Ab-resistant alleles capable of conferring ECB survival on a Bt corn plant are very rare (Andow et al. 2000; Bourguet et al. 2003, Stodol a et al. 2006). The highest estimate of Cry1Ab resistance allele frequency in U.S. ECB populations was

<4 X 10 -4 at the 95% confidence level. The Cry1 Ab resistance allele frequency has not increased significantly in frequency even withthe ten years of widespread use of Cry1Abexpressing corn products. There have been no instances of Cry1Ab re sistance capable of conferring survival on Cry1Ab-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 frequenc y of Cry1Ab-resistance is very low and that itBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

76 has not increased significantly in over ten years of widesp read use of Cry1Ab-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 with Cry1A toxins. These authors summarized Cry toxins for which a putative receptor has been identified (see Table 2 in Pillar & Ella r 2007). The Cry1A proteins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, Cry1Fa) all have am inopeptidase N receptors (APNs) that can serve as Cry-binding proteins that mediate pore formation, but their relevance to toxin susceptibility has not been demonstrated. Cr y1Aa, Cry1Ab, and Cry1A c also have cadherinlike receptors that have been shown to mediate Cry1A toxicity. Other putative receptors, i.e., alkaline phosphatases, glycolipids, BT R-270, P252, may also play a role in Cry1A toxicity, but further study is needed. While there has been progress in what is known about Cry1A 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 Cry1A.105 and Cry1Ab proteins to ECB brush border membrane vesicles using Biacore. These studies (Li & English 2006) indicated that Cry1A.105 and Cry1Ab occupy different binding sites on the ECB midgut epithelial membrane and therefore have distinct membrane binding mechanisms. Cry1Ab binding data suggest that the binding pa tterns are much more complex for Cry1Ab than for Cry1A.105 despite these two proteins having 90% amino acid sequence homology. BPPD agrees with Monsanto that differences in binding mech anisms lessen the likelihood of Cry1A.105 and Cry1Ab cross-resistance.

Monsanto also summarized the laboratory studi es examining ECB colonies selected for resistance to the Cry1Ab protein. While thes e 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, Monsan to discussed a series of studies conducted on

three ECB colonies selected for resistance to Cry1Ab 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 fro m larval collected in Nebraska. A third colony was created from survivors of dia gnostic bioassays from both the Europe and Nebraska populations. All thr ee colonies, along with two suscep tible colonies, were assayed for their response to purified Cry1Ab, Cry1A c, Cry2Ab2, and a version of the Cry1A.105 protein. Results of the bioassays indicated th at all three Cry1Ab-resistant colonies were resistant to Cry1Ab and Cry1Ac, but remain ed susceptible to Cry1A.105 and Cry2Ab2 with

no evidence of cross-resistance.

As noted earlier, Cry1A.105 is a chimeric prot ein consisting of domains I and II and the Cterminus of Cry1Ac and domain III of Cry1Fa. Se veral pieces of evidence suggest that there

is at least some likelihood of cross-resistance of Cry1Fa a nd Cry1Ac/Cry1Ab. Denolf et al.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

77 (1993) conducted Cry1Ab, Cry1Ac, and Cry1B pr oteins binding experiments with isolated brush border membrane vesicles (BBMV) and gut tissue sections from ECB. These studies indicated that Cry1Ab and Cry1Ac proteins r ecognized the same membrane receptor with different binding affinities while the Cry1B protein recognized a separate receptor. More recent binding studies with BBMV from ECB conduc ted by Hua et al. (2001) indicated that there was limited shared binding between Cry1F a and Cry1Ab/Cry1Ac proteins. Pereira et al. (2008) showed that there wa s little cross-resistance to Cry1Ac (6.9-fold) in a Cry1Faresistant line of ECB (>3,000-fold). Jurat- Fuentes & Adang (2001) demonstrated that Cry1Fa (and Cry1Ja) share the Receptor A binding site with the Cry1A toxins in Heliothis

virescens (tobacco budworm), but they also have unique binding sites. These researchers proposed a model that suggests that Cry1Fa, Cry1Ab, and Cry1Ac all bind to the Cry1Aa binding site (called Receptor A, although with different binding affinities) as well as to unique binding sites. An alte red Cry1Aa binding site may cause resistance to Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Fa proteins, but the un ique binding sites al so play a role in toxicity. Competition binding experiments pe rformed by Hernández & Ferré (2005) showed the occurrence of a common receptor for Cry1Ac, Cry1Fa, and Cry1Ja in Helicoverpa armigera, H. virescens , and Spodoptera exigua . So far, all available information on binding site competition suggests that Cry1Aa, Cr y1Ab, Cry1Ac, Cry1Fa, and Cry1Ja share a common binding site in most, if not all, Lepi doptera. These authors suggest that Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, and Cry1Ja protein bi nding to a common site explains, perhaps, the biochemical basis of multiple resistance a nd cross-resistances among these five proteins in some insect species. Jurat-Fuentes & Ada ng (2006) recently demonstrated that a cadherinlike protein, HevCaLP, is the functional recep tor for Cry1Ac binding in a highly-resistant (>300,000-fold) tobacco budworm colony (YHD2) al though it is not a receptor for Cry1Fa (130-fold resistant). These re sults suggest that the Cry1Fa and Cry1Ac 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 Cry1Fa and Cry1A b/Cry1Ac proteins through modification of a single shared receptor site. Hernández & Ferré (2005) suggest that neither transgenic plants expressing stacked combin ations of Cry1Ac (and by extension Cry1Ab), Cry1Fa, and Cry1Ja nor rotations of Bt crops containing single genes of these three (four) proteins would be a good resist ance management strategy. In the case of corn, primary pests susceptible to Cry1Ab and Cry1Fa, such as ECB (and SWCB and CEW), would necessitate the importance of establishing the binding site m odel for this species in order to develop an appropriate resistance management strategy.

Monsanto has shown, using the weight-of-evid ence approach, that th ere is a low likelihood

of cross-resistance between Cry1A.105 and Cry2Ab2 (see section a., “ Assessment of the Probability of Cross-Resistance to the Cry1A.105 and Cry2Ab2 Proteins” of this IRM assessment). It is assumed that the primary m echanism of resistance will be that of binding site modification, which is a reasonable assumption based on studies with other Bt -resistant insect populations (laboratory and field) (see Ferré & Van Rie 2002). Similarly, Monsanto has adequately demonstrated that there is a low likelihood of cross-resistance of Cry1A.105Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

78 and Cry1Ab. On the other hand, Monsanto ha s not addressed the likelihood of crossresistance of Cry1A.105 and Cry1Fa and Cry1A c. The Cry1A.105 protein is a chimeric protein consisting of Domains I and II and th e C-terminus of Cry1Ac and Domain III of Cry1Fa.

It was recommended that Monsanto provide BPPD with additional information on crossresistance of Cry1A.105 and Cry1Fa and Cry1Ac (including binding site models and use of resistant colonies) for the target pests and de termine how such cross-resistance may impact the durability of MON 89034.

d) Proposed IRM Plan for MON 89034

Monsanto stated that the intr oduction of MON 89034 would signifi cantly 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 Cry1A.105 and Cry2Ab2 is expected to be at least par tially recessive; (2) the probability of crossresistance between Cry1A.105 and Cry2Ab2 is low; and (3) the level of both Cry1A.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% contro l). Should these assumptions be met, MON 89034 would have si gnificantly more durability th an all existi ng single-gene products for lepidopteran control in the U.S., including MON 810 (Cry1Ab), BT 11 (Cry1Ab), Herculex I (Cry1Fa), and their respec tive 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 pe sts 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 consiste d of the following

elements.

1. A 5% structured non-lepidopteran Bt corn refuge for the Corn Belt based on two independent (minimal cross-resistance), hi ghly effective modes of action of Cry1A.105 and Cry2Ab2;

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

3. Annual resistance monitoring, grower educa tion, and compliance monitoring programs;

and

4. A remedial action plan that describes a se ries 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 Co rn Uses of MON 89034 in the Corn BeltBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

79 The critical question is whether Monsanto has provided sufficient data/information to

indicate that the durabil ity of a 5% structured refuge (as Monsanto has proposed) is equal to or greater than durability of a 20% structured refuge (the curr ent 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 whether MON 89034 has consistently high mortality of susceptible homozygotes for all of the primary target species al so has to be considered.

In the case of MON 89034, two Bt genes, cry1A.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 protei ns are expressed in MON 89034 corn plants: Cry1A.105, a chimeric protein consisting of dom ains of Cry1Ac and Cry1Fa; and Cry2Ab2, the same protein that is expre ssed in Bollgard II cotton (a Monsan to product). Monsanto has provided sufficient efficacy data to dem onstrate that MON 89034 pr ovides 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 (Cry1Ab) trait corn product).

Monsanto’s first assumption was that Cr y2Ab2 and Cry1A.105 have different modes of action and therefore the potential for cross-resistance is low. As discussed earlier in this review, Cry2Ab2 and Cry1A.105 have low sequ ence homology (14%), different activation mechanisms and binding characteristics, uniqu e antibody binding sites, and resistant insects to one protein will be controlled by the other pr otein. Therefore, it can be concluded that Cry2Ab2 and Cry1A.105 have different modes of action and therefore it is expected that

there will be a low likelihood of cross-resist ance (see earlier discussion in Section a. “Assessment of the Probability of Cross- Resistance to the Cry1A.105 and Cry2Ab2

Proteins” ). Lack of cross-resistance would increa se the durability of MON 89034. These two proteins, Cry2Ab2 and Cry1A.105, therefore, seem to be good candidate proteins for pyramiding. BPPD agrees with Monsanto that the probability of cro ss-resistance between Cry1A.105 and Cry2Ab2 is low and these two protei ns have different modes of action.

On the other hand, Roush (1998) cautions that pr oteins that have al ready 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 Cry1A, Cr y1Fa and Cry1J proteins, should not be used in pyramiding. This same warning was also given by Hernández & Ferré (2005)

. Cry1A.105 is a chimera that consists of binding domains of Cry1Ac and Cry1Fa

. There are

commercial Bt crops that express Cry1Ac, Cry1Ab, Cr y1Fa proteins and these products have been in the marketplace for nearly a decade. Should there be insect populations resistant to Cry1Ac, Cry1Ab, and/or Cry1Fa th at are cross-resistant to Cr y1A.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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

80 Given that there is very high amino aci d similarity between Cry1Ab, Cry1Ac, and Cry1A.105 proteins then the potential for cross-resistance between Cry1Ab, Cry1Ac and Cry1A.105 is an important consideration. Cry1A.105 and Cry1Fa have about 76% amino acid similarity. However, what is really important is the similarity of the binding domain III of Cry1Fa and Cry1A.105 which is presumed to be very high. Cross-re sistance is a real possibility for these two proteins. There are several lines of evidence that indicate that Cry1Fa and Cry1Ab/Cry1Ac share a common bi nding receptor although each of these proteins has unique binding receptors as we ll (Denolf et al. 1993, Hua et al. 2001, JuratFuentes & Adang 2001; Hernández & Ferré, 2005) . Evidence for a shared binding receptor

would increase the likelihood of cross re sistance should resi stance evolve through modification of the shared binding receptor.

Evidence provided by Monsanto indicates that th ere is little cross- resistance of Cry1A.105 and Cry1Ab. One cannot, however, infer much a bout the likelihood of cr oss-resistance of Cry1A.105, Cry1Fa, and Cry1Ac based on the binding patterns of Cry1A.105 and Cry1Ab because binding patterns are unique to each species (e.g., ECB, SWCB, and CEW) and each protein. Monsanto did not address the lik elihood of cross-resi stance of Cry1A.105 and Cry1Fa, a protein already in existing Bt corn and Btcotton products, and what impact crossresistance would have on the durability of MON 89034. BPPD recommends that Monsanto provide additional information on cross-resi stance of Cry1A.105 and Cry1Fa and Cry1Ac (including binding site models and use of resistant colonies) for the target pests and

determine how such cross-re sistance may impact the durability of MON 89034.

Monsanto’s second assumption was that resistan ce will be recessive. Ten years of resistance monitoring data indicate that the frequency of Cry1Ab 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 ev idence of field resistance to Cry1Ab and Cry1Fa 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 cas es of field resistance to Bt crops after a decade of use indicates that any relatively common Bt -resistant alleles must be recessive (Tabashnik et al., 2003). This evidence provides a strong indicator that re sistance to the Bt proteins expressed in MON 89034 would also be recessive. BPPD agr ees with this line of reasoning. Pyramids are considerably more effective when resist ance frequencies are lo w 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 Cry1A.105 and Cry2Ab2 produced in M ON 89034 confers a high level of control of susceptible target pests (defined as at least 90% and preferably >95% mo rtality). It is this third assumption that is the mo st difficult to prove.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

81 Resistance simulation models predict that the grea test benefits of comb ining toxins in single plants by “pyramiding” or “stacking” are achi eved when no cross-resistance occurs, when there are no fitness costs, when resistance to each toxin is rare a nd recessive, and when a refuge of plants without toxi ns are present. Modeling simu lations of two-gene products predict that the resistance risk associated w ith a two-gene product wi ll 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. Modeli ng simulations predict th at 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 in sects that are resistant to the companion protein (Roush 1998). This has been de scribed as “redundant k illing” in the sense that most of the population is sus ceptible to both proteins and thus is killed twice. The extent

to which the individuals that ar e resistant to one protein are ki lled by the other is central to the effectiveness of the pyramiding strategy.

Monsanto relied on the Roush (1998) model to s upport the need for a 5% structured refuge rather than a 20% structured refuge in the Corn Belt. Roush’s m odel (figure 2 in the publication) indicated that a 5% st ructured refuge is equal to or greater than a 20% structured refuge for a highly effective, high dose si ngle-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 assumi ng there is no cross-resistance. Monsanto’s dose studies, as discussed earlier, presented a mixe d 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 Cry1A.105 and Cry2Ab2 proteins in MON 89034 each provide essentially 100% contro l of ECB; (2) the CrylA.105 protein in MON 89034 provides approximate ly 95% control of SWCB, while the Cry2Ab2 protein provides 80-90% control; (3) the Cry1A .105 and Cry2Ab2 proteins in MON 89034 each provide >95% control of FAW; and (4) th e 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 w ould likely preclude deve lopmental completion. The target pests appear to be somewhat more sensitive to Cry1A.105 than to Cry2Ab2.

Monsanto’s dose testing indicate d that MON 89034 has a high leve l 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 th an that of a single-gene Bt corn product expres sing 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 individu als. This is important for prediction of the durability of MON 89034: R oush’s simulations (1998; Figure 3) showed that the greatest gains of pyramiding two prot eins are when the mortality of susceptible insects is considerably greater than 95%, espe cially if resistance allele frequencies are quite

low.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

82 On the other hand, Monsanto’s dose studies did not directly evaluate whether MON 89034 kills at least 70% of the heterozygotes, the othe r threshold needed to support a 5% structured refuge for a two-gene pyramided Bt corn product. Monsanto did not provide enough information to determine the mortality of su sceptible (SS) homozygot es and heterozygotes (RS) on MON 89034 plants. It is important to know whether Cry1A.105 and Cry2Ab2 are: 1) both high dose proteins; 2) one high dose a nd one moderate dose protein (and which one is high and which one is moderate); or 3) two moderate dose pr oteins 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 Cry1A.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 inve stigate heterozygote mortality for MON 89034. BPPD recognizes that direct evaluations of heterozygote effects can be difficult, particularly if resistant colo nies for the target pests are unavailable. However, the 1998 SAP suggested several ways to estimate mortality for less susceptible larvae (i.e., heterozygotes). Th ese techniques included testing larger, later instar larvae that may be less susceptible to the either the Cry1A.105 or Cry2Ab2 protei ns or with PIPs expressing less protein (less Cry1A.105 or Cry2Ab2) than MON 89034. As Roush (1998) cautioned, “...small refuges remain risky...” when mortalities of hete rozygotes 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 Cry1A.105 and Cry1A c and Cry1Fa is not known, but published

studies indicate that there is at least some potential for cross-resistance between Cry1A and Cry1Fa proteins in a number of insect species (see earlier disc ussion). The impact of this potential cross-resistance on the dur ability 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 ste p. 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 Co rn Belt), spatially-e xplicit, 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 re sistance (or SWCB) to MON 89034. This new

model would need to cons ider the impact of other Bt proteins in which there may be some cross-resistance. This is analogous to th e 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 Cry1Ac and Cry2Ab2 proteins expressed in Bollgard II cotton. In conclusi on, Monsanto’s initial data and modeling do not support a 5% structured refuge for MON 89034 for fiel d 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 less er pests in the Corn Belt), cross-resistance likelihood ofBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

83 Cry1A.105, Cry1Ac, and Cry1Fa, and limitati ons of the simulation modeling, BPPD

recommended that the current 20% structured re fuge requirement for field corn uses of MON

89034 in the Corn Belt be maintained until such time as Monsanto could address these uncertainties associated with the durability of a 5% structured re fuge. There are many Bt

corn and Bt cotton products in the landscape. Cro ss-resistance conferred by any of these proteins may negatively affect the durability of MON 89034. Studies indi cate that there is at least some potential for cr oss-resistance between Cry1A.105, Cry1Fa, and Cry1Ac proteins in a number of insect species (see earlier di scussion). Monsanto needs to examine the potential of Cry1A.105, Cry1Ac, and Cry1Fa 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 re fuge 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 ma jor 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 al so sporadic corn pests in cotton-growing

areas. As described earlier in this assessment (Section b “Dose” ), Cry1A.105 and CryAb2 proteins have at least 90% control of CEW. Previous studies submitted by Monsanto (Head & Reding 2001; reviewed in BPPD 2007) de monstrated the low likelihood of crossresistance between the Cry2Ab2 and Cry1Ac pr oteins. Both Cry1A.105 and Cry2Ab2 have

a low likelihood of cross-resi stance with Cry1Ab (see earlie r discussion in Section c,

“ Impact of Prior Use of Cry1Ab-Expressing Bt Corn Products on MON 89034 ”).

Monsanto used its deterministic, non-spatia l 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 cons isted of Bollgard II cotton, with no non- Bt cotton in the system, and that 80% of the corn plante d in the region consiste d of MON 89034 and 20%

non- Bt corn. The modeling was focused on estima tion of the likelihood of CEW resistance in the Mississippi region because of the relativ ely higher risk of CEW resistance evolution in

this review. Monsanto estim ated 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 CE W, 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 esti mates were used as parameter values in the

model. One model scenario assumed that M ON 89034 is fully cross-resistant with Bollgard II cotton (i.e., Cry1A.105 and Cry1Ac are fully cro ss-resistant). Resistance was assumed to be complete with no associated fitness costs. Using these assumptions, the simulation modeling predicted that a 20% non- Btcorn refuge for MON 89034 in the southern cottongrowing areas would be suffi cient to manage the risk of resistance evolution to Bt corn and

Bt cotton products. Resistance to Cry2Ab2 pr otein evolved first and took >24 modeling years to evolve (modeling time was 25 years). It is not clear from Monsanto’s discussionBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

84 whether Cry1A.105 and Cry1Fa cross-resistance was included in the modeling. The current landscape has both Cry1Fa- and Cry1Ab-cor n and Cry1Fa- and Cry1Ac- and Cry2Ab2 + Cry1Ac-cotton products. Should there be substantia l cross-resistance, th en the value of MON 89034 would be dramatically reduced.

A 20% non- Bt corn refuge for MON 89034 in the sout hern 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 Cry1A.105, Cry1Fa, and Cr y1Ac in the cotton-growing landscape and how such cross-resistance may impact the durability of MON 89034. Should crossresistance be of concern then the durabil ity 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 fo r sweet corn production.”

Monsanto has proposed the use of MON 89034 as a sweet corn product to control certain lepidopteran insect pests in c onjunction with no structured ref uge. 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 grai n is allowed to mature and dry in the field. For sweet corn, the ears are harves ted while still wet and placed in cold storage for fresh market corn or pro cessed immediately. Shortly afte r harvest, corn stalks are typically destroyed in the fiel d by disking, chopping or plowing. Previous work by Lynch et al. (1999), show that these ha rvest and post-harvest practices make it unlikely that any surviving/resistant larvae could survive, co mplete 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, typica lly grow sweet corn in small plots along with many other vegetables that serve as altern ative hosts for these polyphagous lepidopteran pests. Therefore, sufficient non-corn refuge s hould be present due to th e 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 suppor t the sweet corn use. Monsan to responded to BPPD’s requestBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

85 for supplemental data on March 9, 2007. M onsanto provided data that compared the estimated Cry1A.105 and Cry2Ab2 protein levels in leaf tissues collected from MON 89034

sweet corn varieties with field corn vari eties (see Table 4 in Bogdanova 2007; MRID# 470794-02). Sweet corn data came from one site a nd field corn data came from five sites. The mean levels of the Cry1A.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 pot ential 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 ar e 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 fiel d 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 e fficacy data to what was provided for field corn to support the use of MON 89034 on popc orn. Without these data, the popcorn use cannot be supported.

g) Other Elements of IRM for MON 89034

Monsanto proposed to have resistance m onitoring, grower education and compliance monitoring as necessary parts of the IRM progr am. They proposed to implement a program

similar to what is curr ently carried out for MON 810 and other single-gene Bt corn products. In particular, the educational and complia nce assurance programs for MON 89034 would

follow the structure establis hed through consultations betw een EPA and the industry, and will involve working closely with NCGA and other interested stakeholders.

Similarly, post-commercial resistance monitori ng programs would be established as an extension of existing programs to track the suscep tibility of the key lepidopteran corn pests to the Cry1A.105 and Cry2Ab2 proteins. In the m onitoring program, insect populations would be collected and each protein will be tested se parately, 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 susceptibilit y studies are planned for the Cry1A.105 protein against ECB (through Dr. Blair Siegfried at th e University of Nebraska), and for the Cry1A.105 and Cry2Ab2 proteins against SW CB (through Dr. Qisheng Song at the University of Missouri), a nd CEW (through Bruce Lang of Custom Bio-Products). The baseline susceptibility of ECB to Cry2Ab2 has already been assessed ov er a two year period (see Appendix 4 - Siegfried & Spencer 2001 in Monsanto’s submission, MRID# 469514-30).Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

86 In the case of CEW, baseline studies a nd annual monitoring have been conducted for Cry2Ab2 protein as part of the Bollgard II cott on IRM program, and the resulting data will

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 develope d 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 act ual resistance, and mitigate the resistant population. The basis of this plan also is appropriate for MON 89034. However, because MON 89034 contains both the Cry1A.105 and Cr y2Ab2 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. Ther efore, Monsanto indicated that the remedial action plan should only be implemented for MON 89034 if a fiel d population evolves re sistance 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 progra m was determined to be “acceptable." No Cry1A.105 baseline susceptibility studies have b een 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 th e results of the resist ance 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 re quirements as part of the Remedial Action plan).

h) Conclusions for Initial Registration 12

MON 89034 field corn uses in the Corn Belt

1) Pyramids can reduce the need for large refuges. Monsanto had origin ally proposed that a 5% structured refuge, rather than the current 20% structured refuge, be used with the field corn uses of MON 89034. However, Mons anto’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 determ ination for ECB, SWCB, CEW, FAW (SS and RS mortality), cross-resi stance likelihood of Cry1A.105, Cry1Ac, and Cry1Fa and its impact on the durability of MON 89034, and lim itations of the simulation modeling. The

12 The assessment for the amendment to reduce lepidopter an refuge requirements modified these conclusions. (See

section II. E. 2. )Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

87 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.

2) Monsanto relied on the Roush (1998) model to support the need fo r 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 re fuge is equal to or greater than a 20% structured refuge for a highly effective, high dose single-gene pr oduct when a two-gene product (MON 89034 in this case) achieves at least 95% contro l of susceptible homozygotes and 70% control of heterozygotes assu ming 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 M onsanto 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 s uggested several ways to estimate mortality for less susceptible larvae (i.e., heterozygotes) (EPA 1998). These techniques included testing larger, later in star larvae that may be less susceptible

3) Monsanto has demonstrated that Cry1A.105 a nd Cry2Ab2 have different modes of action and, therefore, a low likeli hood of cross-resistance. Cry1A.105 and Cry2Ab2 would be suitable partners in a pyramided product. M onsanto has also shown that there is a low likelihood of cross-resistance between Cry1A .105 and Cry1Ab. Monsanto has previously

demonstrated that there is a low likeli hood of cross-resistance between Cry2Ab2 and Cry1Ac.

4) However, Monsanto did not address the likelihood of cross -resistance of Cry1A.105, Cry1Ac, Cry1Fa, 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 cr oss-resistance of Cry1A.105 and Cry1Fa and Cry1Ac (including binding site models and use of resistant colonies) for the target pests and determine how such cross-resistance may impact the durabil ity of MON 89034. The Cry1A.105 protein is a chimeric protein co nsisting of Domains I and II and the Cterminus of Cry1Ac and Domain III of Cry1Fa. It is important to address not only the

likelihood of cross-resist ance potential of Cry1A.105 and Cry1Ab and, similarly, Cry1A.105 and Cry2Ab2 (which was done by Mons anto), but also that of Cry1A.105 and Cry1Ac and Cry1Fa.

5) Additional species-specific (e.g., ECB and SWCB for the Corn Belt), spatially-explicit, landscape modeling is recommended to expl ore 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 conf er some cross-resistance (to Cry1A.105, in particular) and how such cr oss-resistance would impact th e durability of MON 89034 in the Corn Belt (use of simulation modeling). This is analogous to the species-specific simulation modeling that EPA re quired Monsanto do to support the use of na tural refugeBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

88 (instead of a structured refuge) for management of H. virescens and H. zea to the Cry1Ac and Cry2Ab2 proteins expre ssed in Bollgard II cotton.

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 woul d be sufficient to manage the risk of

resistance evolution to Bt corn and Btcotton products assuming there is no crossresistance . However, Monsanto did not sufficien tly address the cross-resistance ofCry1A.105, Cry1Fa, and Cry1Ac in the cotton-growing landscape and how crossresistance may impact the durability of MON 89034. Should cross-resistance be of concern then the durability of MON 89034 in th e 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 poten tial 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 gr ower guide/product use guide and/or in supplements to the grower guide/product use guide:

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

8) Popcorn. 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 educati on and compliance monitoring as part of the MON 89034 IRM program is “acceptable." No Cry1A.105 baseline susceptibility studies have been conducted, but are planned by Monsanto. Monsanto has indicat ed 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 report ing to the Agency of the results of the resistance monitoring, grower education, a nd compliance monitoring is needed (as is required for all other Bt PIPs). If there is confirmed resi stance 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 Manageme nt Plan for MON 89034 X MON 88017 Bt CornBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

89 MON 89034 x MON 88017 expresses the Cry1A.105, Cry2Ab2, and Cry3Bb1 Bt toxins and is targeted against lepidopteran corn pe sts including European corn borer (ECB), southwestern corn borer (SWCB), corn earwo rm (CEW), and fall armyworm (FAW) as well as the coleopteran corn rootworm sp. pe st complex (CRW). MON 89034 (Cry1A.105 and Cry2Ab2) provides activity against the lepidopter an corn stalk and ear insects while MON 88017 (Cry3Bb1) is active against root-feeding CRW. The product was created by conventional breeding in which the previ ously-registered MON 88017 (EPA Reg. No. 524- 551) was crossed with MON 89034 (EPA Reg. No. 524-LTL). The Cry3Bb1 toxin in MON 88017 is the same as expressed by MON 863 co rn (Yieldgard Rootworm, EPA Reg. No. 525- 528), which was registered by Monsan to for the 2003 growing season.

1) Monsanto has provided information to de monstrate 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 IR M considerations (dose, refuge, cross resistance) for MON 89034 and MON 88017 are a pplicable to the stacked MON 89034 x MON 88017 product.

2) Monsanto has proposed a 5% lepi dopteran refuge as part of the “Separate Refuge” option

for MON 89034 x MON 88017 corn. Due to uncer tainties 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% re fuge has been previously required.

3) Monsanto’s proposal for a combined refuge (covering both coleopter an 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 supporte d in southern cott on-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 m onitoring (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 preced ing 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).Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

90 As part of the IRM proposal for MON 89034 corn, Monsanto proposed a 5% lepidopteran structured refuge for non-cott on growing regions instead of th e 20% refuge that has been required for all other Bt corn registrations. Monsanto re asoned that the combination of two toxins targeting lepidopteran corn pests with no cross resistance allowe d for a reduced refuge

with little risk of resistance. As describe d in section 1 above, BPPD’s review of the IRM proposal (BPPD 2007) agreed with much of Monsanto’s justificat ion but determined that there were a number of uncertainties in the request fo r lower refuge. Specifically, there were three areas of concern: (1) Cry1A.105 and Cry2Ab2 dos e determination for the major target pests (ECB, CEW, SWCB, and FAW); (2) cross re sistance potential between Cry1A.105 and Cry1F and Cry1Ac (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 uncer tainty of the reduced refuge request, EPA registered MON 89034 with a 20% stru ctured refuge requireme nt, similar to other

Bt corn products. Separately, EPA did agree withMonsanto’s request to reduce refuge in cottongrowing areas from 50% to 20% (see discussion in section 1 above and in BPPD 2007). As a condition of registration, Monsanto was require d to address cross resistance in existing Bt corn and Bt cotton products for Cry1A.105, Cry1Fa and Cry1Ac.

Monsanto subsequently provided mate rials to address these three areas of uncertainty as part of a new amendment request for a reduced 5% re fuge for non-cotton regions. The response, including a discussion of cross resistance and a new model, is incl uded 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 Amendmen t to Support a 5% Refuge for MON 89034

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

Each of these sections is describe d and reviewed individually below.

In lieu of submitting new dose determination data for Cry2Ab2 and Cry1A.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 Cry1A.105 and Cry2Ab2, wh ich target the same lepidopteran corn pest complex. The Cry1A.105 toxin is a “chimeri c” protein containing domains I and II and the C-terminal from Cry1Ac and domain III from Cry1F a while the Cry2Ab2 protein is the same as that currently expressed in Monsanto’s Bo llgard II cotton. Monsanto has sufficientlyBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

91 demonstrated that the cross resistance potenti al between these two proteins should be low, primarily due to differing modes of action (s ee discussion in BPPD 2007). In evaluating new PIP traits, the landscape of prev iously registered toxins in th e same crop must be taken into 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 bo llworm, CBW, when a pest on cotton), is a pest of both crops. As a condition of registration, Mons anto was required to a ddress cross resistance in existing Bt corn and Bt cotton products for Cry1A.105, Cry1Fa and Cry1Ac.

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) Cry1Ab vs. Cry1Ac; 2) Cry1F vs. Cry1Ab and Cry1Ac; 3) Cry2Ab2 vs . Cry1 proteins; and 4) Cry1A.105 vs. Cry1Ab and Cry1Ac. New data were presented fo r comparisons between Cry1A.105 and Cry2Ab2 vs. Cry1F.

Cry1Ab vs. Cry1Ac : Based on a literature review of bi nding studies with numerous lepidopteran species, Cry1Ac is known to have strong cross re sistance with Cry1Ab. Bo th toxins share a high affinity binding site in ECB, CEW/CBW, SWCB, FAW, and others (references cited in MRID# 474748-01).

Cry1F vs. Cry1Ab and Cry1Ac : Cry1F also shares a binding site with Cry1Ab/Cry1Ac, though the level of cross resistance between Cry1F and Cry1A is not as strong as Cry1Ab vs. Cry1Ac. ECB resistant to Cry1Ab have been shown to be partially resistant to Cry1F although Cry1F resistant ECB were not cross resistant to Cry1Ab and only slightly resistant to Cry1Ac. Similar trends have also been shown with tobacco budworm ( Heliothis virescens , TBW) (references cited in MRID# 474748-01). Overall, Cry1F can be considered partially cross resistant to Cry1Ab and Cry1Ac. The availability of binding sites may explain the partial cross resistance: Cry1Ab and Cry1Ac could have more different sites to bind with than Cry1F so that resistance to Cry1F still allows for some binding of Cry1Ab or Cry1Ac.

Cry2Ab vs. Cry1 proteins : A literature review suggests that Cry2Ab has no cross resistance potential with any of the currently registered Cry1 proteins includi ng Cry1Ab and Cry1Ac.

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 Cry1Ab or Cry1Ac protei ns. Additional studies with Cry1Ac-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 Cry1Ab-resistant E CB were not found to be cross resistant with Cry2Ab while Cry2Ab2-resistant H. armigera were not cross resist ant with Cry1A.105 or Cry1Ac.

Cry1A.105 vs. Cry1Ab and Cry1Ac : For Cry1Ab, a previously submitted binding study with ECB (Head 2006; reviewed in BPPD 2007) showed that the protein has a distinct binding siteBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

92 from Cry1A.105. This was confirmed by studies with Cry1Ab-resistant ECB and sugarcane borer ( Diatraea saccharalis , SCB) that showed no cross re sistance with Cry1A.105. Monsanto argues that due to similar characteristics between Cry1Ab and Cry1Ac (i.e., mode of action), it is reasonable to assume that Cry1Ac should not be cross resistant with Cry1A.105. However, no binding studies or experiments with resistant colonies were descri bed to verify that assumption.

Cry1A.105 and Cry2Ab2 vs. Cry1F : New data were cited by Mons anto (Schlenz et al. 2008) to assess the cross resistance potential betw een Cry1A.105/Cry2Ab2 and Cry1F using Cry1Fresistant ECB and FAW colonies. Artificial diet bioassa ys were used to test Cry1A.105, Cry2Ab2, and control groups against ECB and FAW co lonies previously se lected for high-level Cry1F resistance as well as unselected control coloni es. A range of five concentrations was used and the test was conducted over a seven day period to determine growth inhibition (GI 50 ) for each colony. The results showed that, as e xpected, Cry1F-resistant ECB and FAW were not cross resistant with Cry2Ab2 -- the GI 50 resistance ratios (Cry1F-resistant : Cry1F-susceptible) were 1.4 for ECB and 0.11 for FAW. With Cry1A.105, the GI 50 resistance ratios were > 3.9 for ECB and 7.0 for FAW, indicati ng low level cross resistance.

Table 7 : Cross resistance potential of MON 890 34 (Cry1A.105 and Cry2Ab2) with previously registered Bt corn toxins.

Bt toxins in MON 89034 Existing Bt toxins Cry1A.105 Cry2Ab2

Cry1Ab No cross resistance (ECB, SCB) No cross resistance (ECB)

Cry1Ac Unlikely cross resistance, but unverified experimentally No cross resistance (TBW, PBW, CEW/CBW) Cry1F Low level cross resistance (ECB, FAW) No cross resistance (ECB, FAW)

BPPD Review - Cross Resistance

BPPD agrees with Monsanto’s characterizati on of the cross resistance potential for the Cry1A.105 and Cry2Ab2 toxins with (1) each other (previously demonstrated in Head 2006), (2) Cry1F, and (3) Cry1Ab. Binding and resistant colony work conducted by Monsanto and other researchers clearly show that no cross resist ance can be expected between Cry1A.105, Cry2Ab2 and Cry1Ab (see Table 7 above). New data refere nced in Monsanto’s amendment request also experimentally demonstrate the cross resistance potential between Cry1F and Cry2Ab2 (no cross resistance) and Cry1A.105 (low cross resistance).

Nonetheless, BPPD still has reservations about Cr y1Ac. While Monsanto has made the case that Cry1Ac should be expected to behave like Cr y1Ab due to a similar mode of action, no experimental data (i.e., binding st udies or bioassays with resistan t insect colonies) were providedBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

93 either in the original MON 89034 IRM submi ssion (Head 2006) or the follow-up amendment request (MRID# 474748-01). BPPD notes that Cry1A .105 (a chimeric protei n) contains domains I and II and the C-terminal from Cry1Ac. Cross- resistance could result wh en proteins share key 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 pres ent time there are no registered Bt corn products containing Cry1Ac. Therefore, exposure to ECB and SWCB to Cry1Ac 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 Cry1Ac. 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 Cry1Ab) and Bt corn (including Cry1A.105) and increased selection pressure for resistan ce, particularly if there is a risk of cross resistance.

Given that Monsanto has propos ed to substantially reduce refuge for MON 89034 from 20% to 5%, cross resistance is an important consider ation even for Cry1Ac. Although improbable, BPPD cannot rule out that a CEW/CBW populati on could develop Cry1Ac resistance in cotton and then encounter MON 89034 corn. [Tabashnik et al. (2008) have argued that Cry1Ac resistance has already evolved in CBW in the s outh, although this conclusion has been disputed (Moar et al. 2008).] Should there be a degree of cross resistance between Cry1Ac and Cry1A.105, MON 89034 might functiona lly have only Cry2Ab2 remaining as an effective toxin against CEW. With a reduced refuge (5%), selec tion 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 resistan ce potential of Cry1A .105 with Cry1Ac in CEW/CBW, it is recommended that Monsanto provide additional in formation either experimentally (e.g., binding studies or with resistant colonies) or using a nother analysis. Altern atively, 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 be tween Cry1A.105 and Cry1Ac; 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 M onsanto’s initial IRM plan for MON 89034, BPPD identified the need for additional species-specific (e.g., ECB and SW CB for the Corn Belt), spatially-explicit, landscape modeling to explore the durabi lity of MON 89034 versus single-protein Bt corn products (BPPD 2007). Previousl y, Monsanto had cited the mode ling 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 hom ozygous susceptible insects and 70% of theBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

94 heterozygotes (see BPPD 2007). BPPD recomme nded that Monsanto further characterize the dose expression for the MON 89034 t oxins for the major target pests of the Corn Belt (ECB and SWCB). Given the dose uncertainties, BPPD could not at the time of regi stration 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 Cry1A.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 fr om other registered Bt corn products (Cry1Ab, Cr y1F) and had a number of assumptions and parameters:

• Dose mortality for ECB: 99.9% for Cry1 (Cry1Ab, Cry1F, Cry1A.105) and Cry2Ab2 toxins (one mortality scenario was modeled);

• Dose mortality for SWCB: 99 - 99.5% fo r Cry1 and 85 - 95% for Cry2Ab2 (six dose mortality scenarios were modeled);

• Complete resistance to Cry2Ab2 and Cr y1A.105 (i.e., survival probability of heterozygote resistant individuals = 1) with no fitness costs;

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

• Three cross resistance scenar ios: 1) Cry1A.105 and Cry1Ab fu lly cross resistant (but not Cry1F) (the “base case” scenario); 2) Cry1A .105 and Cry1F fully cross resistant (but not Cry1Ab) (alternate “base cas e” scenario), and 3) Cry1A.105, Cry1Ab, and Cry1F all fully cross resistant (worst case scenario);

• All resistance alleles (Cry1, Cry1A.105, and Cr y2Ab2) have initial frequencies of 0.005. Cry1Ab and Cry1F are modeled as one output (i.e., estimate d time to resistance for Yieldgard/Herculex);

• 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., wi ld hosts or other cultivated crops that

could serve as a source of su sceptible insects) and have tw o 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 m odel simulations. MKT 1 = 100% MON 89034; MKT 2 = 50% MON 89034, 25% MON 810, 25% TC1507; MKT 3 = 0% MON 89034, 50% MON 810, 50% TC1507.

Most of the assumptions above were conservativ e estimates, with the pos sible exception of the dose mortality parameters for SWCB (see discus sion 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. W ithin each cross resistance scen ario, model runs were conducted for three different market adoption con tingencies of MON 89 034, MON 810 (Cry1Ab Yieldgard) and TC1507 (Cry1F Herculex).Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

95 ECB Results

For ECB, the results of the model runs were relatively consistent among the different cross resistance and market adoption sc enarios. In almost all cases, the durability of the MON 89034 toxins (Cry1A.105 and Cry2Ab2; assu ming a 5% refuge) exceeded th e 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 Cry1A.105 less than 30 ye ars (29 years) for ECB -- Cry2Ab2 remained effective in all model simulations (> 30 years) . For the other Cry1 toxins (Cry1Ab and Cry1F) that are expressed in other Bt corn products, resistance develope d in less than 30 years for some of the cross resistance and market adoption scenarios. In the “base case” (Cry1Ab and Cry1A.105 cross resistant), the durability of Cry1Ab/Cry1F lasted 26 years (0% MON 89034, 50% MON 810, 50% TC1507) and 29 years (5 0% MON 89034, 25% MON 810, 25% TC1507). However, for the alternate base case (Cry1F and Cry1A.105 cross resistance), resistance to Cry1Ab/Cry1F did not evolve within 30 years. In the worst case scenario (a ll three toxins cross resistant), resistance to Cry1Ab/ Cry1F 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 Cry1A.105 and Cry1F than those with higher dose mortalities. As with ECB, Cry2Ab2 remained durable (> 30 years) in all but one of the simulations regardless of the cr oss resistance or market adoption

scenario.

For the “base case” cross resistance scenario, th e time to resistance was lowest in the market adoption scheme (MKT 3) without MON 89034 (50% MON 810, 50% TC1507) ranging from 17 years (lower dose mortalities for Cry1 a nd Cry2Ab2 toxins) to 20.5 years (higher dose mortalities). Once MON 89034 was added to the m odel (MKT 1 and 2), the time to resistance with the Cry1 toxins increased by 2 -2.5 year s for all simulations. Cry1A.105 and Cry2Ab2 did not evolve resistance in any of the model r uns for MKT 2, although there were two instances with MKT 1 (100% MON 89034) in which resistance e volved within 30 years. In both of these cases, lower dose mortality values for SWCB (85% for Cry2Ab2; 99% for Cry1A.105) were

included in the model.

Time to resistance in the “alternate base cas e” (Cry1F and Cry1A.105 cross resistant) was > 30 years in almost all cases. Only in the simula tion that incorporated the lowest dose mortality values (85% for Cry2Ab2 and 99% for Cry1A.105) did resistance evolve to one of the toxins (28.5 years for Cry1A.105).

In the “worst case” (Cry1Ab, Cry1F and Cry1A.105 ar e all cross resistant), resistance developed in all scenarios for both the Cry1 toxins and Cry1A.105. Conversely, Cry2Ab2 remainedBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

96 durable (> 30 years) for all of the simulati ons. Time to resistance in the Cry1 and Cry1A.105 toxins was lowest (17 years) in the model run using the lower SWCB dose mortality values (85% for Cry2Ab2 and 99% for Cry1A.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 Tabl e 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 simu lations of MON 89034 (5% refuge), MON 810, TC1507 (20% refuge) expressed in years to resistance (30 year ma ximum). Derived from data reported in MRID# 474748-01.

Cross resistance scenario

Base case 1 Pest

MKT 1 MKT 2 MKT 3 Alt. base

case 2 Worst case 3

ECB Cry1A.105 Cry2Ab2 Cry1Ab/Cry1F >30

>30

N/A >30

>30

29 N/A

N/A

26 >30

>30

>30 29

>30

29

SWCB Cry1A.105 Cry2Ab2 Cry1Ab/Cry1F 22.5 - >30

25 - >30

N/A >30

>30

19 - 23 N/A

N/A

17 - 20.5 28.5 - >30

>30

>30 17 - 22

>30

17 – 22

1 Base case = Cry1Ab and Cry1A.105 cross resistant; thre e 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 = Cry1F and Cry1A.105 cross resistant (only Mkt 2 simulated).

3 Worst case = Cry1A.105, Cry1Ab, and Cry1F all fully cross resistant (only Mkt 2 simulated).

Based on the model work, Monsanto concluded th at the durability of the MON 89034 proteins (Cry1A.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 Cry2A b2 in the model was particularly robust in almost all simulations for ECB and SWCB (onl y one simulation predicted less than 30 years durability). Resistance to Cry1A.105 was also ra re in most simulations, although the “worst case” modeling (assuming complete cross resistance with Cry1Ab and Cry1F) showed resistance developing in less than 30 years. Monsanto also noted that in the simulations with different market adoption scenarios, the addition of MO N 89034 increased the time to resistance for the previously registered Cry1 toxins (Cry1Ab and Cry1F).

BPPD Review - Modeling

BPPD agrees with Monsanto’s overall conclusions that the model simulations demonstrate the effectiveness in delaying resistance of M ON 89034 and provide support for the use of a 5%Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

97 refuge in the Corn Belt. However, BPPD notes that some of the parame ters and assumptions of the model could be revised to improve and expand the overall analysis.

For ECB, the model clearly predicts that resi stance is unlikely to evolve to Cry1A.105, Cry2Ab2, or the previously-registered Cry1 toxins. Ev en under the worst case scenario that assumed complete cross resistance, the durability of all to xins 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 (rev iewed in BPPD 2007) showed that the Cry1A.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 varie d, largely due to the different simulations run with the range of dose mortality assumptions. Not surprisingly, the simu lations that were run with the lower mortality estimates (i.e., 85% fo r Cry2Ab and/or 99.0% for Cry1) resulted in less

time to resistance than those usi ng the higher dose values. In the worst case simulations with the lower dose estimates, SWCB resistance e volved in 17 years to both Cry1A.105 and Cry1Ab/Cry1F while with the higher doses resist ance 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 m odel results. BPPD agr ees with Monsanto that

the addition of MON 89034 in the simulations testing various mark et adoption scenarios delayed resistance in the other previously-registered Cry1 toxins. Likely, these results were due to less selection pressure on each indi vidual toxin because of a dive rse mosaic of toxins in the landscape. Cross resistance was also an important variable. Monsanto’s “base case” for cross resistance assumed cross resistance between Cry1 Ab and Cry1A.105. This re sulted in resistance always developing in Cry1Ab/Cry1F (i.e., within 30 years), although Cry1A.105 and Cry2Ab2 durability remained strong. On the other ha nd, when cross resistance between Cry1A.105 and Cry1F was assumed, resistance rare ly developed in either the M ON 89034 toxins or the existing Cry1 toxins. In the worst case scenario (all th ree toxins cross resist ant), the durability of Cry1A.105 to SWCB was clearly imp acted relative to the other cross resistance simulations. Conversely, Cry2Ab remained durable in almost a ll cases regardless of the varying assumptions

and scenarios included in the mode l. Since Cry2Ab is not cross re sistant to the Cry1 toxins, this result was not unexpected.

BPPD generally agrees with Monsanto that conser vative assumptions were used in the model. BPPD notes, however, that several of the paramete rs could have been expanded or have included an additional degree of conservatism or additi onal 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 no t 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 Cry1 toxins occurred in 17 - 20.5 years.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

98 When MON 89034 with a 5% refuge was included, th e time to Cry1 resistance was 19 - 23 years -- indicating that the addition of MON 89034 provide s some delay in resistance development (2 - 2.5 years). It would have been interesting to observe the impact of a doption of MON 89034 with a 20% refuge on Cry1 resistance. In all likelihood, the time to resistance would be increased, although the magnitude of such an increase is u nknown. 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) wa s another limiting parameter. Many of the

simulations resulted in no resistance within th e 30 year time period of the model, so it was

difficult to discern the effects of certain variab les (i.e., cross resistance, market adoption, dose mortality) between model runs. Had the ti me horizon been extended (e.g. to 50 years),

differences between the various model scenarios may have been apparent.

For the SWCB simulations, Monsanto used dos e mortality range of 85-95% for Cry2Ab2 and 99-99.5% for Cry1 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 Cry1A.105 protein in MON 89034 provided approximately 95% c ontrol in mortality a ssays, though the other registered Cry1 proteins (Cry1A b and Cry1F) 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 scen arios. The extent of this effect is unknown, although BPPD notes that the differences be tween the lower Cry2A b2 dose (85%) and the highest dose (95%) in the range appeared to be ne gligible 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 Cry1 proteins and model simulations which demons trate strong durability of Cry1A.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 eith er MON 89034 or the other previously-registered Cry1Ab or Cry1F corn hybrids.

Despite a good case for a refuge reduction, BPPD not es 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 Cry1Ac and Cry1A.105. Cry1Ac is registered in Bt cotton products and the chimeric protein Cry1A .105 has two Cry1Ac domains. CEW feed on both corn and cotton and successive genera tions may have exposure to both Cry1A.105 and Cry1Ac during the same growing season;Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

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

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

• SWCB model simulations included dose mortal ity 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). Cry1A.105 caused 95% mortality in submitte d dose studies, though a range of 99-99.5%

was used in the model.

As a condition of registration of MON 89034, Monsan to was required to addr ess cross resistance in existing Bt corn and Bt cotton products for Cry1A.105, Cry1Fa and Cry1Ac. Monsanto has sufficiently addressed cross resistance for Cry1A.105 and Cr y1Fa, but there are lingering questions regarding Cry1Ac and Cry1A.105. The amendment submission included only a circumstantial discussion of Cry1Ac cross resi stance with an assumption that the protein will behave similarly to Cry1Ab. But, since Cry1A .105 contains domains I and II and the C-terminal from Cry1Ac, BPPD is still concerned about the potential for cross resistance. As such, BPPD recommends additional work (as described in th e cross resistance section above) to satisfy the condition of registration. Should additional cross resistance work (as previously described) demonstrate little or no cross resistance pot ential between Cry1A.105 and Cry1Ac, further support could be provided for the use of a 5% refuge in the Corn Belt.

In terms of resistance risk for MON 89034, cros s resistance between Cry1Ac and Cry1A.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 Cry1A.105 (in corn) and then Cry1Ac (in Bollgard cotton) later in the gr owing season. Theoretically, CE W could develop resistance to Cry1Ac due to exposure in cotton -- should there be a degree of cross resistance between Cry1Ac and Cry1A.105, MON 89034 could func tionally have only Cry2Ab2 re maining 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 ove rall resistance risk fo r 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 overwin ter in the Corn Belt. On th e 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 ar eas, 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 conducte d to support the use of a 20% refuge for CEW in southern cotton-growing regions (see discussion in BPPD 2007).Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

100 A second mitigating factor is that CEW is a hi ghly polyphagous insect and is known to feed on a wide variety of plants including weeds, wild hos ts, and other cultivated crops (unlike ECB and SWCB which feed primarily on corn). Analys is conducted for Bollgard II cotton determined 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 likel y 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 cott on-growing regions and that host utilization patterns in the Corn Belt are speculative.

The other modeling parameter uncertainties deta iled above are relatively minor, though a more expanded model analysis could have provided str onger 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 sin ce most of the simulati ons 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 pr ovided 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 registered as an expiring registration (expira tion date: September 30, 2010) and BPPD recommends reevaluating 5% refuge if warranted by cross re sistance data or other information during this interim period.

3. Conditional IRM Data Submitted fo r 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 MRID 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 Cry1A.105 and Cry1Ac proteins in tobacc o budworm and cotton bollworm 477912-01Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

101 6/29/09 Resistance monitoring: baseline susceptibility data for Cry1A.105 and ECB 477912-02

6/29/09 Resistance monitoring: baseline susceptibility data for Cry1A.105 and CEW 477912-03

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, Monsan to requested additional time to fulfill some of the conditional data requirements. One of these requirements is baseline susceptibility (resistance monitoring) data for s outhwestern corn borer. This report is now due to be submitted by August 31, 2010.

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

Cross Resistance

For the original registration of MON 89034 cor n, Monsanto demonstrat ed low likelihood of cross resistance between the expressed toxi ns Cry1A.105 and Cry2Ab2 (see review in BPPD 2007). BPPD was concerned, however about po tential cross resistance between Cry1A.105 in MON 89034 and Cry1F and Cry1Ac toxins that are expressed in other registered Bt corn and cotton PIPs. A condition of registration was im posed 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 unlik ely between Cry1A.105 and Cry1F, though BPPD still had uncertainties about Cry1Ac (see review in BPPD 2008a). Gi ven this concern, the condition

to conduct cross resistance data with Cry1A.105 and Cry1Ac 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 Cry1A.105 and Cry1Ac Proteins to Cotton Bollworm ( Helicoverpa zea ) and Tobacco Budworm ( Heliothis virescens ) Brush Border Membranes” (MRID# 477912-01).

Summary of Monsanto’s Cross Re sistance Submission (MRID# 477912-01)

Monsanto addressed the question of potential cross resistance be tween Cry1A.105 and Cry1Ac by conducting binding assays using brush border membranes (BBM) from two cotton pests, tobacco budworm (TBW) and cotton bollworm (C BW). To accomplish this objective, BBMs were obtained from homogenized and filtered w hole body third instar larvae (centrifuged into pellets) using established pro cedures (English et al. 1991). The BBM proteins were thenBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

102 suspended onto nitrocellulose membranes and separated using gel elec trophoresis (SDS-PAGE) techniques. Membranes containing the separate d BBMs were exposed to incubations containing either Cry1A.105 or Cry1Ac. Bound protein was detected us ing a mammalian (goat) antibody capable of recognizing both Cry1A.105 and Cry1Ac.

An analysis of the BBMs of TBW and CBW reveal ed that both insects have similar protein components (as detected by SDS-PAGE). Becau se of this, the binding experiments with Cry1A.105 and Cry1Ac showed similar patterns be tween the insects. The specific binding

characteristics for each insect are summarized in Table 10 below.

Table 10. Binding profiles for Cry1A.105 and Cry1Ac in TBW and CBW (preferential binding sites as analyzed by SDS-PAGE). (Created from data submitted to EPA in MRID# 477912-01)

TBW CBW Cry1A.105 unique binding sites (kDas) 20, 42, 50, 95 20, 42, 50, 90

Cry1Ac unique binding sites (kDas) 57, 63 57, 63

Common binding sites for both toxins (kDas) 40, 120, 150 40, 120, 150

Although there are shared binding sites for Cry1A.105 and Cry1Ac in both TBW and CBW,

Monsanto concluded that “...these proteins have different insect icidal 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 pr esence of three common binding sites could indicate the potential for some cross resistance between Cry1A.105 and Cry1Ac. 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 suppor ting data (e.g., assays with to xin-resistant colonies) the possibility cannot be eliminated. To BPPD’s knowledge, no additiona l studies have been conducted with Cry1A.105 and Cry1Ac. As noted in BPPD (2008a), Cry1A.105 is a chimeric protein that contains domains I and II and the C-terminal from Cry1Ac. Cross-resistance can result when proteins share key structural feat ures, which allows one resistance mechanism to confer resistance to more than one protein (T abashnik 1994; Gould et al . 1995). Because of the structural similarities, some shared binding sites may be expected. Since there are a number ofBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

103 unique binding sites for each toxin, any cross re sistance due to common binding sites will likely

be at low levels.

Cry1Ac could be a potential conc ern due to the presence of Bt cotton PIPs that express the toxin (Cry1Ac is not found in a ny 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. Si milarly, corn pests such as ECB and SWCB are unlikely to be exposed to Cry1A c 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, how ever that there are several mitigating

factors that could lower the impact of any cross resistance development in CBW to Cry1A.105/Cry1Ac. First, CBW is generally a lesser pest in the Corn Belt than ECB, primarily due to poor overwintering capabi lity in much of the region (i.e ., north of Virginia, Tennessee, and Missouri). Therefore, the potential for resi stance 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 lepidopter an pests (ECB, CEW, and SWCB) and the two expressed toxins (Cry1A.105 and Cry2Ab2). In a ddition to the lepidopteran pests, monitoring for corn rootworm (CRW) and the Cry3Bb1 t oxin was also mandated with the MON 89034 x MON 88017 registration.

Monsanto was directed to utili ze 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 ar eas of high risk of re sistance development, bioassays to detect resistant indi viduals, and investigations of re port of unexpected pest damage. To support these objectives, baseline suscepti bility data were required to be submitted for Cry1A.105 and Cry2Ab2 activity against E CB (Cry1A.105 only), CEW, and SWCB.

Resistance monitoring was also required for fall armyworm (FAW) with sw eet corn uses of the

MON 89034 and MON 89034 x MON 88017. FAW mon itoring 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 Cr y1A.105 and Cry2Ab2 were required as terms of registration.

Monsanto’s Responses to Resist ance Management RequirementsBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

104 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 Cry1A.105 onl y -- 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 (to April 1, 2012) to submit FAW baseline da ta (letter to EPA dated June 3, 2010).

Monsanto also provided a re vised 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) su bmitted for SmartStax corn (EPA Reg. No. 524- 581), a separate Bt corn product that targets rootworm.

ECB Baseline Susceptibility Da ta - Cry1A.105 (MRID# 477912-02)

Baseline susceptibility studies for ECB and Cr y1A.105 were conducted by Dr. Blair Siegfried and Dr. Terrence Spencer of the University of Nebraska. Dr. Sieg fried’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 Cry1 A.105 testing was similar to the procedures employed for the annual ABST C 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 larv ae from five states (Illinois, Iowa, Minnesota, Nebraska, and South Dakota). Collected ECB were reared in the lab to produce progeny for testing (F 0 , F 1 , or F 2 neonate larvae were tested). A susceptible laboratory

ECB strain was tested as we ll. Bioassays were conducted wi th dilutions (range 0.06 to 4.0 ng/cm 2 ) of Cry1A.105 toxin overlaid onto artificial di et. The toxin was microbially-produced (by E. coli ) and supplied by Monsanto. Results were tabulated after seven days by assessing mortality (non-molting 1 st 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 Cry1A.105/cm 2 and 100% mortality with all concentrations exceeding 6.25 ng/cm 2 . Of the field collected populations, re sults were reported for 10 populations; the remaining six populations collected as diapausing la rvae were delayed. The susceptibility assays (Table 3) for the reported populations resulted in a LC 50 range of 0.52 - 1.02 ng Cry1A.105/cm 2

and a LC 90 range of 1.66 - 4.04 ng/cm 2 . EC 50 values (determined from larval weights) ranged from ~ 0.17 to 0.46 ng/cm 2 . By comparison, the laboratory colony LC 50 was 0.40 ng/cm 2 , the

LC 90 was 1.06 ng/cm 2 , and the EC 50 was 0.19 ng/cm 2 . 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 Cry1A.105 was not reported in the results.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

105 CEW Baseline Susceptibility Data - Cry1A.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 fo r assessing CEW susceptibility to Cry1A.105.

CEW were collected from 24 locations in nine states (Alabama, Arkansas, Georgia, Illinois, Iowa, Louisiana, Mississippi, North Carolina, an d 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 (F 2 to F 5 ) to Custom Bio-Products for the bioassays. A su sceptible laboratory colo ny was also included in the experiments. Susceptibility was assessed with diet bioassays incorporating dilutions of toxin overlays (concentration range of 0.1 to 6 μg Cry1A.105/cm 2 ). Cry1A.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 we re included in the testing. The baseline susceptibility assays (Table 11) for the field-collected populat ions resulted in the following ranges: LC 50 : 0.010 - 0.540 μg/cm 2 ; LC 90 : 0.042 - 2.118 μg/cm 2 ; LC 99 : 0.114 - 6.457 μg/cm 2 ; EC 50 : 0.0016 - 0.0190 μg/cm 2 ; EC 95 : 0.0184 - 1.1092 μg/cm 2 ; EC 99 : 0.0482 - 10.8520 μg/cm 2 . For the laboratory colony, the susceptibility results were: LC 50 : 0.256 μg/cm 2 ; LC 90 : 1.296 μg/cm 2 ; LC 99 : 4.857 μg/cm 2 ; EC 50 : 0.0034 μg/cm 2 ; EC 95 : 0.2255 μg/cm 2 ; EC 99 : 2.3628 μg/cm 2 . The study author did not suggest a potential value for a CEW dia gnostic concentration based on these results.

Table 11. ECB and CEW baseline susceptibility to Cry1A.105 (Created from data submitted to EPA in MRID# 477912-02 and -03)

Susceptibility to Cry1A.105

LC 50 1 LC 90 1 EC 50 1 EC 95 1

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 Cry1A.105/cm 2 for ECB and μg Cry1A.105/cm 2 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 lepidopter an and corn rootworm pests. Regarding the lepidopteran pests, the Agricu ltural Biotechnology Stewardshi p Technical Committee (ABSTC), a consortium representing Bt corn registrants, has been re sponsible for c onducting resistanceBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

106 monitoring activities. ABSTC submitted a unifiedplan 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 sin ce its submission including insect sampling, bioassays, procedures for unexpected pest damage, definitions of pest resistance, and steps to confirm cases of suspected resistan ce. ABSTC amended the monitoring plan in 2008 (MRID# 474070-01; see review in BPPD 2008b) to adjust the sampling strategy for ECB and SWCB and modify the procedures for determining resistance (m ainly in CEW). This revised monitoring program was integrated into the amended registration terms for both products after the approval of 5% lepidopteran refuge (l etters 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 di fferences from the ABSTC plan and some components have been adapted to Cry1A.105 and Cry2Ab2. For ECB, Monsanto indicated that diagnostic concentrations have been deve loped (“upper 95% confidence limit of the LC 99 or EC 99 ”) for the toxins and will be used in the a ssays, although the specific values were not reported. Survival of >1% on the diagnostic con centration will trigger fo llow-up investigations of the population. For SWCB, Monsanto reported that a diagnostic concentration has not been developed. Rather, dose-response assays (i.e., LC 50 and EC 50 ) will be used to assess field collected populations re lative to historical data for su sceptible populations. With CEW, Monsanto stated that “high diagnostic concentrat ions are not practical or relevant,” but that diagnostic concentrations will be developed for Cry1A.105 and Cry2Ab2 since they have higher activity. Dose-response parameters (LC 50 and EC 50 ) will also be used for comparisons with

historical data.

BPPD believes that diagnostic conc entrations are an integral part of pest monitoring as a means to distinguish susceptible and potentially resi stant individuals. Monsanto submitted acceptable Cry1A.105 baseline susceptibility studies for ECB and CEW but diagnostic concentrations were not proposed in the reports and ad ditional work may be needed to develop functiona l standards. For ECB, 96.9% mortality was achieved at a concentration of 2.0 ng/cm 2 and 100% mortality with all concentrations exceeding 6.25 ng/cm 2 , so a diagnostic concentration based on an LC 99 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 LC 99 and EC 99 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 Mons anto continue to worktowards developing susceptibility data and diagnostic concentrations for these pests and repor t 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 th e revised ABSTC program. To confir m 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) su rvival on a laboratory diagnosticBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

107 concentration that demonstrates a genetic basis fo r the tolerance and a resi stance allele frequency

≥ 0.1; 3) a LC 50 in a standardized laboratory bi oassay that exceeds the upper 95% LC 50 confidence interval for a suscepti ble 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 Monsan to dated December 15, 2008).

For CRW resistance monitoring with the Cr y3Bb1 toxin (expressed in MON 89034 x MON 88017), Monsanto cited to the plan devel oped 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 diagnosti c dose assay and rootworm damage guidelines (for unexpected pest damage). These conditions were also re quired for the existing Monsanto

rootworm Bt corn registrations and were addr essed 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 mainta ining 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 re quired to utilize existin g paradigms to address remedial action plans for lepidoptera and corn ro otworm. 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 incorporat ed into the terms of registration for both MON 89034 and MON 89034 x MON 88017 with the approva l 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 submitte d by Monsanto contains a description of procedures to confirm the heritabi lity and field relevancy of resist ance, estimate the frequency of resistance alleles, determine the geographic boun daries of the resistan ce, and, in cases where resistance allele frequencies ar e increasing or proliferating, creat ion of “an appropriate remedial action plan based on the knowledge of the genetics and level of resistance it c onfers in the field.”

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

• Notify EPA, affected customers, and extens ion agents within 30 days of resistance confirmation;Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

108 • Increase resistance monitoring in the affected area;

• Utilize alternate control measures in the ar ea 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;

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

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

Although Monsanto’s submitted plan does not addr ess 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 te rms of registration for MON 89034 x MON 88017) referenced the remedial action plan previously developed for Cry3Bb1 regi strations. This plan was originally developed for MON 863 and was subsequently carried over to MON 88017 and SmartStax, both of which also e xpress Cry3Bb1 for CRW control.

Conceptually, the CRW remedial action plan (submitted in MRID# 473547-01; reviewed in BPPD 2009) is similar to the st rategy for lepidoptera. Activi ties are centered on assessing the genetics (heritability, r-allele fr equency) 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 M ON 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 assura nce 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 grower s, include annual surveys (an onymous and on-farm) to assess compliance, and provide a means to investigat e “tips and complaints” of out-of-compliance growers. In addition, Monsanto was required to utilize (and provi de copies to EPA) a grower agreement to contractually bi nd 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 (2002Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

109 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 co rn rootworm PIPs (as

well as stacked Bt corn PIPs targeting both pest complexes).

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 re presentative sample of growers from various corn-growing regions in the U.S. Results fr om the survey can assess not only levels of grower compliances with refuges but also grow er 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, complia nce with refuge requirements is assessed and growers out of compliance ar e 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 establis hed for “significant deviati ons” (or <2/3 refuge fields

within 1⁄2 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 1⁄2 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 warni ng letters, compliance assistance visits, educational efforts, and ot her 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 incl uding 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 re gistration, 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 revise d 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 se veral modifications to address the reduced refuge. Monsanto

removed the distinctions between “significant” and “other” devi ations in the phased complianceBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

110 approach; instead, all in stances 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 pr evented from purchasing MON 89034 varieties for at least one growing season.

BPPD agrees that the modification to eliminat e tiered levels of non- compliance for MON 89034 is reasonable given the small (5% refuge). A ny deviation from such a refuge will likely be significant -- a 1% refuge devia tion for a 5% requirement functiona lly results in 20% less refuge. Further, Monsanto’s plan to address non-co mpliant growers in the MON 89034 CAP is the practical equivalent of the appr oach for growers with “significan t 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 re quirements as well as other conditions imposed by the registrant (and not under the purview of EP A). Two sets of grower agreements were submitted: one (for the 2009 growing season) in re sponse 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 biotechno logy products. Portions of the 2010 co ntract pertinent to IRM are quoted

below: In the “Grower Agrees” section:

• “To implement an Insect Resistance Manage ment (‘IRM’) program as specified in the applicable......YieldGard® corn secti ons 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 sectio ns 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/Grow er 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 th e IRM/Grower Guide, including anyBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

111 supplemental amendments. Grower may lose grower’s limited use license for these products if grower fails to follow the IR M program required by this Agreement.”

In the “General Terms” section:

• “Grower acknowledges that grower has re ceived 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 registrati on 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 communi cations, 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 biotec hnology line including MON 89034 and MON 89034 x MON 88017 (trade name YieldGar d VT). A second submission was made in 2009 that includes a more complete description of the grow er 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 wh en 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 in cluded with the TUG. The 2009 TUG was a comprehensive document (50 pg.) that addresse d topics such as bi otechnology, stewardship, IRM, weed resistance, and crop-specific inform ation for corn, cotton, a nd herbicide-resistant soybean, alfalfa, canola, and sugarbeet. In te rms 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. Sp ecific 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 insect icide treatments of re fuges and other aspects of refuge management. Each Monsanto Bt corn platform (corn borer control only, rootworm only, and stacked corn borer/rootwo rm control) was addressed separately in the TUG. For theBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

112 stacked YieldGard Triple VT Triple pr oduct (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.

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

described:

• Grower Agreements: Contractual arra ngements 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 ref uge 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 developmen t 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 representati ves and communications with seed

dealers.

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

Subsequent to the registra tion of MON 89034 and MON 89034 x MON 88017, EPA approved an amendment to lower the refuge to 5% for lepido pteran 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 part ner with ABSTC and the National Corn Grower

Association to harmonize educat ional 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 provide d in the submission. Seed catalogs and bags will be clearly marked to display the required re fuge size and training will be provided for the seed distribution network (incl uding 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 vers ion 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 prov ides 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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

113 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 determ ine that such conditional registration will, inter

alia , be in the public interest. EP A determines whether conditional registration of a pesticide is in the public interest in a ccordance with the criteria set forth at 51 Fed. Reg. 7628 ( Conditional Registration of New Pesticides , March 5, 1986). There is a presum ption 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 pestic ide that is of continuing concern to EPA; (iii) the use is one for which an emergency exempti on under FIFRA Section 18 has been granted (the basis for the exemption was lack of a registered alternative product); or (i v) the use is against a pest of public health significan ce. Notwithstanding whether a regi stration of a pesticide may be presumed to be in the public interest, EPA may de termine that such a regi stration is in the public

interest on the basis of one of th e following criteria: (i) there is a need for the new chemical that is not being met by currently registered pesticid es; (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 pes ticides 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 dete rmined that MON 89034 and MON 89034 x MON 88017 are in the public interest based on criteria (ii) and (iii) mentione d above. Specifically, under criteria (ii), both MON 89034 and M ON 89034 x MON 88017 should allow growers the opportunity to reduce the use of higher risk, and often less ef fective and more expensive, conventional pesticides. A reduction in use of c onventional pesticides equa tes 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 prot ection against primary and secondary corn pests, which should facilitate greater grain quality , a reduction of mycotoxin contamination, increased yield and ultimately have pos itive implications for human health.

1. Agricultural Benefits

MON 89034

BPPD recognizes that MON 89034’s unique comb ination of Cry1A.105 and Cry2Ab2 proteins expands the spectrum of protecti on for corn against lepidopteran pests - past that offered by already-registered MON 810 (BPPD, 2007a). In a ddition 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 ( Spodoptera frugiperda , 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 a pplications on acreage infested with secondary pests, although most growers do not use conventional pestic ides to treat pests that are not part of the soil pest complexBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

114 (BPPD, 2001). Finally, yield appear s to be comparable to other Bt insect-protected corn. In situations of increased lepidopteran pre ssure, 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.

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 hi gher-risk conventional pesticides currently utilized for CRW control (BPPD, 2003). Additionally, MON 89034 x MON 88017, which has tolerance for glyphosate, should allow corn grow ers to utilize a conventional chemical, Roundup, that is recognized by the Agency as a Categor y E chemical (i.e., there is evidence of noncarcinogenicity for humans). Finally, yield appears to be comparable to other Bt insect-protected

corn. In situations of increased lepidopteran and/or coleoptera n pressure, yield could be higher

than other Bt insect-protected corn because of the pres ence 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 sp ectrum 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 pest icides that may be used agai nst particular non-soil complex corn pests. It is reasonable to believe that all of these characteristics should result in increased yield, increased grain quantit y, 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 sl ight advantage over the single event for growers that require protection against lepid opteran pests and CRW.

3. Human Health and Environmental Benefits

MON 89034

Human HealthBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

115 The Cry1A.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 re duce the use of some c onventional pesticides (BPPD, 2001). Finally, because the Cry1A.105 and Cry2Ab2 protei ns target secondary corn pests - such as FAW and CEW - and protect th e ear from damage caused by these pests, decreased amounts of mycotoxin co ntamination 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). A recently published laboratory study shows reduced growth in shedding caddis flie s exposed to anti-lepidopteran Cry1A protein corn litter (Rosi-Marshall, et al. 2007), therefore, BPPD will require additional aquatic invertebrate data. A 7-14-day Daphnia study, per the 885 Series Office of Prevention, Pesticides, and Toxic Substances (OPPTS) Gu idelines, will be submitted as a condition of registration. Finally, use of MON 89034 should encourage a sm all reduction in the use of conventional pesticides. Fewer chemical insectic ide applications genera lly result in increased populations of beneficial organisms that control secondary pests, su ch as aphids and leafhoppers.

MON 89034 x MON 88017

Human Health

In addition to Cry1A.105 and Cry2Ab2 proteins produced in MON 89034, the introduction of MON 88017 in the pyramided product results in production of Cry3Bb1 pr otein. 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 Cry3Bb1 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 880 17 should result in the reduction of many conventional pesticides that are currently used, which have significant adverse effects on human health (BPPD, 2003).

Environment

Cry3Bb1 protein, produced in MON 88017, posed no si gnificant risk to te st organisms (BPPD, 2003). The only potential concern, brought to B PPD’s attention by a recently published study,

relates to MON 89034 and will be dea lt with by submission of 7-14-day Daphnia study. Additionally, use of MON 89034 x MON 88017 s hould reduce the amount of conventional pesticides used in the environmen t. All of the conventional pest icides used for CRW control orBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

116 suppression currently cause signifi cant adverse environmental eff ects under conditions of normal use (BPPD, 2003). Fewer chemical insecticide applications generally result in increased populations of beneficial organisms that control secondary pests, su ch as aphids and leafhoppers.

4. Insect Resistance Management (IRM) Benefits

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

BPPD concludes that MON 89034’s two modes of ac tion 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 li kely 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 th ey will be required to plant less structured refuge. In addition, the Cry1A .105 and Cry2Ab2 toxins are new pr oteins targeting lepidopteran pests in corn. These additional modes of acti on will likely provide a benefit to IRM programs (i.e., a toxin “mosaic” in corn-growing regi ons may reduce the like lihood 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 pot ential 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 20 06 corn crop was 33.7 billion dollars. The corn industry suffers substantial economic losses fr om damage caused by specific lepidopteran and coleopteran pests.

Two primary corn pests of particular concer n 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 th at 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% reducti on, 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 generationBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

117 causes leaf sheath, collar, and ear damage. Monsanto estimates that the average annual U.S. yield loss from ECB infestation is within the ra nge of 3-7%. Deviations from the range are

attributed to level of infestation and region.

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 ove rwinters only in extr eme southern Texas and Florida. Monsanto provides an estimate th at 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 Monsan to cites its economic damage as being low and dependent on timing of infestation, regi on, 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 Cry1A.105 and Cry2Ab2. The Cry1A.105 toxin is a “chimeric” protein containing domains I and II and the C-te rminal from Cry1Ac and domain III from Cry1F. The Cry2Ab2 protein is functionally equivalent to that curren tly expressed in Monsanto’s Bollgard II cotton. MON 89034 is protected from damage caused by larval feeding of ECB, southwestern corn borer ( Diatraea grandiosella

, 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 Cry3Bb1 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 Cry3Bb1 protein produced in MON 88017 and MON 863 is a variant of the wild-type Cry3Bb1 protein from Bt subsp. kumamotoensis . When compared by amino acid sequencing, the Cry3Bb1 protein expressed in MON 88017 has been reported to be 99.8% similar to the Cry3Bb1 protein expressed in MON 863. The primary differen ce 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 expresse s the Cry1A.105, Cry2Ab2, and Cry3Bb1

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 Ass ertions for MON 89034 and MON 89034 x MON 88017Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

118 In the introduction of their publ ic interest document (PID), M onsanto outlines the following reasons why MON 89034 and MON 89034 x MON 88017 ar e in the public inte rest according to some of the criteria set forth in 51 Fed. Reg. 7628:

• Enhanced spectrum of control . MON 89034 provides protection against

a n 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 bot h products improves overa ll 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

c ontrol 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 in creases the efficiency of

b reeding multiple traits into new corn hybr ids, was utilized in the creation of

MON 89034 and MON 89034 x MON 88017.

• Compatibility with integrated pest management (IPM) systems. Both MON 89034

a nd MON 89034 x MON 88017 provide two different modes of action in a single plant and reduce the probability of lepidopter an pests developing resistance to the Bt proteins. This allows for a smaller refuge, helps th e 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

r educe the use of conventional chemicals, which saves costs and protects human

health and the environment.

• Easy implementation . No additional labor or machiner y is needed to plant, grow,

o r harvest MON 89034 and MON 89034 x MON 88017 relative to conventional

corn.

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

5 -enolpyruvylshikimate-3-phospha te synthase protein from Agrobacterium sp. Strain CP4, which confers tolerance to gl yphosate. Therefore, the agricultural herbicide, Roundup, can be utilized on MON 89034 x MON 88017 corn to control weeds and enhance the ability of the co rn plants to access soil nutrients. EPA has classified glyphosate as a Category E Chem ical, meaning there is evidence of noncarcinogenicity 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 ar e in the public interest.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

119 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; (i i) economic (grower) benefits; (iii ) human health benefits; (iv) environmental benefits; and (v) insect resistan ce 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 le vels in grain of MON 89034 corn exposed to lepidopteran insect infesta tion and inoculation with Aspergillus flavus or F usarium

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, Cry1A.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 improve d protection (as compared to MON 810, other Bt corn products, and non- Bt corn products) from feeding dama ge caused by particular lepidopteran pest larvae. The spectrum of protection agains t lepidopteran insects in cludes the following: European corn borer ( Ostrinia nubilalis , ECB), southwestern corn borer ( Diatraea grandiosella

, SCWB), Sugarcane borer ( Diatraea saccharalis , SCB), fall armyworm ( Spodoptera frugiperda

, FAW), black cutworm ( Agrotis ipsilon , BCW), and corn earworm ( Helicoverpa 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 contro l of ECB, SWCB, and SCB was found to be comparable to MON 810 (See Table 11). Because of the production of Cry1A.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 hi gher 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 pr otein produced by MON 89034 provided improved control from CEW, when compared to the activity of MON 810’s Cry1Ab protein (See Table 11).Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

120 Table 9. Summary of arthropod LC 50 values for the Cry1A.105 protein exposure in diet bioassays
In the event that no adverse effect was observed, the LC 50 value is considered to be greater than the

maximum concentration tested.

*Table from page 15 of MRID 472797-01

1 LC 50 values with a greater than sign represent the highest dose tested.

2 Assay was performed with lyophilized leaf tissue from MON 89034.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

121 Table 9. (cont). Summary of arthropod LC 50 values for the Cry1A.105 protein exposure in diet bioassays
In the event that no adverse effect was observed, the LC 50 value is considered to be greater than the

maximum concentration tested.

*Table from page 16 of MRID 472797-01

1 LC 50 values with a greater than sign represent the highest dose tested.

2 The no observed effect concentration wa s determined to be 120 μg/g diet.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

122 Table 10. Summary of arthropod LC50 values fo r the Cry2Ab2 protein exposure in diet bioassays
In the event that no adverse effect was observed, the LC 50 value is considered to be greater than the

maximum concentration tested.

*Table from page 17 of MRID 472797-01

1 LC 50 values with a greater than sign represent the highest dose tested.

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

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

4 Significant mortality was not observed at the highest te sted 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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

123 Table 10. (cont). Summary of Arthropod LC 50 Values for the Cry2Ab2 Protein Exposure in Diet Bioassays
In the event that no adverse effect was observed, the LC 50 value is considered to be greater than the

maximum concentration tested.

*Table from page 18 of MRID 472797-01

1 LC 50 values with a greater than sign represent the highest dose tested.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

124 Table 11. Summary of field efficacy of MON 89034, MON 810 and control corn against major lepidopteran pests during the 2003-2004 growing season

F Field

Location Infestation method Damage

measured f Infestation

level Trait performance 1

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 improv ed performance compared to other treatment = represents no statistically significant difference in performanceBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

125 MON 89034 x MON 88017

In addition to producing the Cry1A.105 and Cry2Ab2 proteins, MON 89034 x MON 88017 also produces the insecticidal protein, Cry3Bb1, th at controls damage caused by CRW.

The efficacy of MON 89034 x MON 88017 against CR W 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 wa s comparable to MON 88017. The average root damage rating (RDR) for MON 88017 and M ON 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-CRWprotected control corn against corn rootworm tested in 2005 and 2006

Entry RDR 1,2

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 E CB 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 provid ed a high level of control against leaf damage and stalk tunneling by ECB.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

126 a) Pest Spectrum and Efficacy – BPPD’s Response

MON 89034

BPPD agrees with Monsanto that MON 89034 target s lepidopteran pests sp ecifically. In two studies conducted by Monsanto, both the Cr y1A.105 protein and Cry2A b2 protein exhibited insecticidal activity in the order Lepidoptera but not in th e orders Coleoptera and Hemiptera (BPPD, 2007c; BPPD, 2007d). The Cr y1A.105 protein, administered at 50 μ g/mL and 100

μ g/mL concentrations to insects in the orders Lepidoptera, Cole optera, and Hemiptera, caused a range of mortality of 32% to 96% in CEW, EC B, and FAW (BPPD, 2007c). Additionally, all four lepidopteran insects (CEW, ECB, FAW, an d BCW) had a range of 32% to 100% growth stunting (BPPD, 2007c). On the other hand, the Cry2Ab2 protein, administered at 50 μ g/mL and

100 μ g/mL concentrations to insects in the orde rs Lepidoptera, Coleoptera, and Hemiptera, caused at least 61% mortality (corrected) ag ainst CEW, ECB, and FAW (BPPD, 2007d). All four lepidopteran insects (CEW, ECB, FAW, an d 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 Yi eldGard Corn Borer (MON 810), a lepidopteran control corn produc t that expresses the Cry1Ab protein. However, MON 89034 did offer a broader spectrum of insect protecti on activity than MON 810 a nd 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 bene fits of MON 89034 x MON 88017 are similar to the efficacy be nefits 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 lepid opteran pests. Although no SCB, WBCW, or

BCW field trials were conducted with MON 890 34 x MON 88017, based on the efficacy against the other pests, it is reasonable to assume co mparable efficacy to MON 89034 for these pests as well (BPPD, 2007b).Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

127 b) Yield – Monsanto’s Summary (MRID 472797-01)

MON 89034 and MON 89034 x MON 88017

In 2006, Monsanto conducted fiel d 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 yi eld across several hybrid s tested for insectprotected hybrids and higher yields comp ared to hybrids that did not produce Bt proteins (See Table 13). Additionally, the assumption is made that under intense lepid opteran pressure, the yield benefit from MON 89034 woul d 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 60

MON 810 180.6 29

MON 89034 x MON 88017 189.3 80

MON 810 x MON 88017 185.7 36

Non- Bt 171.5 2
*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, Cry1A.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 re sult mostly from the characteristics of MON 89034: expanded pest spectrum and the pres ence of two toxins instead of one.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

128 2. Economic (Grower) Benefits

a) Monsanto’s Summary (MRID 472797-01)

MON 89034

For economic benefits, Monsanto cites to Na tional Center for Food and Agricultural Policy statistics that are based on pl anted acreage of MON 810 corn in 2005. Monsanto estimated that MON 810 increased corn production by 103.9 million bushe ls because of the corn borer resistant trait. Net returns, decreased costs (fuel, labor, and conventio nal 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 m illion pounds. In percentage terms, MON 810 planted in 2005 resulted in a 24% increase in yield, a 27% de crease 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 resu lts in increased grain quality and increased yield; (ii) reduction in mycotoxi n contamination levels which c ontributes to economic recovery; (iii) protection that is more effective in control ling corn borers and therefore results in increased yield, grain quality, and grain qua ntity; and (iv) reduction in c onventional 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 a ddition 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 to lerance trait that limits yield loss from weed pressure. Overall, MON 89034 x MON 88017 will lim it yield losses from corn borer insects, CRW, and weed pressure, reduce conventi onal pesticide use, and reduce mycotoxin contamination while increasing yiel d, grain quantity, and grain quality.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

129 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 ec onomic benefits may be affected by factors including pest pressure, clima tic 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 conclude d that a slight decrease in pesticide use should

be realized w ith the use of Bt corn products similar to MON 89034 (BPPD, 2001); therefore, BPPD agrees with Monsanto’s assertion of po ssible conventional pestic ide use reduction and

associated reduced costs.

BPPD agrees with Monsanto’s conclusion that MON 89034 should reduce mycotoxin contamination. Overall, if primary and seconda ry 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). Furthe rmore, field evidence has demonstrated the ability of Bt corn to reduce the infesta tion rates of certain mycotoxi ns (Wu, 2008). This article specifically associates CEW with aflatoxi n 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 be nefits than MON 89034 for grow ers needing to treat both lepidopteran pests and CRW. The economic be nefits 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’s Summary (MRID 472797-01)

MON 89034

Human Health

Monsanto states that the Cry1A.105 and Cry2Ab2 proteins produced by MON 89034 are structurally and functionally rela ted to Cry proteins that have a history of use both as active ingredients in Bt microbial pesticides and bio-tech deri ved food and feed. Furthermore, theyBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

130 state that Bt has been commercially used in the U. S. since 1958 to produce microbial-derived pesticides and no adverse effect s on humans or animals has been reported during their use on food or feed crops. Additionally, Monsanto as serts that Cry1A.105 and Cry2Ab2 proteins are highly unlikely to create any concer ns of toxicity or al lergenicity 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 t oxicity 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 Cry1A.105 and Cry2Ab2 proteins.

Two of the specific human health benefits that Monsanto attributes to use of MON 89034 are pesticide reduction and mycotoxi n reduction. Using numbers from an article from the National Center for Food and Agricultural Policy, Monsan to 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 pesticid e 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 a nd 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 my cotoxin contamination, particularly from Aspergillus

.

Environmental

Monsanto states that Cry1A.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 applicati on 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 al so apply to MON 89034 x MON 88017. In addition to the presence of the Cry1A.105 and Cry2Ab2 proteins found in MON 89034, the pyramided product also produces Cry3Bb1 protein. Given th at MON 88017 is already a product registered by the EPA, Monsanto states that the Cry3Bb1 protein produced by MON 89034 x MON 88017 already has an exemption from tolerance. Furthe rmore, human risk assessment data reviewed by the EPA for registration of MON 88017 has resulte d in a conclusion that there is reasonable certainty that no harm will result from aggr egate exposure to the U.S. population, including infants and children. As with MON 89034, M ON 89034 x MON 88017 is expected to result inBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

131 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 e ffects also apply to MON 89034 x MON 88017. MON 89034 x MON 88017 also produces the Cry3 Bb1 protein, which is in the previously registered MON 88017. Upon review of the environmental effects data for MON 88017’s registration, EPA concluded th at no unreasonable adverse eff ects 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 Cry1A.105 and Cry2Ab2 proteins produced by MON 89034 should not cause toxi city or allergenicity probl ems in humans. The data submitted and cited regarding potential health effects for the Cry1A.105 and Cry2Ab2 proteins include the characterization of the expressed prot eins 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 suppor t the prediction that the Cry1A.105 and Cry2Ab2 proteins would be non-toxic to humans. When prot eins are toxic, they ar e 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 Cry1A.105 and Cry2Ab2 protei ns, even at relatively high dose levels, the Cry1A.105 and Cry2Ab2 proteins are not considered to xic. Basing this conc lusion on acute oral toxicity data without requiring fu rther toxicity testing or residue data is similar to the Agency position regarding toxicity and the requireme nt of residue data for the microbial Bt products from which this plant-incorporated protectant was derived (See 40 CF R 158.740(b)(2)(i)) (BPPD, 2007e).

Since Cry1A.105 and Cry2Ab2 are prot eins, potential allergenicity wa s also considered as part of the toxicity assessment. Considering a ll of the available information (1) Cry1A.105 and Cry2Ab2 originate from a non-allergenic sources; (2) Cry1A.105 and Cry2Ab2 have no sequence similarities with known allergens; (3) Cry1A.105 and Cry2Ab2 are not glycosylated; and (4) Cry1A.105 and Cry2Ab2 are rapidly digest ed in simulated gastric fluid; EPA has concluded that the potential for Cry1A.105 and Cry2Ab2 to be a food allergens is minimal (BPPD, 2007e).Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

132 The lack of mammalian toxicity at high le vels of exposure to the Cry1A.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 demons trated the ability of

Bt corn to reduce the infestation rates of certain mycotoxins (W u, 2008). This article specifically

associates CEW with aflatoxin accu mulation in corn and claims that Bt corn varieties, perhaps such as MON 89034, are being developed to combat th is insect pest in orde r 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 enda ngered species (BPPD, 2007i; BPPD, 2007j). Based on a recently-published laboratory study showing reduced growth in shedding caddis flies exposed to anti-lepidopteran Cry1A protein corn litter (Rosi-Marshall, et al. 2007), however, BPPD will require additional aquatic invertebrate data. A 21-day Daphnia study, per the 885 Series Office of Prevention, Pesticides, and T oxic Substances (OPPTS) Guidelines, will be submitted as a condition of registration.

In addition to Monsanto’s submitted rationale for environmental benefits, Monsanto could have included a reference to MON 89034 use poten tially reducing the amount of conventional pesticides applied in the environment and th e 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 subs tantially fewer applications of chemical pesticides, compared to production of non- Bt corn. Fewer chemical insecticide applications generally result in increased populations of benefi cial 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 Cry1A .105 and Cry2Ab2 proteins produced in MON 89034, the introduction of MON 88017 results in production of Cry3Bb1Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

133 protein. BPPD has already reviewed human ri sk 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 chil dren (BPPD, 2007g). Additionally, Cry3Bb1 currently has an exemption from tolerance established under 40 CFR 174.518.

BPPD also agrees with the claim that there s hould be a reduction of some pesticide use (BPPD, 2001). Additionally, Cry3Bb1 protein primarily protects corn plants against CRW. Virtually all of the registered conventional in secticides 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 c onventional 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 produc tion of the Cry1A.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-ta rget organisms, including mammalian wildlife species, aquatic species, avian species, non-target insects, and endangere d 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 Cry3Bb1 variant or to an artificial diet containing a Bt -produced Cry3Bb1 variant. Results indi cated that the Cry3Bb1 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 intera ction between Cry1A.105, Cry2Ab2, and Cry3Bb1 proteins in the pyramided product would not ch ange the overall proper ties of each individual component. BPPD concluded that the activ ity of Cry1A.105 and Cry2Ab2 proteins was not significantly altered by the pr esence of Cry3Bb1, and the ac tivity of Cry3Bb1 was not significantly altered by the pres ence of Cry1A.105 and/or Cry2A b2. The study, along with the previously reviewed interaction study between Cry1A.105 a nd Cry2Ab2, indicated that MON 89034 x MON 88017 corn should not result in any unexpected interaction with regards to target and non-target in sects (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 be nefits. BPPD believes that

cultivation of MON 89034 x MON 88017 corn ma y 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, co mpared to production of non- Bt corn. The reduction in conventional pesticide use should essentially be the sa me seen from MON 88017 and MON 863 use (BPPD, 2003). Fewer chemical insecticide applications generally result in increased populations ofBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

134 beneficial organisms that contro l secondary pests, such as aphi ds and leafhoppers. Furthermore, all of the conventional pesticides used for CRW control or suppression ca use significant adverse

environmental effects under condi tions of normal use (BPPD, 2003).

4. Insect Resistance Management (IRM)

a) Monsanto’s Summary (MRID 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 pr oduces Cry1A.105 and Cry2Ab2 proteins, has two different modes of action agai nst lepidopterans, particularly in the way the proteins bind to the midgut. Therefore, based on the distinct m odes 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 bette r than one for reducing the risk of insect resistance to MON 89034. But, due to un certainties in the data submitted to support the MON 89034 IRM plan, a 5% refuge cannot be esta blished until additional data is submitted to support such a reduction. Instead, BPPD recomme nds 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 regi ons of the southeastern U.S. where a 50% refuge has been previously re quired (BPPD, 2007 b; 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 result ing economic benefits to growers). These benefits can likely be achieve d without an unreasonable ri sk of resistance to Cry1A.105 and Cry2Ab2. Additional grower benefits may be realized in th e 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 registra tion of MON 89034. These studies demonstrate the efficacy of MON 89034 corn and the individual Bt proteins (Cry1A.105 and Cry2Ab2) against a range of lepidopteran corn pests incl uding European corn borer (ECB), corn earworm (CEW), southwestern corn borer (SWCB), fall armyworm (FAW), and sugarcane borer (SCB).Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

135 MRID 46951413 In laboratory bioassays, the in secticidal activity of Cry1A.10 5 protein was tested against agronomically important insects from the orde rs 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 di ets containing the appropriate doses of 50 or 100 μg Cry1A.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 μg Cry1A.105/mL of insect diet in diet-incorporation bioassays for western tarnished plant bug and green peach aphid. Mortality a nd a reduction in weight or ho neydew production over a five or seven day period depending on the insect were the endpoints used to indicate insecticidal activity. The 50 μg/mL and 100 μg/mL Cry1A.105 c oncentrations caused a range of 32 to 96% mortality in three (corn earworm, European corn borer, and fall armyworm) of the four lepidopterans. All four lepi dopteran insects had a range of 32-100% stunting. The Cry1A.105 protein had activity against al l four lepidopteran insects, but no activity against the two coleopteran or two hemipteran insects tested. Classification: Acceptable

MRID 46951414 In laboratory bioassays, the insecticidal ac tivity of Cry2Ab2 protein was tested against agronomically important insects from the orde rs 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 di ets containing the appropriate doses of 50 or 100 μg Cry2Ab2/mL of insect diet in the diet-overlay bioassays fo r fall armyworm, black cutworm, European corn borer, corn earworm, Southern corn rootworm, and boll weevil or 40 or 80 μg Cry2Ab2/mL of insect diet in diet-incorporation bioassays for western tarnished plant bug and green peach aphid. Mortality a nd a reduction in weight or ho neydew 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 μg/mL and 100 μg/mL Cry2Ab2 concentrations caused at least 61% mortality (co rrected) 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 mortalit y resulting from testing against the 50 μg/mL Cry2Ab2 concentration failed to meet the study criterion of >30% mortality. The Cry2Ab2 protein had activity against al l four lepidopteran insects, but no activity against the two coleopteran or two hemipteran insects tested. Classification: Acceptable

MRID 46951415

Field trials were conducted in 2003-2004 season s in Puerto Rico, the United States and Argentina to determine the efficacy of M ON 89034 corn (Cry1A.105 and Cry2Ab2) and MON 89597 (Cry1A.105 and Cry2Ab2) corn against Eur opean corn borer (ECB), corn earworm (CEW), southwestern corn borer (SWCB), fall armyworm (FAW), and sugarcane borer (SCB). Across all geographies the efficacy of th e MON 89034 and MON 89597 against ECB, SWCB, CEW, FAW, and SCB was equal to or greater than that of Yi eldGard® Corn Borer (MON 810), a lepidopteran control corn product that expres ses the Cry1Ab protein. For all geographiesBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

136 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 significantly better control of CE W, 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.

III. REGULATORY POSITION FOR Cry1A.105, AND Cry2Ab2

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

Pursuant to FIFRA section 3(c)(7)(C), EPA ma y conditionally register a new pesticide active ingredient for a period of time reasonably su fficient for the generation and submission of required data that are lacking because insuffici ent time has elapsed since the imposition of the data requirement for those data to be devel oped. EPA may grant such conditional registration only if EPA determines that (1) the use of the pesticide product dur ing the period of the conditional registration will not cau se any unreasonable adverse effect on the environment, and (2) the registration and use of the pesticide during the conditiona l 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 im position of the data requirements for:

1) an independent lab validation of the analytical method for th e detection of Cry2Ab2 and/or Cry1A.105 to satisfy residue analytical me thod 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, repres enting the functional group of

a leaf shredder in headwater streams..

3) additional information on cross-resistance of Cry1A.105 and Cry1Fa and Cry1Ac (preferably including binding site models and us e of resistant colonies) for the target pests

and determine how such cross-resistan ce may impact the durability of MON 89034

4) baseline susceptibility studi es and/or a discriminating concen tration assay that are required for the Cry1A.105 protein against ECB, SWCB, and CEW and for the Cry2Ab2 protein against SWCB, CEW.

5) baseline susceptibility studi es to support sweet corn uses that must be conducted on FAWBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

137 populations collected from sweet corn growi ng 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 monitori ng of the collected populations of FAW for changes in susceptibility to the Cry1A.105 and Cry2Ab2 proteins.

The applicants submitted or cited data suffi cient for EPA to determine that conditional registration of Bacillus thuringiensis Cry2Ab2 and Cry 1A.105 proteins and the genetic material necessary for their production in event M ON 89034 field corn and sweet corn under FIFRA 3(c)(7)(C) will not result in unreason able adverse effects to the environment, as discussed above. The applicants submitted and/or cited satisfact ory data pertaining to the proposed use. The

human health effects data and nont arget organism effects data ar e considered sufficient for the period of the conditional registra tion. These data demonstrate that no foreseeable human health hazards or ecological effects are likely to arise from the use of the produc t 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 1A.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 re sult 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 sp ectrum of protection against primary and secondary corn pests, which should facilitate greater grain quality, a reduction of mycotoxin contamination, increased yield and ultimately ha ve positive implications for human health.

In view of these minimal risks a nd the clear benefits related to 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 , EPA believes th at 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 ingred ient are satisfactory, they are not sufficient to support an unconditional registra tion 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 speci fied 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 andBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

138 Cry 1A.105 proteins and the ge netic 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 regist ration under section 3(c )(7)(C) of the Federal Insecticide, Fungicide, and Roden ticide Act (FIFRA) have been re viewed 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 e ffects to man or the environment and is in the public interest.

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

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

Section 3(c)(7)(A) of FIFRA provides for the regi stration or amendment of a pesticide when the pesticide and proposed use “...are id entical or substantially simila r 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 environmen t, 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 FI FRA 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 increa se the risk of unreasonable a dverse effects; and (3) approving the registration would not significantly increase the risk of any unreasonable adverse effect.

The Agency concludes that the following Cry1A.105 and Cry2AB2 corn product registrations, set to expire on September 30, 2010 and describe d in-depth throughout th is BRAD, meet both criteria (1) and (2):

(1) Event MON 89034 with Cry1A.105 and CryAb2 (EPA Reg. No. 524-575) (2) Events MON 89034 with M ON 88017, Cry1A.105, Cry2Ab2, and Cry3Bb1 (EPA Reg. No. 524-576)

All of these Cry1A.105 and Cry2AB2 corn product s are identical in both composition and use (field corn) to plant-incorporated protectants that are currently re gistered. 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 cu ltivation of Cry1A.105 and Cry2AB2-containing

corn will not cause unreasonable adverse effects on the environment. The conditional environmental effects data, submitted in respons e to terms and conditions of registration andBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

139 summarized in section II(C)(2)(b) of this BRAD, strengthen the Agency ’s initial position and also confirm that long-term effects on non-targ et organisms are not anticipated. Lastly, the continued use of these products will likely still provide many of the benefits as were evaluated in section II(E) of this BRAD to support th e 2005 registration of these products.

In conclusion, as the expiring Cry1A.105 and Cry2A B2 products have met the required criteria under section 3(c)(7)(A) of FIFRA, the Agency is proposing to amend these registrations to extend their respective expiration dates as follows:

Product Name (EPA Reg. No.) Proposed Expiration Date

Event MON 89034 with Cry1A.105 and Cry2Ab2 September 30, 2022

Events MON 89034 with MON 88017, Cry1A.105, Cry2Ab2, and Cry3Bb1 September 30, 2015

Although data provided were satisfactory to make the determinations required by section 3(c)(7)(A) of FIFRA, they we re not sufficient to support an unconditional registration under FIFRA section 3(c)(5). Additional data, specifically in relation to aquatic invertebrate testing and 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 proposed amendments.

C) Period of Registration

In the 2001 BtCorn reassessment, EPA determined that it was appropriate to amend the thenexisting registrations to extend the period of regi stration of those products to an expiration date of October 15, 2008. All of the products being a ssessed at that time were efficacious against lepidopteran pests. EPA based this action on the finding that use of Cry1A.105 and Cry2AB2 expressed in corn will not si gnificantly increase the risk of unreasonable adverse effects on the environment "for the limited time period of 7 additional years (to Oc tober 15, 2008)." These registrations were later amended to extend the pe riod of registration to an expiration date of September 30, 2010. EPA subsequently grante d time-limited registrations to products efficacious against coleopteran corn rootworm pe sts. For example, EPA registered Cry3Bb1 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 require ments intended to countermand the potential development of resistance. Registrants similarl y have been busily developing various products, product mixes (i.e., so-called "pyramids" and "st acks"), and resistance strategies, to maximize agronomic benefits and address resistance manageme nt issues. The result has been a vast arrayBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

140 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 lo w as to guarantee that ad equate natural refuges would be available from neighboring non- Bt corn fields. From 1995 to 1997, Bt corn registrations included voluntary re fuge 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 modifi cations, 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: fa rmers were required to plant a 20% refuge that could be treated for insects, or a 50% treated re fuge in cotton-growing ar eas; 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 differe nt combinations of "pyramided" products (i.e., products containing two or more toxins efficacious against the same pest) and "stacked" products (i.e., products combin ing toxins efficacious ag ainst different pests), the refuge requirements have begun to vary. For example, certain products require a 20% external refuge; some products permit a 5% ex ternal refuge; one produc t incorporates a 10% seed blend refuge; we have applications in proc ess for products that prop ose to incorporate a 5% seed blend refuge; and other permutations are possible.

Given the profusion of various toxin combinati ons and refuge options, we can no longer proceed on the basis that, as concerns in sect resistance management, all products are equal. It was a relatively simple proposition when the default requi rement of a 20% sprayed refuge applied to

almost all of the Bt corn crops in the market. Under thos e circumstances, the relative durability of products against the development of resist ance 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 e volving regulatory approach to th e continually evolving product mix wrought by developers, we think it appropriate to revise ou r regulatory requirements in scientifically defensible ways to reflect the actual le vel of risks posed by the products that we regulate. Here, for example, where we've determ ined 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 pr oduct, or category of products, a registration for a period greater than that grante d a corresponding product that poses a greater risk of insect resistance developing. This approach is reflective of complement ary principles: first, to ensure that we apply our limited resources to the products that pose greater risk of adverse effects to theBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

141 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 propose to follow will include registration periods of five, eight, and twelve years; a fifteen year registrati on period will also be available, if adequately supported by our science assessment. In this scheme, (i) a produc t with a single PIP toxin, and a 20% external refuge, qualifies for a five year registration; (ii) a product with pyr amided PIP toxins (i.e., two or more 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 registrati on; (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 base line single toxin product with a 20% external refuge, would qualify for a fifteen year regist ration. 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 regi stration period for such products will be determined on a case-by-case basis consistent with the leve l of risk they pose. Similarly, instances where other risk issues ma y arise, or where novel resistance concerns may be present, would also be dete rmined on a case-by-case basis.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

142 IV. BIBLIOGRAPHY : Citations Considered as part of the Data Base Supporting the Registration of MON 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 Perm it to Test ZMIR245 x MON 88017 Combined Trait Corn Along with ZMIR245 and M ON 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 Cry1A.105 Protein: Final Report. Project Number: EUF 00081, 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 Cry1A.105 Protein Utilizing the AD5, Toxin5, and Allpeptides Da tabases: 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) Assessmen t of the In Vitro Dige stibility of the CryA.105

Protein in Simulated Gast ric 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) Immunode tection of Cry2Ab2 and Cry1A.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 Susceptib ility of Corn Lepidoptera Pests to CrylAb and Cry1F Proteins: 2005 Monitoring Results. Report submitted to EPA by the IRM Technical Subcommittee of the Agricultu ral Biotechnology Stewardship Technical

Committee.

MRID: 46951300Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

143 Citation: Monsanto Company (2006) Submissi on of Product Chemistry, Efficacy, Exposure and Risk Data in Support of the Applicat ion for Registration of MON 89034 x 88017.

Transmittal of 6 Studies.

MRID: 46951301 Citation: Bogdanova, N. (2006) Human Health and Environmental Assessment of the PlantIncorporated Protected Bacillus th uringiensis Cry1A.105, Cry2Ab2 and Cry3Bb1

Proteins Produced in Insect-Protect ed 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 Analys is. Project Number: 06/01/50/03, MSL/20145. Unpublished study prepared by Monsanto Company. 22 p.

MRID: 46951303 Citation: Hartmann, A.; Niemeyer, K.; Silva novich, A. (2006) Assessment of the Cry1A.105,Cry2Ab2, Cry3Bb1, and CP4 EPSPS Protein Levels in Selected Tissues of InsectProtected 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 8903 4 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 Pote ntial for Interactions Between the Bacillus thuringensis Protei ns Cry1A.105, Cry2Ab2, and Cry3Bb1. Project Number: 05/01/39/19, MSL/20270. Unpublished st udy prepared by Monsanto Company. 26 p.

MRID: 46951306 Citation: Head, G. (2006) Insect Resistance Ma nagement Plan for the Combined Trait Product MOON 89034 x MON 88017. Project Numb er: 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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

144 MRID: 46951401 Citation: Bogdanova, N. (2006) Human Health and Environmental Assessment of the PlantIncorporated Protectant Bacillus thur ingiensis Cry1A.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.; Silva novich, A. (2006) Assessment of the Cry1A.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 Grai n of MON 89034 and Comparison of the Physicochemical and Functional Properties of the Plant-Pro duced 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) Ev aluation of the Functiona l Equivalence of the Cry2Ab2 Protein Produce d in E. coli and Bt Against a Sensitive Lepidopteran Species. Project Number: 05/01/39/23, MSL/20132. U npublished 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) Assessmen t 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.

MRID: 46951408Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

145 Citation: Kapadia, S.; Rice, E. (2005) Assessmen t of the in vitro Digestibility of the Cry1A.105

Protein in Simulated Intestinal Flui d. 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 Flui d. 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 Cry1A.105 Protein Utilizing the AD6, T OXIN5, and ALLPEPTIDES Data bases. Project Number: 20351, 06/01/62/04. Unpublished study prepared by Monsanto Company. 550 p.

MRID: 46951411 Citation: McClain, J.; Silvanovi ch, A. (2006) Bioinformatics An alysis of the Cry2Ab2 Protein Utilizing the AD6, TOXIN5, and ALLPEPTIDE S Databases. Project Number: 20307, 06/01/62/01. Unpublished study prepared by Monsanto Company. 361 p.

MRID: 46951412 Citation: Davis, S. (2006) Comparison of Br oiler Performance and Carcass Parameters When Fed Diets Containing MON 89034, Control or Co mmercial 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. (200 6) Spectrum of Insecticidal Activity of Bacillus thuringiensis Cry1A.105 Protein. Proj ect Number: 04/01/39/27, MSL/20230. Unpublished study prepared by Monsanto Company. 18 p.

MRID: 46951414 Citation: Macrae, T.; Brown, C.; Levine, S. (200 6) 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 M ON 89597 Against Major Insect Pest in the United States, Puerto Rico and Ar gentina During 2003-2004 Seasons. Project Number: 05/RA/39/05. Unpublished study prepared by Monsanto Company. 18 p.

MRID: 46951416Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

146 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 Cry1A.105 Protein in an Acute Study with the Earthworm in an Artificial Soil Substrate: Final Report. Proj ect 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 Cry1A.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 Cry1A.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 Po tential Dietary Effects of Cry1A.105 Protein on the Ladybird Bee tle, 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 Po tential 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 Cry1A.105 Protein on Minute Pirate Bugs, Orius insidiosus (Hem iptera: Anthocoridae). Project Number:Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

147 252/6201, SE/2005/074, MSL/20170. Unpublished study prepared by Springborn Smithers Laboratories. 105 p.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

148 MRID: 46951424 Citation: Teixeira, D. (2006) Evaluation of Potential Dietar y Effects of Cry2Ab2 Protein on Minute Pirate Bugs, Orius insidiosus (Hem iptera: 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 Cry1A.105 Protein in an Acute Study with the Parasitic Wasp, Ichneumon promissorius (Hymenoptera : Ichneumonidae). Projec t 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). Projec t 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) Ev aluation of Potential Di etary 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 Cry1A.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 Resistan ce Management Plan for Second Generation Lepidopteran-Protected Cor n, MON 89034. Project Number: 06/RA/39/06. Unpublished study prepared by Monsanto Company. 98 p.

MRID: 47079400 Citation: Monsanto Co. (2007) Submission of E fficacy Data in Support of the Application for Registration of MON 89034 X 88017. Transmittal of 2 Studies.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

149 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 InsectProtected Corn MON 89034, MON 89034 x NK603, and MON 89034 x MON 88017 Against Major Insect Pests in the Field Tr ials 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 Regist ration 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-prot ected 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 Anal ysis. Project Number: 06/CR/172/3, BR/ME/0878/01, BR/ME/0094/01. Unpublished st udy 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. Proj ect Number: 06/CR/172/3, BR/ME/0878/01, BR/ME/0094/01. Unpublished study prepar ed by Monsanto Company. 78 p.

MRID: 47127504 Citation: Hartmann, A.; Niemeyer, K.; Silv anovich, A. (2006) Assessment of Cry1A.105, Cry2Ab2, Cry3Bb1 and CP4 EPSPS protein Levels in Selected Tissues of INsectprotected 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.

MRID: 47127505Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

150 Citation: Hartmann, A.; Niemeyer, K.; Silva novich, A. (2006) Assessment of the Cry1A.105 and Cry2Ab2 Protein Levels in Tissues of Insect-protected Corn MON 89034 Produced in 2005 U.S. Field Trials. Proj ect Number: 06/CR/172/3, AG/EQ/1023/01, BR/ME/1026/01. Unpublished study prepar ed 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. U npublished 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 USDA, 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 USDA, ARS Southern Insect Management Laboratory. 16 p.

MRID: 47243700 Citation: Monsanto Company (2007) Submi ssion of Residue Data in Support of the Experimental Use of Bacillus thuringien sis Cry1A.105 and Cry2Ab2 Proteins and the Genetic Materials Necessary for its Produc tion in Corn. Transmittal of 4 Studies.

MRID: 47243701 Citation: Murphy, J.; Stillwell, L. (2007) Cry1A.105, Cry2Ab2, Cry3Bb1, 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 Su pport of an Application for an Experimental Use Permit. Project Number : 07/CR/185E/2. Unpublished study prepared by Monsanto Company. 13 p.

MRID: 47243702 Citation: Phillips, A. (2007) Cry34Ab1, Cry35Ab1, Cry1F 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: InterimBacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

151 Report. Project Number: 061026/01. Unpublis hed 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 Tole rance for Bacillus thuringiensis Cry1A.105

Insecticidal Protein and the Ge ntic 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 Tole rance for Bacillus thuringiensis Cry2Ab2

Insecticidal Protein and the Ge netic Material Necessary for its Production When Used as a Plant-Incorporated Protectant in Food a nd 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. Proj ect 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 Cry1F-resistant and susceptible European corn borer and fall armyworm colonies to Cry1A.105 and Cry2Ab2. Attached as Appendix 1 of the In sect Resistance Management Plan for MON

89034 x TC1507 x MON 88017 x DAS-59122-7.

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.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

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Additional Insect Resistance Ma nagement Assessment References:

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Additional Benefits Assessment References:

BPPD, 2001. Biopesticides Registration Action Document – Bacillus thuringiensis PlantIncorporated Protectants. Available at: http://www.epa.gov/oppbppd1/biopesticides/pips/ Bt _brad.htm

BPPD, 2003. Benefits review for MON 863 corn. E. Brandt, S. Matten, and A. Reynolds memo to M. Mendelsohn, 02/24/2003.

BPPD, 2005. Review of proposed in sect resistance management pl an and benefits information submitted by Monsanto for MON 88017 corn. A. Reynolds and S. Matten memo to M. Mendelsohn, 10/13/2005.

BPPD, 2007a. Review of Monsanto Company’s assessment of the efficacy of lepidopteranprotected corn MON 89034 and MON 89597 during the 2003 and 2004 seasons. S. Matten memo to S. Cerrelli, 10/30/2007.

BPPD, 2007b. Review of proposed insect resist ance management plan submitted by Monsanto

for MON 89034 x MON 88017 Bt corn. A. Reynolds memo to S. Cerrelli, 11/29/2007.

BPPD, 2007c. Product performance of Bt Cry1A.105 protein (M RID 469514-13). Data

Evaluation Record from E. Lewis to S. Matten, 07/23/2007.

BPPD, 2007d. Product performance of Bt Cry2Ab2 protein (MRID 469514-14). Data Evaluation Record from E. Lewis to S. Matten, 07/20/2007.

BPPD, 2007e. Review of human health and produ ct characterization data for registration of B. thuringiensis Cry1A.105 and Cry2Ab2 proteins and the ge netic material necessary for their production in MON 89034 corn. R. Edelstein memo to S. Cerrelli, 11/07/2007.

BPPD, 2007f. Review of “Evaluation of Potential Interactions between the Bacillus thuringiensis Proteins Cry1A.105, Cry2Ab2, and Cry3Bb1” for Monsanto’s MON 89034 x MON

88017 maize. M. Hunter memo to S. Cerrelli, 12/14/2007.

BPPD, 2007g. Cry3Bb1 Corn Biopesticide Regi stration Action Document. Available at: http://www.epa.gov/oppbppd1/biopesticides /ingredients/tech_docs/brad_006484.htm

BPPD, 2007h. Technical review of Monsanto Company’s submissions regarding insect resistance management for MON 89034 corn. S. Matten and A. Reynolds memo to S. Cerrelli, 11/06/2007.Bacillus thuringiensis Cry1A.105, and Cry2Ab2 Protein in Corn Biopesticide Registration Action Document

165 BPPD, 2007i. Review of non-ta rget invertebrate studies for MON 89034 maize containing Cry1A.105 and Cry2Ab2 proteins. M. Hunter memo to S. Cerrelli, 12/14/2007.

BPPD, 2007j. Review of broiler, quail, soil fate , and endangered species data packages submitted in support of MON 89034 Bt corn, T. Milofsky to S. Cerrelli, 12/14/2007.

Crawford, A. and Bogdanova, N., 2007. Public in terest document supporting registration of Bacillus thuringiensis Cry1A.105, Cry2Ab2 and Cry3Bb1 pr oteins and the genetic material necessary for their productions in insect-protected corn MON 89034 and MON 89034 x MON 88017. Report submitted by Monsanto Company MRID 472797-01.

Rosi-Marshall E.J., J.L. Tank, T.V. Royer, M.R. Whiles, M. Evans-White, C. Chambers, N.A. Griffiths, J. Pokelsek, and M.L. Stephen. (2007). Toxins in transgenic crop byproducts may affect headwater stream ecosystems. PNAS, vol. 104, no. 41, 16204–16208.

Wu, F. (2008). Field Evidence: Bt Corn and Mycotoxin Reduction. Available at: http://www.isb.vt.edu/news/2008/feb08.pdf