BIOPESTICIDES REGISTRATION ACTION DOCUMENT

Modified Cry3A Protein and the Genetic Material Necessary for its Production (Via Elements of pZM26) in Event MIR604 Corn SYN-IR604-8

U.S. Environmental Protection Agency Office of Pesticide Programs Biopesticides and Pollution Prevention Division2 Table of Contents

I. Overview .....................................................................................................................1

A. Executive Summary ........................................................................4

B. Use Profile ....................................................................................8

II. Science Assessment ....................................................................................................7

A. Product Characterization ......................................................................................10

B. Human Health Assessment ...................................................................................23

C. Environmental Assessment ...................................................................................34

D. Insect Resistance Management .............................................................................69

E. Benefits and Public Interest Finding ........................................................... .........121

III. Regulatory Position for Event MIR604 Corn ............ ................................136Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

3 Modified Cry3A protein and the Genetic Material Necessary for its Production (Via Elements of pZM26) in Event MIR604 corn SYN -IR604-8 Corn Regulatory Action Team

Product Characterization and Human Health

John Kough, Ph.D. Annabel Waggoner

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. Tessa Milofsky, M.S.

Mika Hunter

Benefit Assessment

Tessa Milofsky, M.S. Sharlene Matten, Ph.D.

Registration Support

Mike Mendelsohn Matt Thompson

Ann Sibold

Biopesticides Registration Ac tion Document Team Leader

Mike Mendelsohn

Office of General Council

Angela Huskey, Esq.4 I. Overview

A. Background

On October 3, 2006, EPA conditionally registered (w ith an expiration date of September 30, 2010), Syngenta Seeds Inc.’s active ingredient, modified Cry3A protein and the genetic material necessary for its production (via elements of pZM26) in event MIR604 corn SYN-IR 604-8. Additionally, on January 24, 2007, the Agency conditionally registered (with an expiration da te of October 15, 2008, subsequently extended to September 30, 2010) S yngenta Seed Inc.’s stacked product MIR 604 and Bt11 with mCry3A and Cry1Ab. The Agency determined that the use of these pesticides was in the public interest and that they would not cause any unreasonable adverse effects on the environment during the time of conditional registration.

Results of efficacy trials conducted in 2002, 2003, and 2004 indicated that MIR604 corn provides effective control of key rootworm pests of field corn. MIR604 corn has unique biochemical properties which may benefit insect resistance management fo r this and other CRW-protected corn products. MIR604 and Bt 11 with mCry3A an d Cry1Ab effectively controls ke y rootworm pests and European corn borers. The availability of multiple CRW- protected corn products and CRW/CB-protected corn products will increase grower choice and price competition, resulting in lo wer seed prices for consumers and higher adoption rates. Registration of these products is expected to result in further

reduction of chemical insecticide us e by growers. This is of special importance since many pesticides registered for CRW-control are highly toxic to humans and the environment, while mCry3Aexpressing corn poses no foreseeable human health or environmental risks.

In order to reduce the possibility of corn rootworm or European corn borer developing resistance to Bt, EPA required Syngenta Seeds, Inc. to ensure that 20 percen t of the planted acreage of this product be set aside where non-CRW-protected Bt corn will be gr own to serve as a “refuge .” These refuge areas will support populations of corn root worm not exposed to the Bt cor n. The insect populations in the refuges will help prevent resistance development when they cross-breed with insects in the Bt fields. This resistance management strategy was develope d as a condition of the registration, and EPA will require routine monitoring and documentation that these measures are followed. The submitted insect resistance management data support a registration until 2010.

A tolerance exemption (40 CFR Part 174.505) was established for Bacillus thuringiensis modified Cry3A protein and the genetic material ne cessary for its production in corn.

B. Executive Summary

On October 1, 2009, EPA announced a new policy to pr ovide a more meaningful opportunity for the public to participate on major regist ration decisions before they occur. According to this new policy, EPA intends to provide a public comment period pr ior to making a registration decision for, atModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

5 minimum, the following types of applications: new active ingredients; first food uses; first outdoor uses; first residential uses; and other actions for which the Agency anticipates that there will be significant public interest.

Consistent with the policy of ma king registration actions more tran sparent, the proposed amendments to the expiring mCry3A corn products are subjec t 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 decision on amending the expiring mCr y3A corn products is c ontingent upon review and consideration of public comments received, the Agency maintains that, based on all data submitted in support of the expiring mCry3A corn registrations (both for initial registrations and as responses to conditions of registration), it is in the best interest of the public and the e nvironment to amend the currently existing mCry3A registrati ons by extending the current expira tion dates. The basis for this preliminary decision can be found in the risk a ssessment for the expiring mCry3A corn products, which is characterized throughout this Biopesticid es Registration Action Do cument (BRAD). After

scientific reviews of mCry3A conditional data were comple ted, this BRAD was updated appropriately

to reflect the most current information availabl e to and evaluated by the Agency regarding these registrations.

All data and findings for the expi ring mCry3A corn products are pr esented 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 th is should be the most familiar format to outside stakeholders interested in providing comments on th ese actions. In addition to the expiring mCry3A corn products, there are other Bt corn PIPs, expressing diffe rent proteins effectiv e in controlling corn borers or corn rootworm, that expire in 2010, and fo r which the associated registrants have formally requested an extension to the cu rrent expiration dates. Therefore, within the same docket (EPAHQOPP-2010-0607) as this document, the following information a is also available for public

examination:

• CrylF and Cry lAb BRAD • Cry34/35Abl BRAD • mCry3Bb1 BRAD • Cry l A.105 and Cry2Ab2 BRAD • Optimum® AcreMax TM 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 expiration dates. We have conducted comprehensive assessments of

a Each of the Biopesticides Registration Action Documents available for public comment in this action is modified from previous versions to account for data/information su bmitted to fulfill terms and conditions of registration6 each of these registrations, considering all toxicity and environmental effects data, data from insect resistance monitoring, and insect resistance refuge compliance reports, received and obtained since the last comprehensive evaluation of these products in 2001. Based upon our comprehensive assessment, we have reached significant conclusions regarding th e positive environmental impact of Bt corn PIPs, and we are proposing several actions to strengthen the in sect 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-registration fi eld studies required to be submitted to the Agency, have demonstrated that non-target invert ebrates are generally more abundant in Bt cotton and Bt corn fields than in non-transgenic fiel ds managed with chemical insec ticides. Thus, these published and registrant-produced studies dem onstrate that, not only are the Bt crops not causing any unreasonable adverse effects in the environment, but, arthr opod prevalence and dive rsity is greater in Bt crop fields. Although no effects have been observed in the envir onment, the Agency is requiring additional aquatic invertebrate data in light of pub lished laboratory studies showing re duced growth in shredding caddis flies exposed to Cry1A protein corn litter.

To strengthen insect resistance management of thes e corn PIPs and to address reports that compliance with the mandated refuge requirements has been decreasing, EPA is proposing to require enhanced compliance assurance programs (CAPs), and a phased requirement for seed ba g labeling that clearly shows the refuge requirements. Al so, given the increasing variety of PIP products and combinations, and the differing risk of resistance evolution that the various products represent, we are proposing to grant registrations for the corn PIP products for different timeframes, based on assessments of their likelihood of forestalling the evolut ion of insect resistance. We are registering differing categories of products for differing time periods to reflect the assessed level of risk of resistance posed by the various corn PIP products. The scheme that we propose to follow will include registration periods generally of five, eight, and twelve years; with the possibility of a fifteen year registration period for products that are demonstrated to m eet specified criteria. We retain, however, the discretion to register products for time periods differing from thes e defaults where circumstances warrant.

Product Characterization

The modified Cry3A (mCry3A) Bacillus thuringiensis ( Bt ) insect control protein is produced in transgenic corn plants derived from transforma tion Event MIR604 and has activity against certain

beetles. A cry3A gene from Bt subsp. tenebrionis was recreated synthetically to optimize for expression in corn. Additional changes in this corn-optimized gene were made, such that the encoded mCry3A protein has enhanced activity against larvae of the western corn root worm and northern corn

rootworm.

Protein characterization data dem onstrate that the plant-produced pr otein is of sufficiently similar biological activity to that of the two modified Cry3A protein variants produced in the recombinant E. coli system (designated as test material MCRY 3A-0102) for the purposes of human health and ecological effect risk assessments. Although the MCRY3A-0102 test material was not as active towards target pests as the plant-produced modified Cry3A protein, the doses in submitted studies wereModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

7 much higher than would occur via the modified corn.

Bt corn Events MIR 604 and Bt11 with modified Cry3A and Cry1Ab is a stacked product that has activity against the rootworm vari eties previously mentioned and Eu ropean corn borer. The product characterization and protein expressi on analyses of Bt Cry1Ab and mCr y3A insect control proteins and the genetic material necessary fo r its production in maize (corn) pl ants derived from Event Bt11 x MIR604 hybrid cross via traditi onal plant breeding were found to be similar and functionally equivalent to the Cry1Ab protein expressed in Bt11 and to mCry3A protein expressed in MIR604.

Human Health Assessment

There is a reasonable certainty that no harm will result from aggregate exposure to the U.S. population, including infants and children, to the mCry3A protein and the gene tic material necessary for its production, or MIR 604 and Bt11 with mCry3A and Cry1Ab.

An acute oral toxicity study was submitted fo r the mCry3A protein and MIR 604 and Bt11 with mCry3A and Cry1Ab. Both studies support the prediction that the products w ould be non-toxic to

humans.

Amino acid sequence comparisons showed no similar ity between the mCry3A protein or MIR 604 and Bt11 with mCry3A and Cry1Ab to known toxic proteins available in public protein databases.

Amino acid sequence comparisons showed no similar ity between the mCry3A protein or MIR 604 and Bt11 with mCry3A and Cry1Ab to known allergens available in public alle rgen databases.

Environmental Assessment

The Agency is aware of no identi fied significant adverse effects of mCry3A proteins or MIR 604 and Bt11 with mCry3A and Cry1Ab on the abundance of non- target beneficial organisms in any population in the field environment, whether they are pest para sites, pest predators, or pollinators. Further, the EPA believes that cultivation of these products may have fewer adverse impacts on non-target organisms than use of chemical pesticides for corn production, because under normal circumstances, these products require substantially fewer applications of chemical pesticides, compared to production

of non-Bt corn. And fewer chemical insecticide applications genera lly result in increased populations of beneficial organisms that control secondary pests, such as aphids and leaf hoppers, in corn fields.

In addition, no adverse effect on e ndangered and threatened species lis ted by the US Fish and Wildlife Service is expected from the proposed MIR604 CRW resistant corn registration. Further, the EPA has determined that there is no significa nt risk of gene capture and expr ession of mCry3A protein or MIR 604 and Bt11 with mCry3A and Cry1Ab by wild or weedy relatives of co rn in the U.S., its possessions, or territories, availabl e data do not indicate th at Cry proteins have any measurable adverse8 effect on microbial populations in th e soil, nor has horizonta l transfer of genes from transgenic plants

to soil bacteria be en demonstrated.

Insect Resistance Management

The proposed IRM strategy and data to support it are “acceptable” except that the in-field strip refuge

must be at least 4 rows wide based on recent larval movement data. If resist ance is recessive, then the proposed IRM plan using a 20% structured refuge will be adequate to delay resistance for at least 15 years given the assumptions of Syngenta’s model. If MIR604 maize is planted in areas with observable rotation-resistance in WCRW, then planting tr ansgenic corn only in rotated maize fields is a good IRM strategy that will delay the evolution of resistance by at least 15 years regardless of gene expression.

Benefits

Registration of modified Cry3A pr otein and the genetic material necessary for its production (via elements of pZM26) in event MIR604 corn S YN-IR604-8 is in the public interest because:

1. Results of efficacy trials conducted in 2002, 2003, and 2004 indicate that MIR604 corn provides effective control of key rootworm pests of field corn.

2. MIR604 corn has unique biochemical propert ies which may benefit insect resistance management for this and other CRW-protected corn products.

3. If MIR604 corn is registered, it will be the third CRW-protected Bt corn product on the market. The availability of multiple CRW-protected corn products will increase grower choice and price competition, resulting in lower seed prices for consumers and higher adoption rates.

4. Registration of MIR604 corn is expected to resu lt in further reduction of chemical insecticide use by growers. This is of sp ecial importance since many pestic ides registered for CRW-control are highly toxic to humans and the environm ent, while mCry3A-expressing corn poses no

foreseeable human health or environmental risks.

B. Use Profile

1. Pesticide Name : Modified Cry3A protein an d the genetic material nece ssary for its production (via elements of pZM26) in event MIR604 corn SYN-IR604-8

Trade and Other Names: Agrisure RW Root worm-Protected Corn; MIR 604 corn

EPA Registration Number: 67979-5

OPP Chemical Code: 006509Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

9 Basic Manufacturer: Syngenta Seeds, Inc,

Type of Pesticide: Plant-Incorporated Protectant

Uses: Field Corn

Target Pest(s): Corn Rootworm.

2. Pesticide Names: Modified Cry3A protein and the genetic material necessary for its production (via elements of pZM26) in event MIR604 corn SYN-IR604-8

and Bacillus thuringiensis Cry1Ab delta-endotoxin and the gene tic material necessary for its production (Plasmid Vector pZ01502) in corn

Trade and Other Names: Bt11 x MIR 604 Corn Seed; Agrisure CB/LL/RW, Bt11 x SYN-IR6Ø4-5

corn seed Bt11 x SYN-IR6Ø4-8 corn seed

EPA Registration Number: 67979-8

OPP Chemical Code: 006509 and 006505

Basic Manufacturer: Syngenta Seeds, Inc,

Type of Pesticide: Plant-Incorporated Protectant

Uses: Field Corn

Target Pest(s): European Corn Borer and Corn Rootworm.

II. Science Assessment

The classifications that are found for each data submission are assigned by the EPA science reviewer

and are an indication of the useful ness of the information contained in the documents and if the data meet the intent of the test guidelines. A rating of “ACCEPTABLE” indicates the study is scientifically valid and has been satisfactorily performed accordi ng to accepted EPA guidelines or other justified criteria. A “SUPPLEMENTAL” rating indicates the data provide some information that can be useful for risk assessment. However, the studies may e ither have certain aspects not determined to be scientifically acceptable (SUPPLEMENTAL, but UPGR ADABLE) or that the studies have not been done to fulfill a specific EPA guideline requirement. If a study is rated as “SUPPLEMENTAL, but UPGRADABLE,” EPA always provides an indication of what is lacki ng or what can be provided to change the rating to “ACCEPTABLE.” If there is simply a “SU PPLEMENTAL” rating, the reviewer10 will often state that the study is not required by current EPA guidelines or does not need to be

reclassified as “ACCEPTABLE.” Both ACCEPT ABLE and SUPPLEMENTAL studies may be used in the risk assessment process as appropriate.

II.A. Product Characterization

1. 2007 Position.

The Modified Cry3A (mCry3A) Bacillus thuringiensis ( Bt ) insect control protein is produced in transgenic corn plants derived from transformati on Event MIR604. The Agency’s detailed assessment of the product characterization for Bt mCry3A is found in Fellman (2005a, b, e and 2006a and c). A cry3A gene from Bt subsp. tenebrionis was recreated synthetically to op timize for expression in corn. Additional changes in this corn-optimized gene were made, such that the encoded mCry3A protein has enhanced activity against larvae of the western corn rootworm (WCRM; Diabrotica virgifera virgifera ) and northern corn rootworm (NCRW; D. longicornis barberi ). Introduced via transformation vector pZM26, the mcry3A gene, consisting of 1797 base pairs (bp), was incorporated between an MTL promoter (2556 bp) from the Zea mays metallothionein-like gene and a terminator sequence from the nopaline synthase (NOS) gene of Agrobacterium tumefaciens used to provide a polyadenylation site. An Escherichia coli manA gene (1176 bp) encoding a phosphomannose isomerase (PMI) was incorporated between a promoter region from the Zea mays polyubiquitin gene (ZmUbInt (1993 bp)) and the same NOS terminator sequence described above . This PMI gene, which was introduced along with the mcry3A gene via the same pZM26 transformation vector, encodes the enzyme phosphomannose isomerase (PMI), which is employed as a selectable marker during the process of regenerating plant material followi ng transformation. The PMI protein is a common enzyme involved in carbohydrate metabolism and allows for selection of transf ormants in cell culture by enabling only transformed corn ce lls to utilize mannose as a sole carbon source, while corn cells lacking the pmi gene fail to grow.

The native Cry3A protein of Bt subsp. tenebrionis is a ca. 73 kDa polypeptide of 644 amino acids. By comparison, the mCry3A protein expr essed in Event MIR64 corn is a ca. 67 kDa polypeptide of 598 amino acids. The amino acid sequence of the mCry3A protein corresponds to that of the native Cry3A protein, except: 1) Its N-terminus corresponds to methionine-48 of the native protein; and 2) A cathepsin-G protease recognition si te has been introduced, beginning at amino acid residue 155 of the native protein. This cathepsin-G recognition si te has the sequence alanine-alanine-prolinephenylalanie, and has replaced the amino acids vali ne-155, serine- 156, and serine-157 in the native protein (MRID No. 461556-01, reviewed in Fellman, 2005a).

The molecular properties of the mcry3A gene and the pmi gene, both present in Event MIR604 corn, were evaluated by utilizing Sout hern blotting analyses, DNA sequenc ing of the T-DNA insert, and by studying the Mendelian inheritance of the tran sgene insert (MRID No. 461556-02, reviewed in Fellman, 2005a). Maize genomic DNA was digested with Kpn I restriction enzyme and hybridized to probes for mcry3A (1797 bp) , pmi (1176 bp) and plasmid backbone (5309 bp; not inserted in the host genome). The T-DNA insert (via the pZM 26 plasmid) in MIR604 was analyzed via Southern blot analyses and single copies of mcry3A and the pmi genes were present and cl osely linked in MIR604.Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

11 The results also verified that the backbone DNA se quence from the transformation plasmid pZM26 in

MIR604 was not inserted into MIR604.

DNA sequencing revealed that there was a 44 and 43 bp truncation at the right a nd left break points of the T-DNA insert, respectively, during the transformati on process that resulted in MIR604. Therefore, the overall integrity of the insert and the contiguous ness of the functional elements were confirmed. In addition, T 5 generation plants generated from T 4 generation plants known to be heterozygous for the mcry3A and pmi genes were analyzed by an enzyme-linke d immunosorbent assay (ELISA) of mCry3A and Polymerase Chain Reaction (PCR) analysis for both the mcry3A and pmi genes. Results showed that the two genes were closely linked with the expected Mendelian inheritance ratio.

Expression levels of the mCry3A pr otein in transgenic corn tissues were also provided for both inbred and hybrid varieties (MRID 461556-04, reviewed in Fellman, 2005a) and were found comparable. The data on expression levels in two hybrid s lines are summarized in Table 1.

TABLE 1. mCry3A Levels on a Dry Weight Basis in Event MIR604-Derived Corn Plants

Tissue Genotype Whorl Anthesis Seed Maturity Senescence

mean μ g mCry3A/g dry weight ± S. D. (range)

Leaves MIR604-B Hybrid 18.64 ± 2.08 (16.27 - 20.90) 15.93 ± 3.65 (11.40-20.87) 13.29 ± 2.37 (9.61 - 15.40) 5.05 ± 2.37 (1.69 - 8.24)

MIR604-C Hybrid 25.76 ± 4.11 (22.23 - 31.86) 16.83 ± 2.88 (13.16-21.18) 24.49 ± 3.34 (19.58-28.87) 6.57 ± 2.97 (3.65 - 10.33)

Roots MIR604-B Hybrid 25.50 ± 1.36 (23.55 - 27.07) 15.57 ± 4.35 b
(10.58 - 18.57) 7.29 ± 1.96 (4.99 - 9.03) 10.31± 6.34 (1.68 - 19.36)

MIR604-C Hybrid 19.41 ± 2.17 (16.29 - 21.18) 14.46 ± 2.57 (11.56 - 18.30) 12.96 ± 5.36 (7.10 - 19.46) 9.87 ± 5.43 (5.45 - 17.60)

Kernels MIR604-B Hybrid N/A N/A 1.09 ± 0.45 (0.74 - 1.83) 0.77 ± 0.20 (0.59 - 0.99)

MIR604-C Hybrid N/A N/A 1.95 ± 0.74 (1.26 - 3.13) 0.94 ± 0.47 (0.43 - 1.59)

Silk MIR604-B Hybrid N/A <0.34 (ND-DNQ) 0.99 d N/A

MIR604-C Hybrid N/A <0.56 (DNQ) 3.04 ± 0.78 a (2.31 - 3.92) N/A

Pollen MIR604-B Hybrid N/A ND e N/A N/A

MIR604-C Hybrid N/A ND e N/A N/A

Whole MIR604-B 7.31 ± 3.40 11.16 ± 5.48 16.97 ± 8.24 8.96 ± 3.9612 Plant Hybrid (1.33 - 9.51) (4.23- 16.99) (5.88 - 28.88) (5.03 - 15.40)

MIR604-C Hybrid 9.83 ± 1.83 (8.56 - 13.01) 11.22 ± 2.87 (9.05 - 14.41) 23.77 ± 4.91 a (16.82 - 28.35) 11.85 ± 3.51 (7.44 - 16.78)
Except where noted otherwise, five samples were used to determine the means and standard deviations.

a Four samples were used to determ ine the mean and standard deviation.

b Three samples were used to determ ine the mean and standard deviation.

c Two samples were used to determine th e mean and standard deviation.

d One sample analyzed.

e Result of one pooled sample. N/A Not analyzed at this stage.

ND Not detectable. DNQ. Detectable but not quantifiable

Production of bacterial-derived mCry3A protein was chosen in order to obtain sufficient material for testing, therefore, its equivalen ce to the plant-derived protein wa s determined. The mCry3A protein was produced in recombinant E. coli by over-expressing the same modified cry3A gene that was introduced into Event MIR604 corn plants.

The similarity of mCry3A pr otein expressed in corn event MIR604 and the recombinant E. coli test system (designated as test material MCRY3A -0102) were evaluated (MRID 461556-03, reviewed in Fellman, 2005a). The mCry3A proteins from both sources had the same approximate molecular weight (ca. 67,700 Da.) based on mass spectral analysis. Moreover, West ern blot analysis of the test material showed a single immunoreactive band corresponding to the predicted molecular weight of ca. 67,700 Da. SDS-PAGE showed the purity of mCry3A in the test material to be ca. 90.3% by weight. The mCry3A protein from both sources was imm unologically cross-reactiv e with the same antimCry3A antibody. Both proteins produced comparab le toxicities toward Western Corn Rootworm larvae, based on LC 50 values. There was also no evidence of post-translational glycosylation of mCry3A protein from either source . Therefore, it was concluded that the mCry3A proteins from corn

event MIR604 and from recombinant E. coli were equivalent.

However, further testing revealed that the test material, MCRY3A -0102, contained two closely related components in a ratio of ca. 2:3 via SDS-PAGE and MALDI TOF mass spectrometry (MRID 461556- 06, reviewed in Fellman, 2005a). The lesser of the tw o components, with the lower molecular weight, corresponded to the intended mCr y3A protein with 598 amino acids. The other component contained the same 598 amino acids as the first component but also contained an additional 16 amino acids at the N–terminal end of the protein. To satisfy Agen cy’ concerns with the long form component of mCry3A, Syngenta conducted an in-depth examin ation of the nucleotide sequence of the cry1Ac promoter in the cassette utilized for expression of the mCry3A protein in the MCRY3A-0102 test substance. The registrant reported that an “ATG ” initiation codon sequence was identified within the cry1Ac promoter indicating the presence of an add itional putative open reading frame (ORF). This demonstrated that the resultant putat ive ORF would be in-frame with the mcry3A gene and would

result in an additional 16 amino acids on the N- terminus of the intended mCry3A protein (MRID 464667-01, reviewed in Fellman, 2005b).Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

13 Visual confirmation of the molecular weights for th e long and short forms of mCry3A protein was also provided via SDS-PAGE and western blot analysis. These gels indi cated single intense bands consistent with the predicted molecular weights of ca. 69,500 Da and ca. 67,700 Da for the mCry3ALF (long form) and mCry3A-SF (short form) samples, respectively. Therefore, the identity of the two components was conclusively determined by peptide mapping using tandem (MS/MS) mass spectrometry and by MALDI TOF mass spectrometry of the intact proteins (MRID 461556-07). The molecular weight data showed the two prot eins to have masses of 67,519 and 69,138 Da.

Because both forms of mCry3A were insecticidally active against WC RW and taking into account the high degree of structural homology (97.4% amino acid identity), the two forms of mCry3A in test

material MCRY3A-0102 were consid ered equivalent. Moreover, the te st material was re-analyzed ca.

9 months after its initial characterization and found to be substan tially stable when stored at -20 °C (MRID 461556-05, reviewed in Fellman, 2005a).

On March 14-15, 2006, EPA held a FIFRA Scie ntific Advisory Panel (SAP) meeting, http://www.epa.gov/scipoly/sap/ meetings/2006/index.htm#march to address the scientific issues that arose during the risk assessment of mCry3A. The SAP Report (SAP, 2006) is reviewed in Fellman (2006c). EPA asked the SAP to comment on the equiva lence of the mCry3A proteins from corn event

MIR604 and from recombinant E. coli - specifically the presence of two forms in the bacterially produced mCry3A protein and the di fferences in bioactivity in the WCRW bioassay. The majority of

the Panel concluded that the two fo rms of the mCry3A are of relative ly comparable biological activity for the purposes of the human health assessments based on the amino acid seque nce identity, lack of glycosylation, and general stability.

2. Product Characterization Data. The product characterization data is summarized in Table 2.

Table 2. Product characterization studies (Updated)

MRID Title Summary14 MRID Title Summary

461556-

01 Review of

Characterization and Safety of

Modified Cry3A protein

and maize (corn) plants

derived from

Event MIR604

with comparison to native Cry3A protein The mCry3A protein cont ains 598 amino acids ( ca . 67 kDa), whereas the native Cry3A protein is ca . 73 kDa polypeptide of 644 amino acids. The amino acid sequence of the mCry3A pr otein corresponds to that of the native Cry3A protein, except: 1) Its N-terminus corresponds to methionine-48 of the native protei n; and 2) A cathepsin-G protease recognition site has been introduced, beginning at amino acid residue 155 of the native protein. The susceptib ility of insect pest species and insecticidal properties, such as solubilization, prot eolytic processing, receptor binding, and membrane pore forming properties, were also determined for mCry3A protein and differentiated with native Cry3A protein. The mCry3A protein has a si milar spectrum of activity to the native Cry3A, but with enhanced toxicity to NCRW and WCRW.

Classification: Acceptable

461556-

02 Molecular

characterization

of event

MIR604 maize (corn) expressing a modified Cry3A

bacillus thuringiensis protein Corn Event MIR604 does not contain any of the backbone sequences from the transforming plasmid pZM26. Three nucleotide changes were identified, one in a regulator y region associated with the mcry3A gene and

two in the pmi coding sequence. The mcry3A and pmi genes are closely

linked.

Classification: Acceptable

461556-

03 Characterization

of modified Cry3A protein produced in

event MIR604-

derived maize (corn) and comparison

with modified Cry3A protein expressed in

recombinant E.

coli This study evaluated the similarity of modified Cry3A (mCry3A) insecticidal protein expressed in co rn event MIR604 and mCry3A protein expressed in a recombinant E. coli test system. The mCry3A protein

derived from corn event MIR604 and recombinant E. coli had the same approximate molecular weight (c a. 67,700 Da.) based on mass spectral analysis (from MRID 461556-06). The mCry3A protein from both sources was immunologically cross-reactive w ith the same anti-mCry3A antibody. Both proteins produced comparable toxicities toward Western Corn Rootworm larvae, based on LC 50 values. There was no evidence of posttranslational glycosylation of mCry3A protein from either source. It was concluded that the mCry3A proteins from corn event MIR604 and from

recombinant E. coli were substantially the same.
Classification: AcceptableModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

15 MRID Title Summary

461556-

04 Quantification

of modified Cry3A and PMI proteins in transgenic maize (corn) tissues, whole plants, and silage derived

from

transformation

event MIR604 The plant extracts (including leaves, r oots, kernels, sil k, pollen, silage, and whole plants) from inbred and hybrid corn varieties derived from MIR604 field plants were quantitatively an alyzed for mCry3A by ELISA. The magnitude of expression for tissue ty pes was as follows in descending order: leaves, roots, silage, and kern els. All control tissues were negative for the expression of mCry3A. The mean extraction efficiency for mCry3A over all tissues (except for pollen or si lk tissue, where levels were too low to be determined) was 76.6%. Low, but quantifiable, levels of PMI protein

were found in most of the Event MI R604-derived plant tissues analyzed including pollen. The mCry3A and PMI proteins were stably expressed in four backcross generations in leaf tissue analyzed at anthesis stage. Mean levels across all backcross generations were ca. 11.8 - 15.5 μ g/g dry weight

and 1.1 - 1.3 μ g/g dry weight for mCry3A and PMI proteins, respectively.

Classification: Acceptable

461556-

05 Characterization

of modified Cry3A test

substance mCry3A-0102

and certificate of analysis This study characterized test ma terial MCRY3A-0102, a microbially produced protein preparation cont aining a modified Cry3A (mCry3A) protein. The purity of mCry3A in the test material was shown to be ca. 90.3% by weight using SDS- PAGE analysis. Western blot analysis of the test material showed a single immu noreactive band corre sponding to the predicted molecular weight of ca. 67,700 Da. The test material was insecticidally active and had a 144-hour LC 50 of 1.4 μg/mL diet (95% confidence interval: 0.7 - 2.2 μg/mL) against Western corn rootworm (WCRW) larvae. Two forms of mCry3A were found in the test material, designated mCry3A-SF and mCry3A-L F, respectively, and were both insecticidally active against WCRW. On this basis, and taking into account the high degree of structural homol ogy (97.4% amino acid identity), the two forms of mCry3A in test materi al MCRY3A-0102 were considered to be equivalent. The test material wa s re-analyzed ca. 9 months after its

initial characterization and found to be substantially stable when stored at -

20 °C.

Classification: Acceptable16 MRID Title Summary

461556-

06 Further

Characterization

of Modified Cry3A Test

Substance

MCRY3A-0102 The test material, MCRY3A-0102, cont aining mCry3A protein was shown, by SDS-PAGE and MALDI TOF mass spectrometry, to contain two closely related components in a ratio of ca. 2:3. The lesser of the two components, with the lower molecular weight, corresponded to the intended mCry3A protein with 598 amino acids. The other component

contained the same 598 amino acids as the first component but also

contained an additional 16 amino acids at the N–terminal end of the protein. The identity of the two components was conclusively determined by peptide mapping using tandem (MS/MS) mass spectrometry and by MALDI TOF mass spectrometry of the intact proteins. The molecular weight data showed the two prot eins to have masses of 67,519 and 69,138

Da.

Classification: Acceptable

461556-

09 Analysis for the presence of modified Cry3A protein in wet and dry milled fractions, corn

oil and corn chips from corn (maize) event

MIR604 Among the wet-milled fractions, the me dium fiber (0.46 μg mCry3A/g), fine fiber (0.26 μg mCry3A/g), a nd gluten meal (0.24 μg mCry3A/g) fractions yielded quantifiable amount s of mCry3A. Among the dry-milled fractions the highest con centrations were found in the flaking grits (2.12 μg mCry3A/g), the corn hulls (1.42 μg mC ry3A/g), and the coarse grit (0.92 μg mCry3A/g) fractions. Levels of mCry3A found in the other dry-milled fractions, including fine grits, corn m eal, corn cone and corn flour, were between 0.32 and 0.69 μg mCry3A/g . Although the concentration of mCry3A protein measured in the flour used to prepare the corn chips was 0.32 μg mCry3A /g, no mCry3A protein was detected in the corn chips. Similarly, mCry3A protein was not dete ctable in oil, whereas the starting material, flaking grits, c ontained 2.12 μg mCry3A/g.

Classification: AcceptableModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

17 MRID Title Summary

465974-

01 Analytical

Method for the

Detection of the

PlantIncorporated

Protectant

Modified Cry3A Protein

in Event

MIR604 Corn

Grain and Independent, Third-Party

Validation of

Said Method A monoclonal antibody based enzy me-linked immunosorbent assay (ELISA) method was developed by Synge nta to detect modified Cry3A (mCry3A) protein expressed in MI R604 seed and leaf tissues. An independent, third party la boratory (EnviroLogix, Inc.) utilized the assay methodology, Event MIR604 seed, negative isoline seed, and three lots of mCry3A monoclonal antibody for validati on of assay protocol, sensitivity and cross-reactivity, according to the USDA GIPSA directive. The assay sensitivity was estimated at 1 pos itive Event MIR604 kernel in 999 nonMIR604 kernels (0.1%), which was based on a minimum of 120 ground seed samples (with a LOD of 0.33 ppb for seed material). The average quantification was 2.07 ppb (ng/g fresh we ight corn). No cross reactivity with other commercial, conventional and transgenic corn was detected with the exception of products expressi ng the Cry3Bb1 protein. Moreover, extraction efficiency was determined at 59% for ground seed and 75% for leaf tissue. Therefore, the monoc lonal antibody-based commercial ELISA detection assay (tested by EnviroLo gix, Inc.) satisfies the EPA Residue Chemistry Guidelines OPPTS 860.1340(c )(6) Residue Analytical Methods and PR Notice 96-1. EPA’s Analytical Method Laboratory located in Fort Meade (Maryland) will have to inde pendently validate Syngenta’s ELISA protocol for accuracy, preci sion, and sensitivity.

Classification: Acceptable18 MRID Title Summary

467956-

01 Comparative

Southern Analysis of a

Bt11 x MIR604 Maize Hybrid

with the

Parental Event

Bt11 and Event

MIR604 Maize

Inbreds The purpose of this study was to use S outhern blot analysis to confirm the presence of the cry1Ab and pat genes from the parental Event Bt11 and mcry3A and pmi genes from parental Event MIR604 in the hybrid Bt11 x MIR604 in a predictable manner. For the cry1Ab -specific probe, the Nde I digest resulted in a si ngle hybridization band of ~4.6 kb in both Event Bt11

and Bt11 x MIR604. For the pat -specific probe, the Nde I digest resulted in a single hybridization ba nd of ~1.9 kb in both Event Bt11 and Bt11 x

MIR604. For the mcry3A -specific probe, the Kpn I digest resulted in a single hybridization band of ~5.6 kb in both Event MIR604 and Bt11 x MIR604. Likewise, for the pmi -specific probe, the Kpn I digest resulted in a single hybridization band of ~5.2 kb in both Event MIR604 and Bt11 x

MIR604

. The predicted DNA hybridization patterns were retained and stability of the transgenic locus from parent to progeny was demonstrated. However, a more detailed explanation with documentation and/or literature reference for the unexpected cross hybr idization of the Bt11 transformation plasmid control lane (observed on Fi gure 7, lane 8) a nd the commercial DNA ladder is needed to verify the re sults of the Bt11 x MIR604 Southern blot, hybridized with the mcry3A -specific probe.

CLASSIFICATION: SUPPLEMENTAL but UPGRADEABLEpending submission of a more detaile d explanation for the unexpected hybridization band detected in the Bt11 transformation plasmid control

lane on the Bt11 x MIR604 Southe rn blot, hybridized with the mcry3A

specific probe.Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

19 MRID Title Summary

469165-

01 Comparison of Transgenic

Protein Expression in Event Bt11,

Event MIR604

and Stacked

Bt11 x MIR604 Maize (corn) Hybrids The purpose of this study was to compar e expression of the four transgenic proteins (Cry1Ab, mCry3A, PAT an d PMI) in a Bt11 x MIR604 maize (field corn) hybrid with expression in corresponding near-isogenic hybrids

derived from the individual transfor mation events to determine whether any unexpected differences in protein expression had occurred as a result of combining the traits by breeding via ELISA.

Mean Cry1Ab concentrations in l eaves and roots of the Bt11 x MIR604 hybrid ranged from ca . 19.7 to 27.7 μg/gdw and ca. 5.6 to 10.0 μg/gdw, respectively. Mean Cry1Ab con centrations in kernels were ca . 1.7 μg/gdw. The Cry1Ab concentrations in pollen fr om all three locat ions ranged from below the LOQ to 0.06 μg/gdw. Mean mC ry3A concentrations in leaves

and roots of Bt11 x Event MI R604 hybrid ranged from ca. 33.4 to 46.3 μg/gdw and ca. 18.9 to 23.9 μg/gdw, respectively. Mean mCry3A

concentrations in kernels were ca . 0.7 μg/gdw. The mCry3A levels in the pollen collected at anthesis from all three locations range from below the LOQ to ca. < 0.03 μg/gdw. Mean PAT concentrations in leaves and roots of the Bt11 x MIR604 hybrid ranged from ca . <0.05 to 0.17 μg/gdw and

ca . 0.08 to 0.19 μg/gdw, respectively. Mean PAT concentrations in the kernels were both <LOQ to <0.04 μg/gdw . The PAT levels in the pollen collected at anthesis from all three locations range from below the LOQ to

ca. < 0.034 μg/gdw. Mean MIR604 PMI con centrations in leaves and roots of the Bt11 x MIR604 hybrid ranged from ca . 5.7 to 10.4 μg/gdw and ca

. 2.3 to 6.0 μg/gdw, respectively. Mean MIR604 PMI concentrations in

kernels were ca . 1.9 μg/gdw. The PMI levels in the pollen collected at anthesis from all three locations range from 39.3 to 56.9 μg/gdw.

Overall, concentrations of Cry1Ab, mC ry3A, PAT, and PMI protein levels were found comparable and all cont rol tissues were negative for the expression of Cry1Ab, PAT, mCry3A and PMI. Therefore, transgenic protein expression in the Bt11 x MI R604 hybrid are not substantially different from that of the hybrids de rived from the individual Bt11 and

MIR604 transformation events.

Classification: Acceptable20 MRID Title Summary

470062-

01 Response to EPA questions regarding mcry3A

Southern blot in

the Bt11 x

MIR604 Comparative

Southern Analysis Report (MRID No. 467956-01, Figure 7) On November 22, 2006, EPA and representatives fro m Syngenta Seeds Inc., held a conference call

to discuss the deficienci es noted in MRID 467965-01. EPA requi red clarification for the Southern blot (Figure 7) where an unexpected hybridiz ation band (as 6.2 kb) was observed in the Bt11 transformation plasmid (the Not I-digested pZO1502) control lane.

In response, Syngenta s ubmitted a report that explained that the unexpected hybridization band was due to a small amount of pUC plas mid present in the KB ladder (used as the molecular weight marker). Syngenta ascertained this after discussion with a tech service representative of Stratagene (the KB ladder supplier), who indicated trace am ounts of pUC plasmid may re main as contaminants in the KB ladder stock (as a result of the manufac turing process). Due to the pUC plasmid presence in the KB ladder, Syngenta subsequently hybr idized the membrane with KB ladder probe, mcry3A

specific probe, and pUC plasmid probe.

Syngenta provided an additional rationale based on the generated Southern data for another project (Bt11 x MIR604 x GA21) in which they utilized a different ladder (Analytical Marker DNA, Wide Range, Promega Cat. No. DB1931). There was no unexpected hybridization band in the pZO1502 lane on the blot probed with the mcry3A -specific probe. This further confirmed that the unexpected hybridization band (Figure 7, lane 8) was due to the radiolabeled pUC plasmid, present in the KB ladder, hybridizing to COLE1in pZO1502.

EPA also questioned Syngenta’s me thod of radiolabeling the ladder DNA. Syngenta stated a preference to hybridize blots with the ladder and element-specific probes simultaneously so that the ladder can be visualized directly on the blot. The re gistrant utilizes this technique to eliminate the potential for introducing errors when manually tr ansferring the position of stained ladder bands from the gel to the membrane.

Syngenta also stated that they are using a differe nt molecular weight mark er preparation (consisting of a mixture of restriction enzyme digests of lambda DNA and Phi X174 DNA) to avoid any future potential non-specific sequence binding of the labeled ladder probe. Since this ladder is made from phage DNA, no unexpected hybridiza tion bands in the plasmid control lanes is either expected nor observed in other Southern analyses (when pr obing with a combination of the radiolabeled Analytical Marker DNA, Wide Range ladder and a radiolab eled element-specific probe).

Syngenta also noted that choosing different enzymes would not elimin ate the presence of any of the plasmid backbone sequences in the plasmid contro l lanes, because the plasmid controls on the Southern blots contain all the fragme nts of the digested plasmids.

EPA Reviewer’s Comment: The registrant has submitted a sufficient explanation for the nonspecific binding in the Southern blot (Figure 7, lane 8). However, visual ve rification is needed to confirm the lack of any unexpected hybridization band in the pZO1502 lane on the blot probed with the mcry3A-specific probe. Ther efore, Syngenta should submit a Sout hern blot containing genomic DNA from Bt11, MIR604, Bt11 x MIR 604 and a negative control as we ll as the plasmid controls pZO1502 and pZM26 hybridized with a combination of the mcry3A-specific and the newly employed Analytical Marker DNA, Wide Range la dder, which does not contain the COLE1 origin of replication vector sequence.

Classification: Supplemen tal, But Upgradeable- pending submission of an additional Bt11 x MIR604 Southern blot probed with the mcry3A -specific probe to confirm the lack of non-specific sequence binding in the Bt11 transformation pZO1502 pl asmid control lane as confirmatory data to complete the Bt11 x MIR604 database.Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

21 MRID Title Summary

472648-

01 Supplement to Report Titled: Comparative

Southern Analysis of a

Bt11 x MIR604 Maize Hybrid

with the

Parental Event

Bt11 and Event

MIR604 Maize

Inbreds The original submission of Southern blot data fo r the registration of this combination PIP product expressing both Cry1Ab and mCry3A proteins did not have clear results for the reactivity of the molecular weight standards with labeled plasmid components. Syngenta was required as a condition of the registration to submit new Southern blot data to clarify the cross reactivity and other issues. The study presents Southern blot data with appropri ate plasmid maps to explain the expected fragment banding patterns. The combination of several endonuclease digestions and probes gives the necessary background to judge the results from th e single parental lines as well as the hybrid cross Bt11 x MIR604. There was no aberrant probe binding to the molecular weight markers. The control samples in each gel were free from probe reactivity except in the case of positive controls. These data demonstrate stability and presence of both the Cry1Ab and mCry3A proteins and PAT and PMI marker genes in the parental line and the hybrid cross. Classification: Acceptable

none Provide to the EPA Laboratory (Ft. Meade, MD) methodology and/or reagents necessary for

validation of mCry3A analytical

method. More recently, the Agency decided to a llow this requirement to be satisfied when the Grain Inspection, Packers and Stockyards Administration (GIPSA) of the United States Depart ment of Agriculture (USDA) has verified the performance of a qualitative rapid test kit for detecting the presence of the biotechnology event in grains and oilseeds. In the case of mCry3A, the Agency has confirmed that a test kit has been verified by GIPSA and, therefore, the aforementione d requirement has been satisfied. Classification: Acceptable

3. 2010 Update: Terms and Conditions of the Event MIR 604 expressing modified Cry3A

1. Validation of analytical me thod for mCry3A. When Event MI R 604 expressing modified Cry3A Corn (EPA Reg. No. 67979-5) was initially regi stered on October 03, 2006, the Agency issued registration notices to Syngenta Seeds, Inc. that contained the following requirement for further product characterization information:

“Provide to the EPA laboratory (Ft. Meade, MD) methodology and/or reagents necessary for validation of a mCry3A anal ytical method within 6 months from the date that the Agency requests them.”

More recently, the Agency decided to allow this requirement to be sa tisfied when the Grain Inspection, Packers and Stockyards Administra tion (GIPSA) of the United States Department of Agriculture (USDA) has verified the performance of a qualitative rapid test kit for detecting the presence of the22 biotechnology event in grains and oilseeds. In the case of mCry3A, the Agency has confirmed that a test kit has been verified by GIPSA and, therefore, the aforementione d requirement has been satisfied.

2. Product Characterization for Events MIR 604 x Bt 11, expressing the proteins mCry3A and Cry1Ab

The product Agrisure CB/LL/RW contains Bt corn events MIR 604 x Bt11 expressing the proteins mCry3A and Cry1Ab. This stacked product targets both lepidopteran corn pests that are susceptible to Cry1Ab as well as corn rootworm that is susceptible to Cry3A. Both Bt11 and MIR 604 have been previously registered as single gene products. Specific product characterization information on this stacked product is presented below.

The purpose of the Transgenic Protein Expression Study was to compare expression of the four transgenic proteins (Cry1Ab, mC ry3A, PAT and PMI) in a Bt11 x MI R604 maize (field corn) hybrid with expression in corresponding ne ar-isogenic hybrids derived from the individual transformation events to determine whether any unexpected differen ces in protein expression had occurred as a result of combining the traits by breeding via ELISA.

Overall, concentrations of Cry1A b, mCry3A, PAT, and PMI protein le vels were found comparable and all control tissues were negative for the expression of Cry1Ab, PAT, mCry3A, and PMI. Therefore, transgenic protein expression in th e Bt11 x MIR604 hybrid are not substa ntially different from that of the hybrids derived from the individual Bt 11 and MIR604 transformation events.

The Southern blot analysis is used to confirm the presence of the cry1Ab and pat genes from the parental Event Bt11 and mcry3A and pmi genes from parental Event MIR604 in the hybrid Bt11 x MIR604 in a predictable manner. The predicte d DNA hybridization patterns were retained and stability of the transgenic locus from parent to progeny was demonstrated. However, a more detailed explanation with documentation and/ or literature reference for the unexpected cross hybridization of the Bt11 transformation plasmid control lane and th e commercial DNA ladder was needed to verify the

results of the Bt11 x MIR604 Sout hern blot, hybridized with the mcry3A -specific probe.

On November 22, 2006, the EPA and representatives fr om Syngenta Seeds Inc., held a conference call

to discuss the deficiencies noted in the Southern Blot data (M RID 467965-01). The Agency wanted

further clarification for the S outhern blot where an unexpected hybridization band (as 6.2 kb) was

observed in the Bt11 tran sformation plasmid (the Not I-digested pZO1502) control lane.

In response, Syngenta submitted a report that expl ained that the unexpected hybridization band was due to a small amount of pUC plasmid present in the KB ladder (used as the molecular weight marker). Syngenta ascertained this after disc ussion with a tech serv ice representati ve of Stratagene (the KB ladder supplier), who indicated tr ace amounts of pUC plasmid may rema in as contaminants in the KB ladder stock (as a result of the ma nufacturing process). Due to th e pUC plasmid presence in the KB ladder, Syngenta subseque ntly hybridized the membra ne with KB ladder probe, mcry3A -specific probe, and pUC plasmid probe.

Syngenta provided an additional rationale based on th e generated Southern data for another projectModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

23 (Bt11 x MIR604 x GA21) in which they utilized a different ladder (Analy tical Marker DNA, Wide Range, Promega Cat. No. DB1931). There was no une xpected hybridization ba nd in the pZO1502 lane on the blot probed with the mcry3A -specific probe. This further confirmed that the unexpected hybridization band was due to the ra diolabeled pUC plasmid, present in the KB ladder, hybridizing to COLE1in pZO1502.

EPA also questioned Syngenta’s method of radi olabeling the ladder DNA. Syngenta stated a preference to hybridize blot s with the ladder and elem ent-specific probes simultaneously so that the ladder can be visualized directly on the blot. The registrant utilizes this technique to eliminate the potential for introducing errors when manually tran sferring the position of stai ned ladder bands from the gel to the membrane.

Syngenta also stated that they are using a different molecular weight marker preparation (c onsisting of a mixture of restriction enzyme digests of la mbda DNA and Phi X174 DNA) to avoid any future potential non-specific sequence bind ing of the labeled ladder probe. Since this ladder is made from phage DNA, no unexpected hybridization bands in the plasmid control lanes is either expected nor observed in other Southern analyses (when probing with a combination of the radiolabeled Analytical Marker DNA, Wide Range ladder and a radiolabeled element-specific probe).

Syngenta also noted that choosing different enzymes would not eliminate the pr esence of any of the plasmid backbone sequences in the plasmid contro l lanes, because the plasmid controls on the Southern blots contain all the fragme nts of the digested plasmids.

EPA found that the registrant submitted a sufficien t explanation for the non-specific binding in the Southern blot. Visual verification was needed, ho wever, to confirm the lack of any unexpected hybridization band in the pZO1502 lane on the blot probed with the mcr y3A-specific probe. The registrant should submit a Southern blot co ntaining genomic DNA fr om Bt11, MIR604, Bt11 x MIR604 and a negative control as well as the plas mid controls pZO1502 and pZM26 hybridized with a combination of the mcry3A-specific and the newl y employed Analytical Ma rker DNA, Wide Range ladder, which does not contain the COLE1 origin of replication vector sequence

.

Syngenta submitted a supplemental study which showed Southern blot data with appropriate plasmid maps to explain the expected fragment banding pa tterns. The combination of several endonuclease digestions and probes gave the necessary background to judge the results from th e single parental lines as well as the hybrid cross Bt11 x MIR604. There was no aberrant probe binding to the molecular weight markers. The control samples in each gel were free from probe reactivity except in the case of positive controls. These data demonstrated stabi lity and presence of both the Cry1Ab and mCry3A proteins and PAT and PMI marker genes in the parental line and the hybrid cross.

II. B. Human Health Assessment24 1. Mammalian Toxicity and Allergenicity Assessment

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

Data have been submitted demonstrating the lack of mammalian toxicity at high levels of exposure to the pure mCry3A protein. These data demonstrate th e safety of the products at levels well above maximum possible exposure levels that are reasonably anticipated in the crops. This is similar to the Agency position regarding toxicity and the re quirement of residue data for the microbial Bacillus thuringiensis products from which this plant-incorporated protectant was derived (See 40 CFR Sec. 158.2130 and 158.2140).158.740(b)(2)(i)). For microbial products , further toxicity testing and residue data are triggered by significant acute effects in studi es such as the mouse oral toxicity study, to verify the observed effects and clarify the source of these effects (Tiers II and III).

An acute oral toxicity study was submitted fo r the mCry3A protein (MRID 461556-10, reviewed in Fellman, 2005a). The acute oral toxicity data subm itted support the prediction that the mCry3A protein

would be non-toxic to humans. Male and fema le mice (5 of each) were dosed with 2,377 milligrams/kilograms bodyweight (mg/kg bwt) of mCr y3A protein. With the exception of one female in the test group that was euthanized on day 2 (due to adverse clinical signs consistent with a dosing injury), all other mice surv ived the study, gained weight, had no test material-related clinical signs, and

had no test material-relat ed findings at necropsy.

When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad, Roy D., et al. 1992). Therefore, since no effects were shown to be caused by the plantincorporated protectants, even at relatively high dose levels, the mC ry3A protein is not considered toxic. Amino acid sequence comparisons showed no similarity between the mCry3A protein and known toxic proteins available in public protein data bases (MRID 461556-11, reviewed in Fellman, 2005a). According to the Codex Alimentarius guideli nes, the assessment of potential toxicity also includes stability to heat (FAO/WHO Standards Pr ogramme, 2001). Further data demonstrate that mCry3A is inactivated against WCRW, when heated to 95 ° C for 30 minutes (MRID 461556-08)

Since mCry3A is a protein, allergen ic sensitivities were considered. Currently, no definitive tests for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight of the evidence approach where the following factors are cons idered: source of the trait; amino acid sequence similarity with known allergens; prevalence in food; and biochemical properties of the protein, including in vitro digestibility in simulated gastric fluid (S GF), and glycosylation. Current scientific knowledge suggests that common food a llergens tend to be resistant to degradation acid and proteases; may be glycosylated; and are present at high concentrations in the food.

Data have been submitted that demonstrate that the mCry3A protein is rapidly degraded by gastric

fluid in vitro (MRID 461556-07, review in Fellman, 2005a)

. In a solution of simulated gastric fluid 1Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

25 mg/mL mCry3A test protein mixed with simulate d gastric fluid (pH 1.2, c ontaining 2 mg/mL NaCl, 14 μL 6 N HC1, and 2.7 mg/mL pepsin) resulting in 10 pe psin activity units/ μg protein (complies with 2000 US Pharmacopoeia recommendations), complete degradation of detectable mCry3A protein occurred within 2 minutes. A comparison of am ino acid sequences of know n allergens uncovered no evidence of any homology with mCry3A, even at the level of 8 contiguous amino acids residues (MRID 461556-12). Further data demonstr ate that mCry3A is not glycos ylated and is present in low

levels in corn tissue.

EPA also asked the March 14-15, 2006 FIFRA SAP to comment on EPA’s conclusions regarding the lack of mammalian toxicity and alle rgenicity of the mCry3A protein- sp ecifically the impact of the less potent mCry3A form on the results of the acute oral toxicity tests and the usefulness of in vitro digestibility studies and amino acid sequence homology analysis as part of th e risk assessment (SAP, 2006 and reviewed in Fellman, 2006c). Overall, th e Panel was more concerne d with the quality of data, i.e. inadequately described methods and poor re production of data images. The Panel specifically noted that the amino acid sequence an alysis to known toxins were mi ssing the following data: a full, technical description of each spec ific toxin sequence compared to the mCry3A amino acid sequence in the NCBI database; specification of which version of NCBI database was u tilized; descriptions of parameters utilized; and dates accessed for the BLAST search. EPA recognizes that these are important parameters to include in a description of an amino acid analysis, however, the parameters used and dates of the BLAST search were provided in the reference list and in a footnote, respectively, in the toxin homology report by the registrant. EPA is requiring subm ission of a detailed description of each amino acid sequence of known toxins compared to mCry3A by Syngenta Seeds, Inc. However, the Agency maintains that the conclusions of the amino acid sequence analysis are still valid for the purpose of the risk assessment. EPA reached this decision based on the following: 1) lack of mammalian toxicity of mCry3A protein as shown by th e acute oral mouse study; 2) mCry3A protein is rapidly digested in SGF; 3) mCr y3A protein originates from a non- allergenic source; 4) lack of sequence identity of mCry3A prot ein with 8 contiguous amino acids or more than 35% identity over 80 amino acids with known allergens; and 5) mCry3A prot ein is not glycosylated when expressed in corn.

Therefore, the potential for the mCr y3A protein to be a food allergen is minimal. As noted above, toxic proteins typically act as acute t oxins with low dose levels. Therefor e, since no effects were shown to be caused by the plant-incorporated protectant, even at relatively hi gh dose levels, the mCry3A protein

is not considered toxic.

A summary of the toxicity and allergenicity data is presented in Table 3.

Table 3. Mammalian Toxicity and Allergenicity studies (Updated)26 MRID Title Summary

461556-

10 Acute oral toxicity study of modified Cry3A protein(MCRY3A0102) in the

mouse MCRY3A-0102 was not acutely toxic to mice. There

was no evidence of toxic ity at 2,632 mg MCRY3A0102/kg body weight, representing ca. 2,377 mg mCry3A protein/kg body weight. The estimated LD 50 value for pure mCry3A protein in male and female mice was > 2,377 mg/kg body weight , the single dose used.

Classification: Acceptable

461556-

07 In vitro digestibility of modified Cry3A protein (MCRY3A-0102

and IAPMIR604- 0103) under

simulated

mammalian gastric

conditions The susceptibility of mCry3A protein to proteolytic degradation was evaluated in simulated gastric fluid (SGF) containing pepsin. Modi fied Cry3A protein from transgenic corn and recombinant E. coli (test material M MCRY3A -0102) was readily degraded in SGF. The data support a conclusion that mCr y3A protein expressed in transgenic plants will be read ily digested as conventional dietary protein under typi cal mammalian gastric

conditions.

Classification: Acceptable

461556-

08 Effect of

temperature on the stability of modified Cry3A protein(MCRY3A0102) At 95°C mCry3A protein was completely inactivated. At 4°C, 25°C, and 37° C there was little or no effect on mCry3A bioactivity, while at 65°C there was some reduction in the bioactivity.

Classification: Acceptable

461556-

11 Modified Cry3A protein as expressed in transgenic maize

event MIR604:

assessment of

amino acid homology with

known toxins The National Center for Biotechnology Information (NBI) GenBank Database (NBI, 2003) containing all publicly available protein sequences was queried for proteins with amino acid se quences having significant homology to mCry3A protein and that were toxins. The query found no significant amino acid homology between any protein toxin and the mCry3A protein.

Classification: AcceptableModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

27 MRID Title Summary

461556-

12 Modified Cry3A protein as expressed in transgenic maize

event MIR604:

assessment of

amino acid homology with known allergens No significant similarity was found between any of the mCry3A 80-amino acid peptides and any entries in the SBI Allergen Database. Als o, there were no alignments of eight or more contiguous amino acids between the mCry3A protein and any of th e proteins in the allergen database. Overall, the mCry3A protein showed no significant amino acid homology to any known or putative allergenic protein.

Classification: Acceptable

461556-

13 Phosphomannose

Isomerase as expressed in transgenic maize

event MIR604:

assessment of

amino acid homology with

known toxins Two nucleotide changes we re discovered in the pmi gene sequence inserted in corn Event MIR604. This resulted in two changes in the PM I protein; valine-61 was replaced by alanine, and gl utamine-210 was replaced by

histidine. These substitutions have not resulted in any apparent functional change in the PMI protein. The NCBI GenBank Database containing all publicly available protein sequences wa s queried for proteins with amino acid sequences having significant homology to this modified PMI protein that were toxins. The query found no significant amino acid homology between any protein toxin and the PMI pr otein expressed in corn

Event MIR604.

Classification: Acceptable

464252-

01 Phosphomannose

Isomerase as expressed in transgenic maize

event MIR604:

assessment of

amino acid homology with known allergens No significant similarity was found between any of the PMI 80-amino acid peptides and any entries in the SBI Allergen Database. However, in the eight or more contiguous amino acids homo logy search, there was an alignment between the PMI protein and a recently identified allergen, α -parvalbumin from Rana species CH2001 (a frog of Indonesian origin). However, aserum screening concluded that there is no crossreactivity between PMI and se rum IgE (obtained from an allergic individual who displayed food-induce anaphylaxis from α -parvalbumin). Bovine serum

albumin was also tested as an internal check. The observed low degree of se quence identity between

MIR604 PMI and α -parvalbumin is not biologically28 MRID Title Summary

relevant.

Classification: Acceptable

none “Submit the following data to augment the mCry3A amino acid sequence analysis to

known toxins and allergens

within six

months of the

date of registration: specification of

which version of

NCBI database was utilized; descriptions of

parameters utilized; and

dates accessed

for the BLAST

search.” In a letter dated July 1, 2 010, Syngenta replied with the following information to supplement MRIDs 46155612

and 46155611:

Assessment of Amino Acid Homology with Allergens.

The SBI Allergen Database version 2.0 was utilized for this assessment on March 3, 2003. In all the FASTA searches, the scoring matrix was Blosum 62, the gap extension penalty was two a nd the gap creation penalty

was 12.

Assessment of Amino Acid Homology with Known

Toxins.

The NCBI Database versi on NCBI-GenBank Flat File Release 137.0 was utilized on August 20, 2003. BLASTP Version 2.2.6 was used with the following parameters: No complexity filt er, expect score =10; word size = 3; gap costs: existe nce = 11 and extension = 1. The similarity matrix was Blosum 62.

Classification: ____________

2. 2010 Update: Terms and Conditions of th e Event MIR 604 expressing mCry3A and Events MIR 604 x Bt11 expressing mCry3A and Cry1Ab proteins.

When products containing Event MIR 604 expre ssing mCry3A protein (EPA Reg. No. 67979-5), and events MIR 604 x Bt11 expressing mC ry3A + Cry1Ab proteins (EPA Reg. No. 67979-8) were initially registered on October 03, 2006 and January 25, 2007 respectively, the Agency issued registration notices to Syngenta Seeds, Inc that contained the following requirement:

“Submit the following data to augm ent the mCry3A amino acid sequence analysis to known toxins and allergen s within six months of the date of registration: specification of which ve rsion of NCBI database was utilized; descriptions of parameters utili zed; and dates accessed for the BLAST

search.“

In a letter dated July 1, 2010, Syngenta provided the requested supplemental information.Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

29 3. Aggregate Exposures

In examining aggregate exposure, section 408 of the FFDCA directs EPA to consider available information concerning exposures from the pesticid e 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 buildi ngs (residential and other indoor uses).

The Agency has considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of c onsumers) to the pesticide chemi cal residue and to other related

substances. These considerations include dietary exposure under the tolerance exempti on and all other tolerances or exemptions in effect for the plant-in corporated protectant chem ical residue, and exposure from non-occupational sources. Exposure via the sk in or inhalation is not likely since the plantincorporated protectant is contained within plant cells, which essentially eliminates these exposure routes or reduces these exposure routes to negligib le. Exposure via residential or lawn use to infants and children is also not expected because the use s ites for the mCry3A protein are all agricultural for control of insects. Oral exposure, at very low le vels may occur from ingestion of processed corn products and, potentially, drinking water.

However, oral toxicity testi ng done at a dose in excess of 2 gm/kg showed no adverse effects. Furthermore, the expression of the modified Cry3A prot ein in corn kernels has b een shown to be in the parts per million ranges, which makes the expected di etary exposure several orders of magnitude lower

than the amounts of mC ry3A protein shown to have no toxicity. Therefore, even if negligible aggregate exposure should occur, the Agency concl udes that such exposure would present no harm due to the lack of mammalian toxicity and the rapid dige stibility demonstrated for the mCry3A protein.

4. Cumulative Effects

Pursuant to FFDCA section 408( b)(2)(D)(v), EPA has considered available information on the

cumulative effects of such residues and other substances that have a common mechanism of toxicity.

These considerations included the cumulative effects on infants and children of such residues and other

substances with a common mechanism of toxicit y. Because there is no indication of mammalian toxicity, resulting from the plant-incorporated prot ectant, we conclude that there are no cumulative effects for the mCry3A protein.

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

a) Toxicity and Allergenicity Conclusions The data submitted and cited regarding potential h ealth effects for the mCry3A protein include the characterization of the expressed mC ry3A protein in corn, as well as the acute oral toxicity, heat stability, and in vitro digestibility of the proteins. The resu lts of these studies were determined30 applicable to evaluate human risk, and the validity, completeness, and reliability of the available data

from the studies were considered.

Adequate information was submitted to show that the mCry3A protein test material derived from

microbial cultures was biochemica lly and functionally similar to the protein produced by the plantincorporated protectant ingredient s in corn. Microbially produced protein was chosen in order to obtain sufficient material for testing.

The acute oral toxicity data submitted supports th e prediction that the mCry3A protein would be nontoxic to humans. As mentioned above, when protei ns are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad, Roy D., et al. 1992). Since no effects were shown to be caused by mCry3A protein, even at relative ly high dose levels (2,377 mg mCry3A/kg bwt), the mCry3A protein is not considered toxic. This is similar to the Ag ency position regard ing toxicity and the requirement of residue data for the microbial Bacillus thuringiensis products from which this plantincorporated protectant was de rived. (See 40 CFR 158.740(b)(2)(i)). Moreover, mCry3A showed no sequence similarity to any known toxin and was not efficacious against WCRW when inactivated by

heat.

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

Since modified Cry3A is a protein, its potential allergenicity is also c onsidered as part of the toxicity assessment. Data considered as part of the allerg enicity assessment include that the modified Cry3A protein came from Bacillus thuringiensis which is not a known allergenic source, showed no sequence similarity to known allergens, was readily degr aded by pepsin, and was not glycosylated when expressed in the plant. Therefore, there is a reason able certainty that modified Cry3A protein will not be an allergen.

Neither available information concerning the diet ary consumption patterns of consumers (and major identifiable subgroups of consumers including infa nts and children); nor sa fety factors that are generally recognized as appropriate for the use of animal experime ntation data were evaluated. The lack of mammalian toxicity at high levels of expos ure to the mCry3A protein, as well as the minimal potential to be a food allergen de monstrate the safety of the produc t at levels well above possible maximum exposure levels anticipated in the crop.

The genetic material necessary for the production of the plant-incorporated protectant active ingredients are the nucle ic acids (DNA, RNA) which comprise genetic material encoding these proteins and their regulatory regions. The geneti c material (DNA, RNA) n ecessary for the production of mCry3A protein has been ex empted under the blanket exempti on for all nucleic acids (40 CFR 174.475).Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

31 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 effect s on infants and children of the residues and other substances with a common mechanism of toxicity.

In addition, FFDCA section 408(bfl2 )(C) also provides that EPA sh all apply an additional tenfold margin of safety for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the completene ss of the data base unless EPA determines that a different margin of safety will be safe for infants and children.

In this instance, based on all the available informa tion, the Agency concludes th at there is a finding of no toxicity for the mCry3A protein and the genetic material necessary for their production. Thus, there

are no threshold effects of concern and, as a result, the provision requiring an additional margin of safety does not apply. Further, th e provisions of consumption patte rns, special susceptibility, and cumulative effects do not apply.

c) Overall Safety Conclusion

There is a reasonable certainty that no harm will result from aggregate exposure to the U.S. population, including infants and children, to the mCry3A protein and the gene tic material necessary for its production. This includes all anticipa ted dietary exposures and all othe r exposures for which there is reliable information. The Agency has arrived at th is conclusion because, as previously discussed, no toxicity to mammals has been observed, nor any i ndication of allergenicity potential for the plantincorporated protectant.

6. Other Considerations

a) Endocrine Disruptors

2010 Updated Position. As required under FFDCA section 408(p) , EPA has developed the Endocrine Disruptor Screening Program (EDSP) to determine whether certain substan ces (including pesticide active and other ingredients) may have an effect in humans or wildlife similar to an effect produced by a “naturally occurring estrogen, or other such endocr ine effects as the Administrator may designate.” The EDSP employs a two-tiered approach to making th e statutorily required de terminations. Tier 1 consists of a battery of 11 screeni ng assays to identify the potential of a chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal systems. Chemicals that go through Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP where EPA w ill determine which, if any, of the Tier 2 tests are32 necessary based on the available data. Tier 2 tes ting is designed to identify any adverse endocrine related effects caused by the substance, and estab lish a quantitative relations hip between the dose and the E, A, or T effect.

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

construed as a list of known or likely endocrine disruptors.

Modified Cry3A is not among the group of 58 pesticid e active ingredients on th e initial list to be

screened under the EDSP. Under FFD CA § 408(p) the Agency must screen all pesticide chemicals. Accordingly, EPA anticipates issuing future EDSP orders/data call-ins for all Registration Review cases, including those for which EPA has already ope ned a Registration Review docket for a pesticide active ingredient.

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

.

b) Analytical Method(s)

A method for extraction and ELISA analysis of mCry3A protein in corn was submitted and found acceptable by the Agency.

2010 Updated Position . More recently, the Agency decided to a llow this requirement to be satisfied when the Grain Inspection, Packers and Stockyard s Administration (GIPSA) of the United States Department of Agriculture (USDA) has verified th e performance of a qualitative rapid test kit for detecting the presence of the biot echnology event in grains and oilsee ds. In the case of mCry3A, the Agency has confirmed that a test kit has been ve rified by GIPSA and, therefore, the aforementioned requirement has been satisfied.

c) Codex Maximum Residue Level

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

7. Tolerance Exemptions

The data submitted and reviewed for Modified Cry3A support the petition for an exemption from the requirement of tolerance for Bacillus thuringiensis mCry3A protein and the genetic material necessary for its production in corn.

An exemption from tolerance was established for residues of mCry3Aprotei n when used as plant-Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

33 incorporated protectant in the food and feed comm odities corn, field corn, sweet corn and popcorn. (40 CFR 174.505)

References

Fellman, A., 2005a. EPA Review of Product Characte rization and Expression Analysis for Modified Cry3A (mCry3A) Bacillus thuringiensis insect control protein and ma ize (corn) plants Derived from Event MIR604 in support for a tempor ary exemption from tolerances and an Experimental Use Permit (EUP) Application. Memorandum dated February 11, 2005.

Fellman, A. 2005b. EPA Addendum to Product Characteri zation and Expression Analysis for Modified Cry3A (mCry3A) B.t. insect control protein and maize (c orn) plants Derived from Event MIR604. Memorandum dated February 11, 2005.

Fellman, A., 2005c. EPA Addendum No. 2 to Produc t Characterization and Expression Analysis for Modified Cry3A (mCry3A) B.t. insect control protein and mai ze (corn) plants derived from Event MIR604. Memorandum dated February 23, 2005.

Fellman, A., 2005d. EPA Evaluation of Phosphomannos e isomerase protein (PMI) as expressed in Transgenic maize Event MIR604 with E. coli -derived PMI protein in support for a temporary tolerance exemption for Modified Cry3A B.t. insecticidal protein a nd the genetic material necessary for its production in corn plants derived from Event MIR604. Memorandum dated March 3, 2005.

Fellman, A., 2005e. EPA Review of Product Characte rization and Expression Analysis for Modified Cry3A B.t. insect control protein and maize plants derived from Event MIR604 in support for an exemption from the requirement of tolera nce and Sec. 3 Registra tion. Memorandum dated May 31, 2005.

Fellman, A., 2006a. EPA Review of Product Characte rization and Human Health Data for Modified Cry3A B.t. insect control protein and maize plants de rived from Event MIR504 in support for a an exemption from the requirement of tolera nce and Sec. 3 Registra tion. Memorandum dated February 15, 2006.

Fellman, A., 2006b. EPA Review of Application to Extend/Amend the Experimental Use Permit and existing temporary exemption from the requi rement of tolerance for Modified Cry3A B.t. insect control protein as expressed in Event MIR 604 corn plants. Memorandum dated February 24,

2006.

Fellman, A., 2006c. EPA Evaluation of FIFRA SAP meeting minutes dated June 1, 2006 from March 14 – 15, 2006 meeting on scientific issues asso ciated with the Produc t Characterization and34 Human Health Assessment for Modified Cry3A B.t. insect control Protein and maize plants derived from Event MIR604. Memorandum dated August 31, 2004.

Food and Agriculture Organization of the United Nations and World Health Organization, 2003. Foods Derived from Biotechnology. Codex Alimentarius. Sec. 4, No.38, pg.16.

Kough, J., 2004. EPA Review of Char acterization and Safety of P hosphomannose Isomerase (PMI), a selectable marker expressed in Event 3243M -derived maize plants. Memorandum dated January 30, 2004.

Kough, J., no date. EPA Review of Southern Bl ot data for Bt11 X MIR604 Corn from Syngenta Seeds, Inc. Memorandum to M. Mendelsohn.

Scientific Advisory Panel (SAP), 2006. Minutes of the FIFRA Scie ntific Advisory Panel Meeting Held March 14 – 15, 2006 on Event MI R604 Modified Cry3A Protein B.t. corn- Plant - Incorporated Protectant, Report date d June 1, 2006 (SAP Report No. 2006-02).

Sjoblad, R.D., J.T. McClintock and R. Engler , 1992. Toxicological considerations for protein components of biological pesticide products. Regulatory Toxicol. Pharmacol. 15: 3-9.

USDA. 2004. Performance verification of qualitative my cotoxin and biotech rapid test kits (Directive 9181.2). Available from: http://www.gipsa.usda.gov/GIPSA/docum ents/GIPSA_Documents/9181-2.pdf

.

Waggoner, A., and J. Kough, 2007. Review of Product Characterization, Expr ession Analysis and Human Health Data for Plant-Incorporated Protectant Bacillus thuringiensis Cty1Ab and mCry3A insect control proteins and the geneti c material necessary for their production in Maize (Corn) Plants as expressed in Even t Bt11 x MIR 604 (EPA Reg. No. 67979-I) in support for Sec. 3 Registration, submitted by Syngent a Seeds, Inc. – Field Crops – NAFTA. Memorandum to M. Mendelsohn, dated Jan 10, 2007.

II.C. Environmental Assessment

Background

Syngenta Ltd. has requested a registration for Bacillus thuringiensis mCry3A protein and the genetic material, which includes the PMI inert marker gene, necessary for its production in all corn lines and varieties. This protein is intend ed to control corn rootworm (CRW, Diabrotica spp.), a primary pest of

corn in the United States. Corn rootworm larvae feed on corn roots, resulting in lodging and a reduction in a plant’s ability to absorb water and nutri ents from soil. In areas where the CRW is a pest ( e.g. Corn Belt), significant financial losses are real ized from decreased corn yields and increased expenditures on chemical pest control agents, including organophosphate, car bamate and pyrethroid

insecticides.Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

35 The EPA has conducted an environmental risk as sessment of mCry3A when expressed in corn. General topics covered in this assessment include e ffects on wildlife, gene flow to related wild plants and its potential effects, and fate of mCry3A protein in the environment. This assessment is based on data submitted to EPA during the development of Ev ent MIR604 corn lines, additional data submitted for registration, Federal Insecticid e Fungicide and Rodenticide Act (F IFRA) Scientific Advisory Panel (SAP) recommendations, consultations with scien tific experts, and public comments on PlantIncorporated Protecta nt (PIP) regulation.

A. Environmental Hazard Assessment

I. The Hazard Assessment Process

1. Tiered Testing Hazard Assessment

To minimize data requirements and avoid unnecessary testing, risk assess ments 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 provid e enough confidence that there is no risk and no

further data would be needed. He nce, screening tests c onducted early in an inve stigation tend to be broad in scope but relatively simple in design, and can be used to demonstrate acceptable risk under most conceivable conditions. When screening st udies suggest potentially unacceptable risk additional studies are designed to assess risk under more realis tic field exposure conditions. These later tests are more complex than earlier screening studies. Use of this “tiered” testing framework saves valuable time and resources by organizing the studies in a cohesive and coherent manner and eliminating unnecessary lines of investigation. Lower tier, high dose screening st udies also allow tighter control over experimental variables and exposure conditi ons, resulting in a greater ability to produce statistically reliable results at relatively low cost.

Tiered tests are designed to first represent unrealistic worst case s cenarios and ONLY progress to real

world field scenarios if the earlier tiered tests fail to indicate adequate certain ty of acceptable risk. Screening (Tier I) non-target organism (NTO) hazar d tests are conducted at exposure concentrations several times higher than the highest concentrations expected to occur unde r realistic field exposure scenarios. This has allowed an endpoint of 50% mort ality to be used as a trigger for additional highertier testing. Less than 50% mortality under thes e conditions of extreme exposure suggest that population effects are likely to be negligible given realistic field exposure scenarios.

The EPA uses a tiered (Tiers I-IV) testing syst em to assess the toxicity of a Cry protein ( Bt endotoxin) to representative non-target organisms that could be e xposed to the toxin in the field environment. Tier I high dose studies reflect a screenin g approach to testing designed to maximize any toxic effects of the test substance on the test (non-targ et) organism. The screening test s evaluate single species in a laboratory setting with mortality as the end point. Tiers II – IV generally encompass definitive hazard36 level determinations, longer term greenhouse or fi eld testing, and are implem ented when unacceptable effects are seen at the Tier I screening level.

Testing methods which utilize the tiered approach were last published by the EPA as Harmonized OPPTS Testing Guidelines, Series 850 a nd 885 (EPA 712-C-96-280, February 1996). 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 that are strain-improved, either by natural selection or by deliberate genetic manipula tion. Therefore Bt Cry protein in corn, being a microbial toxin, is also covere d by these testing guidelines.

The Tier I screening maximum hazard dose approach to environmental hazard assessment is based on some factor (whenever possible >10) times the maxi mum amount of active ingred ient expected to be available to terrestrial and aquatic non-target organisms in the environm ent (EEC). Tier I tests serve to identify potential hazards and are conducted in the laboratory at high dose leve ls which increase the statistical power to test the hypotheses. Elevated dos es, therefore, add certainty to the assessment, and

such tests can be well standardized. The Guidelines cal l for initial screening test ing of a single group or several groups of test animals at the maximu m hazard dose level (MHD). The Guidelines call for testing of one treatment group of at least 30 animals or th ree groups of 10 test an imals at the screening

test concentration. The Guidelines further state th at the duration of all Tier I tests should be approximately 30 days. Some test species, notably non-target insects, may be difficult to culture and the suggested test duration has been adjusted accordingly. Control and tr eated insects should be observed for at least 30 days after dosing, or in case s where an insect species cannot be cultured for 30 days, until negative control mort ality rises above 20 percent.

Failing Tier I screening does not nece ssarily indicate the presence of an unacceptable risk in the field but it triggers the need for additional testing. b A less than 50% mortality e ffect at the MHD is taken to indicate minimal risk. However, greater than 50% mortality does not necessarily indicate the existence of unacceptable risk in the field, but it does trigger the need to collect additional dose-response information and a refinement of the exposure estima tion before deciding if th e risk is acceptable or unacceptable. Where potential hazards are detected in Tier I testing (i .e. mortality is greater than 50%), additional information at lower test doses is required which can serv e to confirm whether any effect might still be detected at more realistic field (1X EEC)concentra tions and routes of exposure.

When screening tests indicate a need for additiona l data, the OPPTS Harmoni zed Guidelines call for testing at incrementally lower doses in order to establish a definitive LD 50 and to quantify the hazard. In the definitive testing, 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 repr oductive and behavioral effects such as feeding inhibition, weight loss, etc. In the final analysis , a risk assessment is made by comparing the LOAEC to the EEC; when the EEC is lower than the LOAEC, a no risk conclusion is made. Appropriate statistical methods, and appropriate statistical power, must be employe d to evaluate the data from the

b 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 at 10X must be considered significant.Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

37 definitive tests. These tests offer greater environmen tal realism, but they may have lower statistical power. Higher levels of replication, test species numbers or repetition are needed to enhance statistical power in these circumstances.

Data that shows 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 evalua te adverse effects, making lowe r field exposure dose definitive testing unnecessary. It is also notable that the recommended >10X EEC maximum hazard dose level is a highly conservative factor. The published EPA Level of Concer n (LOC) is 50% mortality at 5X EEC (see footnotes 5 and 6).

The tiered hazard assessment approach was developed for the EPA by the American Institute of Biological Sciences and confirmed, in 1996, as an acceptable method of environmental hazard assessment by a FIFRA Scientific Advisory Panel (S AP) on microbial pesticides and microbial toxins. The December 9, 1999 SAP agreed th at the Tiered approach was suitable for use with Plant Incorporated Protectants (PIP); however, this panel recommended th at, for PIPs with insecticidal properties, additional testing of beneficial invertebrate s closely related to target species and/or likely to be present in GM crop fields should be conducted. Te sting of Bt Cry proteins on species not closely related to the target insect pest was not reco mmended, although it is still performed to fulfill the published EPA non-target species data requiremen ts [40 CFR § 158.740 (d)]. In October 2000, another

SAP also recommended that field te sting should be used to evalua te population-level effects on nontarget organisms. The August 2002 SAP, and some public comments, generally agreed with this approach, with the additional recommendation that in dicator organisms should be selected on the basis of potential for field exposu re to the subject protein. c

The tiered approach to test guidelines ensures, to the greatest extent possible, that the Agency requires

the minimum amount of data needed to make sc ientifically sound regulatory decisions. The EPA believes that maximum hazard dose Tier I testing presents a reasonable approach for evaluating hazards related to the use of biological pesticides and for identifying negative results with a high degree of confidence. The Agency expects that Tier 1 testing for short-term hazard assessment will be

c EPA-SAP. February 4, 2000. Characterization and non-target organism data requirements for protein plantpesticides. 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. March 12, 2001. Bt plant-pesticides risk benefit assessments. SAP report No. 2000-07 for FIFRA Scientific Advisory Panel Meeting held October 18-2 0 at the Marriott Crystal City Hotel, Arlington, VA. EPA-SAP. November 6, 2002. Corn rootworm plant-inco rporated protectant insect resistance management and non-target insect issues. Transmittal of meeting minutes of the FIFRA Scientific Advisory Panel Meeting held August 27- 29 at the Marriott Crystal City Hotel, Arlington, VA. EPA-SAP. August 19, 2004. Product characterization, hu man 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.38 sufficient for most studies submitted in support of plant-incorporated Bt Cry protein registrations. However, if long range adverse effects must be ascer tained, then higher-tier longer-term field testing will be required. As noted above, the October 2000 SAP and the National Academy of Sciences (NAS 2000) recommended direct field testi ng to ascertain long range environm ental effects. This approach, with an emphasis on testing invertebrates found in corn fields, was also recommended by the August 2002 SAP and was supported by several public comments.

Since delayed adverse effects and/or accumulation of toxins through the food chain are not expected to result from exposure to Bt Cry proteins, these protein toxins are not routinely tested for chronic effects on non-target organisms. The 30 da y test duration requirement does, however, amount to subchronic testing when performed at field exposure test doses. Bacillus thuringiensis Cry endotoxins are proteins and proteins do not bioaccumulate. The biological nature of protein makes Bt Cry toxins readily susceptible to metabolic, microbial, and abiotic degradation once they ar e ingested or excreted into the environment. Although there are report s that Cry proteins bind to soil particles, it has also been shown that these proteins are degraded rapidly by microbes upon elution from soil. The same sources also report that Bt proteins present in soil collected from Bt corn fields have no dete ctable adverse effect on

soil invertebrates or culturable microbial flora.

2. MIR604 Scientific Advisory Panel Comments

The FIFRA Scientific Advisory Panel (SAP) Meeting Held March 14 - 15, 2006 on Event MIR604 Modified Cry3A Protein Bt Corn had several panel members specifi cally assigned to evaluate the Agency’s Environmental Risk Assessment. (These) “Panel members expressed a diversity of opinions concerning the adequacy of the Agency’s analysis of the ecological studies submitted by the Registrant. These opinions ranged from nearly unqua lified acceptance to qualif ied rejection.” (p. 9, SAP Minutes).

The panel members who understood the Agency’s c oncept of maximum hazard dose screening (limit) testing (outlined in Section A. I.1 above) concluded that ... “If one takes the NTO (Non-Target Organism) toxicity assays of this Tier I hazard assessment at face value, none of them reported high acute toxicity. Rather, in most instances mortality was low or did not occur at the ETCs (Estimated

Test Concentrations ) achieved. Although the ETCs frequently di d not meet the standard for maximum hazard dose testing, these results do support the conclu sion of the absence of a strong , toxic response

in the NTOs .” (p. 19, SAP Minutes).

In reality, the absence of a strong (50%) mortality response at 5X the field concentration is the Agency standard (endpoint) for maximum h azard dose Tier I screening tests d (see Section A.I.1 above). The results of the testing performed fo r MIR604 were far below this publis hed Agency standard (Level of

d See footnotes 5& 6Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

39 Concern) for maximum hazard dose testing. There is an absence of 50% mort ality in all of the nontarget tests performed, and no mort ality at all attributable to th e test substance was noted.

Tiered tests are designed to first represent unrealistic worst case scenarios and ONLY progress to field exposure scenarios if the earlier tiered tests fail to indicate an adequate certainty of acceptable risk (i.e. show greater than 50% mortality of the test species). Many of the current Panel comments were made as if field exposure scenario testing was being reviewed, when in fact the worst case (screening) scenarios did not trigger a need for field exposure rate (sublethal/definitive) testing. Therefore many of the Panel’s comments are supe rfluous to the establis hed EPA hazard assessment process which is based on the result s of screening tests (w hen these show less than 50% mortality of the test species).

The EPA risk assessment is centered only on adverse effects at the field expos ure rates (1X EEC), and not on adverse effects at greater concentrations. “The Panel was split on the importance of the Registrant not adhering to the 10X (EEC test dose) standard. Several Panel members considered this a serious deficiency but a deficiency th at easily could have been avoided. Other Panel members

considered the test concentrati ons/doses adequate to support the Ag ency’s finding of no likely adverse ecological effects. ” (p. 16 SAP Minutes) e The dose margin can be less than 10x where uncertainty in the system is low or where high con centrations of test material are not possible to achieve due to test organism feeding habits. High dos e testing also may not be necessa ry where many species are tested or tests are very sensitive, although the c oncentration used must exceed 1X EEC.

The purpose of convening the SAP was to comment on the Agency’s conclusion that the MIR604 corn

would not cause unreasonable and/or irreversible adverse effects to the environment as determined by existing EPA risk assessment SOPs. As noted above, some panel members responded positively to this charge, whereas others chos e to comment on the EPA testing and risk assessment SOPs. The MIR604 SAP was not asked to revise the EPA pestic ide program's assessment system which has been developed through many years of evaluation both with in the government and thr ough outside experts.

e It is notable those panel members who perceived any non-adherence to the 10X standard as a serious deficiency did so as a result of an impression that all testing must be done at a 10X “safety” factor. In reality the 10 X EEC screen testing approach is not equivalent to what is commonly known as “testing at a 10X SA FETY FACTOR” where any adverse effect at 10X must be considered significant. In addition, those panel members who perceived non-adherence to the 10X standard did so as a result of an impression that the purified, bacterially produced Cry protein test substance had half of the activity of the plant produced Cry protein. This impression may not be justified because the intrinsically variable insect bioassay results which appear to show a two fold difference in activity were in fact within the range of variability for insect bioassays. In addition, in the event that the test concentration was at half of the plant Cry protein activity level, the test

concentrations are still at or above the EPA Level of Concern (5X EEC) and in all cases above the 1X field exposure rate (EEC) at which the assessment of adverse ecological effects is made. The 1X EEC (based on plant tissue content of Cry protein) is in turn still much greater than any amount which any given non-tar get organism may be actually ingesting in the

field.40 The methods have already undergone several layers of SAP, public, peer a nd EPA Science Advisory Board review and have been found to be adequate. f (See also Section A.I. 1 above and footnote 2)

In addition to not commenting on the adequacy of the Agency’s risk conclusions at field exposure rates (i.e. 1X EEC), some panel members were not prep ared to accept the concep t of screening (limit) testing. Some viewed the high dose screen tests designed to detect LD 50 ’s as if these were definitive determinations where any effect (at a limit test do se) is also a level of concern at field exposure concentrations. Tier I screening test s were also referred to as 10X “ safety factor” tests by some Panel members (p.9, SAP Minutes). Tier I sc reen testing is not ‘safety factor testing’. In a “10X safety factor” test any adverse effect noted is a “level of concern” g , whereas in the EPA risk assessment scenario any adverse effect is viewed as a concern only at 1X the field exposure. As a result the panel comments on the testing procedures were not addr essing screening methods but the definitive (1X) testing procedures that are triggered only when the screening results show greater than 50% mortality. This level of toxicity was not seen in a ny screening test performe d for mCry3A protein. Therefore definitive testing (at 1X EEC) was not necessary and any comments made by the panel applicable only to the definitive (1 X) testing methods are superfluous. h In addition, the EPA Guidelines require three replicates of 10 test animals, or one repli cate of 30 for the tier I screening tests, therefore the SAP comments on insufficient replication and nu mbers of test species are also superfluous.

However, in principle the Agency is in agreem ent with these SAP comment s and the OPPTS Testing Guidelines are already implementing them as much as they apply to definitive testing procedures (See Section A.I.1 above). The SAP methodological comme nts do not, however, apply to Tier I screening tests which in this case did not trigger additional, definitive testing for MI R604 mCry1A proteins There was no effort by some panel members to disti nguish 'screening testing' (which is performed to determine intrinsic toxicity of an insecticidal pr otein) from ‘definitive testing’ triggered by positive

screen test results and which is much more co mplicated and uses more realistic environmental

exposure rates.

A tiered risk assessment as performed by the Agency is recognized as being the most appropriate and rigorous approach to assess non-targ et affects from both scientific and regulatory standpoints. This process is designed to optimize the use of resources and to identify a nd define sources of potential risk.

f Environmental Protection Agency (USEPA) (1998). “Guidelines for Ecological Risk Assessment.” EPA 630/R-95-002F. Washington, DC, USA. (Federal Register, May 14, 1998. 63(93): 26846-26924.]

g The established (see footnote 5 above), peer and USEPA Science Advisory Board reviewed guidance on screening test levels of concern is 50% mortality at 5X environmenta l 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.

h The 1X EEC test dose is based on plant tissue content and is considered a high worst case dose of the Cry protein. This 1X EEC is in turn still much greater than any amount which any given non-target organism may be ingesting in the field. Therefore the actual exposure scenario wo uld have to be further refined in the event that adverse effects are noted at the (1X) EEC based on plant tissue analysis.Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

41 Where no unreasonable hazard is det ected at Tier I limit (screen/high dose) testing, costly and unnecessary testing at field exposur e rates is prevented from taking place. Both hazard and exposure

can be evaluated within different levels or “tiers ” that progress from worst-case hazard and exposure to

more realistic scenarios. Lower tie r tests serve to identify potential hazards, and they are conducted in the laboratory at high dose leve ls which increase the statistical power to test the hypotheses and therefore do not require high replicat ion or large numbers of test spec ies. Where potential hazards are detected in Tier I testing (i.e. test species mortal ity is greater than 50%), additional information at lower test doses is required which can serve to confirm whether any e ffect might still be detected at more realistic field (1X EEC) concentrations and r outes of exposure. The confirmatory tests offer greater environmental realism, but they may have lo wer statistical power. Highe r levels of replication or repetition are needed to enhance sta tistical power in these circumstances. Tier I high dose/screening tests do not require multiple repl ication and repetition as was recommended by some panel members

. In addition, screening tests withou t multiple replications are of publication quality, and numerous screening rests are published in peer reviewed literature.

Some panel members also listed defici encies in the testing procedures of non-target wildlife other than insects. The precision of some assay methods was also criticized. As discussed above, these comments are valid, but they do not apply to screening testing, and additionall y, testing of wildlife other than insects related to the target pest is not essentia l for an environmental ri sk assessment of mCry3A proteins. This fact was recognized by some members of the SAP who state that: “Cry3A is a protein endotoxin produced naturally by the tenebrionis strain of Bacillus thuringiensis subsp. morrisoni (H 8a8b) that upon ingestion and proteo lytic activation exhibits a high le vel of insecticidal activity to certain species of coleopteran insects, but otherwise is apparently non-toxic to othe r types of insects, as

well as vertebrates ” (p.29, SAP minutes). Therefore any defici encies as noted by the panel on testing of non-target organisms other than insects would not have had an a dverse effect on the environmental risk assessment of mCry3A protein in corn because th ey were (a) superfluous tests for Bt Cry proteins and (b) none of the screening tests showed unacceptabl e effects according to the EPA risk assessment guidelines.

In summary, some panelists agreed with the Agency ’s risk assessment. Other panelists recommended a variety of refinements to field exposure rate te sting protocols which do not apply to the high dose screening tests performed for MIR604 corn a nd therefore do not cha nge the “no apparent

environmental risk” assessment for MIR604 corn. Th e Panel’s critical comme nts are valid for field exposure dose definitive testing which was not triggered by the screeni ng rests performed with mCry3A protein. However, the Panel’s commen ts and recommendations are being reviewed for incorporation into the Agency’s definitive testing protocols as may be necessary.

The following comments address additional observations found in the SAP Minutes.

A no “may effect” finding was made by the Agency to the endangered Hunge rford’s crawling water beetle due to low aquatic exposure from corn, a terre strial crop. In addition, the submitted studies with mCry3A protein did not show any a dverse effects at terrestrial fiel d use rates to non-target insect42 species most closely related to the endangered aquatic beetle. The EP A Environmental Risk

Assessment for MIR604 states: “There is no evidence for sensitivity of aquatic (including endangered) species to anti-coleopteran Bt Cry proteins. Furthermore, aquatic exposure to mCry3A is extremely small or non-existent since mCry3A is not expr essed in MIR604 pollen.” and: “... Since mCry3A protein has not been shown to have toxicity effect s on ... aquatic species, insect s and other invertebrate species at the EEC, a ‘may affect’ situation for endangered land and a quatic species is not anticipated.”; and “...Further, se veral of the federally listed insect species are aquatic and consequently, are unlikely to come in contact with MIR604 maize plant material .” Therefore it is the Agency’s position that the MIR604 Risk Assessment has adequately addressed the endangered aquatic beetle species.

Contrary to the perceptions of so me panel members, the testing perf ormed did meet, and in many cases actually exceeded the Agency’s published Tier I te sting standards. Where th e 10X standard was not met it was due to insect biology (f eeding habit) factors that did not permit exposure to 10X EEC

concentrations.

The large variability in re-isolation of Cry protein in test soils or test diets is inherent in such testing, especially when insect bioassays ar e used. It is not realistic to e xpect precision or accuracy in such assays. For that reason the Agency relies on these te sts primarily as a measure that the test material was actually added to, and is pr esent in the test systems.

The determination of a slope (as was done in the so il degradation study) from experimental data points is a standard procedure in OPP and is not considered inadequate.

The maturation rates (weight gain) of male and fema le birds are naturally di fferent in some avian species and therefore the Agency does not consider the different weight gain rate s to be an effect of Cry protein ingestion.

The death of one test animal (fish or bird) in a sc reening high test dose study (where 50% mortality is the end point) is not considered by the Agency to be an indication of hazar d at real world field

exposure rates.

Criticism of the use of agar pour plates is not considered a criti cal issue by the Agency. Agar pour plates have been used for more than 50 years to enumerate microbial and vi ral particles, and the 50 o C temperature used to prepare these plates does not a ffect the function of (protein) enzymes or viability of bacteria or virus particles. Therefore there is no r eason to expect that th e use of the pour plate method to prepare insect diets will have adverse effects on the functionality of Cry proteins.

3. 2010 Update: Terms and Conditions of the Event MIR 604 expressing modified Cry3A and Events MIR 604 x Bt11 expressing the proteins modified Cry3A and Cry1Ab.

When Event MIR 604 expressing mCry3A Corn (E PA Reg. No. 67979-5) and events MIR 604 x Bt11 expressing mCry3A and Cry1Ab (E PA Reg. No. 67979-8) were initia lly registered on October 03, 2006 and January 25, 2007 respectively, the Agency issu ed registration notices to Syngenta Seeds, IncModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

43 that contained the following requirement for fu rther Environmental Assessment information:

“Submit field degradation studies evaluating accumula tion and persistence of mCry3A in several different soils in various strata. Repr esentative fields must have been planted with mCry3A corn and in clude both conventional tillage and no-till samples and be harvested under typical agronomic conditions. Sampling must

continue until the limit of detection is reached. Studies should include soils with high levels of a variety of clays. Both ELISA and insect bioassays need to be conducted and compared to determine if mCry3A is accumulating or persisting in soil samples. A protocol is due within 90 da ys of the date of registration. Should the registration expiration date be extended, a final report regarding data from fields that have had three continuous years of cultivation of Event MIR604 corn is due by January 31, 2011.”

In response to this requirement, Syngenta Seeds, Inc. – Field Crops – NAFTA submitted a request to waive the conditionally-required soil persistence field study for the plant-incorporated protectant (PIP) Bacillus thuringiensis ( Bt. ) mCry3A insecticidal protein and th e genetic material necessary for its production in Event MIR604 corn [EPA Reg. No. 67979-5] in support of the Section 3 registration.

Soil microorganism exposure to Bt proteins can occur thr ough contact with corn plant roots (by direct feeding), corn plant exudates, inco rporation of above-ground plant tissu es into soil following harvest, or by soil-deposited pollen. Scie ntists have generated data on Bt proteins in crop residues and their persistence in soil for long periods of time following exposure to a wi de range of environmental field

conditions.

The results of these studies, summarized in Tabl e 4, show that there is no detectable Cry protein accumulation in agricultural soils during commercial planting of currently registered Cry proteinproducing crops (Icoz and Stotzky, 2007; Sanvido, et. al , 2007). Likewise, no unexpected accumulation of Cry proteins has been seen in numerous studies submitted directly to EPA for currently registered Cry prot eins. There are various publis hed studies that measured Bt protein degradation in a laborato ry setting relating the dissipation time of the protein to bioactivity (DT 50 ); data predict that Cry proteins in th e field degrade rapidly and do not pers ist or accumulate in the soil to any extent that would affect nont arget organisms (NTOs). The DT 50 (based on a simple first-order kinetic model) for mCry3A in an artificial soil environment was 7.6 days (MRID No. 462656-14). The results of this study demonstrate a similar rate of de gradation to other Cry prot eins in artificial soil systems (U.S. EPA, 2001) and, along with the literature cited below, i ndicate that mCry3A is likely to have a similar field degr adation profile to other Bt proteins. In addition, data already exist to support the lack of effects of the mCry3A protein on NT Os (U.S. EPA, 2006; Raybould et al., 2007), which is the primary focus when considering soil fate data.

II. Non-Target Wildlife Hazard Assessment44 Two separate SAP reports (October 2000 and August 2002) recommended that non-target testing of Bt Cry proteins should focus on inverteb rate species exposed to the crop being registered. Following SAP recommendations, the EPA determined that non-targe t organisms with the greatest exposure potential to Cry protein in transgenic corn fields are benefi cial insects, which feed on corn pollen and nectar, and soil invertebrates, particular ly Coleoptera species. Therefore, maximum hazard dose toxicity testing on representative benefici al organisms from several taxa was performed in support of this Section 3 FIFRA registration. The toxicity of the mCr y3A protein has been evaluated on several species of invertebrates including the lady beetle, Carabid beetle, r ove beetle, flower bug, honey bee, and earthworm. Reproductive and developmental observati ons were also made in the lady beetle, rove beetle and honeybee studies.

Although the mCry3A protein is known to be very host specific, conferring toxic effects on corn rootworm, Colorado potato beetle, and closely re lated species, and despite the October 2000 and August 2002 SAP’s recommendations against testing of non-target species not related to susceptible target pests, EPA has done a risk assessment on a ra nge of non-target wildlif e to comply with the Agency’s published non-target data requirements (in the absence of PIP-specific risk assessment guidance, EPA requires applicants for PIP registrati ons to meet the 40 CFR Part 158 data requirements for microbial toxins). These requir ements include birds, mammals, plants and aquatic species. In addition, an earthworm study was voluntarily submitted to the Agency to ascertain the potential effects of mCry3A on beneficial decomposer species.

Test substances ( i.e. source of mCry3A protein) used for st udies submitted in support of the mCry3A registration included bacterially-pro duced purified mCry3A protein (r eferred to as mCry3A-0102) and corn grain. The October 2000 SAP recommended that wh ile actual plant material is the preferred test material, bacterially-derived protein is also a valid test substance, particularly in scenarios where test animals do not normally consume corn plant tissue and where large amounts of Cry protein (Cry protein concentrations that exceed levels present in plant tissue) are needed for maximum hazard dose testing. An insect feeding study, which compared the relative potency of plant produced mCry3A protein to microbe produced mCry3A -0102, indicated that plant produced protein was twice as toxic as microbe produced protein (see Section A.II.1.d.vii). However, since exposure to mCry3A-0102 was at

least several times the EEC for all submitted non-t arget studies, EPA determined that exposure to mCry3A protein was adequate despite the lower potency of microbe produced mCry3A-0102. In accordance with OPPTS Harmonized Testing Guidel ines, adult insect studies were generally conducted for 30 days or until mortality in the ne gative control reached, or exceeded, 20% and larval

studies were carried out thr ough pupation and adult emergence.

The results of ecological effects studies subm itted in support of the MIR604 Section 3 FIFRA registration are summarized in Ta ble 4 and presented in a more de scriptive format in subsequent

sections of this risk assessment document. Full re views of each study can be found in the individual Data Evaluation Reports (DERs) and accompanying memos.

Table 4. Updated Summary of environmental effects st udies and waiver justifications submitted to comply with data requirements published in 40 CFR § 158.740 (d).Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

45 Guideline Study Results MRID

885.4150 Wild

Mammal Testing, Tier

I Mammalian wildlife exposure to mCry3A protein is considered likely; however, mCry3A-0102 toxicity data indicate that, when tested at the maximum hazard dose level, there was no significan t toxicity to rodents.

Therefore no hazard to mamma lian wildlife is anticipated

and data on wild mammal testing is not required. N/A

850.2100 Avian Acute Oral Toxicity Test, Tier 1 (Northern

Bobwhite quail, Colinus
virginianus ) Young adult northern bobwhite quail were administered a single nominal oral dose of 722 mg mCry3A-0102/kg body wt and observed for 14 days. There were no treatment-related adverse clinical signs or mortality. Body weight and feed consumption of the test birds were comparable to those of th e controls. Classification: Acceptable 461556-

16

885.4050 Avian Oral,

Tier 1 (Broiler,

Gallus

domesticus ) In a 49-day feeding study, co mmercial broiler chickens

were fed formulated diets c ontaining one of the following ingredients: MIR604 corn (c ontained mCry3A protein);an MIR604 isoline (no mCry3A protein); or a nontransgenic commercial corn hybrid (no mCry3A protein). No adverse clinical signs we re noted, and carcass yield and mortality were not si gnificantly different among treatment groups. Classification: Acceptable 462656-

15

885.4200 Freshwater Fish Testing,

Tier 1 (Rainbow

trout, Onchorhynch us mykiss ) In a 28-day toxicity study, gr anular fish feed containing 50% by weight Event MIR604 corn grain did not produce statistically significant mort ality or sublethal effects when fed twice daily to juvenile rainbow trout. Classification: Acceptable 461556-

17

462656-

02

885.4280 Estuarine and

Marine

Animal Testing, Tier

I Estuarine and marine animal studies are not required for this product, because mCry3A is not intended for direct application to estuarine or marine environments and there is very low potential that these ecosystems will be exposed to mCry3A pr otein in field corn. N/A46 Guideline Study Results MRID

885.4300 Nontarget Plant Studies,

Tier I The active ingredient is an insect toxin ( Bt endotoxin) that is non-toxic to aquatic and terrestrial plants. Consequently, non-target plant studies have been waived for this product. N/A

885.4340 Nontarget

Insect Testing, Tier 1 (Lady beetle,

Coccinella

septempuncta

ta ) Lady beetle larvae were fed live pea aphids that were dipped in a solution contai ning: 50μg mCry3A-0102/mL Agral 90 solution (a non-ioni c surfactant); Agral 90 solution only (negative cont rol); or 0.5 mL of Nemolt (teflubenzuron)/L Agral 90 solution (positive control). The rate of pupal developm ent was not significantly different between the nega tive control and mCry3A-0102 treatments. However, the number of days to adult emergence was significantly lower in the mCry3A-0102 treatment. Classification: Acceptable 462656-

03

462656-

04

885.4340 Nontarget

Insect Testing, Tier 1 (Carabid beetle,

Poecilus

cupreus ) Twenty-four to 48 hour-old Carabid beetle larvae were fed daily until pupation with bl owfly pupae that had been injected with one of three treatments. Results showed no significant difference, in the percent of pre-imaginal mortality or mean weight of emerged adults, between the mCry3A treatment and the negative control group. Classification: Acceptable 462656-

05

462656-

06

885.4340 Nontarget

Insect Testing, Tier 1 (Rove beetle,

Aleochara

bilineata ) Rove beetles were provided approximately 0.2 g of

minced beef treated with: 50 μg mCry3A protein/g meat; 10 mL deionized water/90 g meat (negative control); teflubenzuron at a rate of 0.01 mg a.i./g meat (positive control). Beetle mortality a nd reproductive capacity were not adversely affected by feed ing on a test diet composed of 45.85 μg mCry3A/g diet fo r 35 days. Classification: Acceptable 462656-

07

462656-

08

885.4340 Nontarget

Insect Testing, Tier 1 (Insidious flower bug,

O rius.

insidiosis ) Nymphal flower bugs were fed, on a daily basis, diet with

one of three treatments: 50 μg mCry3A-0102 /g of diet; 20 mL deionized water/per 180 g diet (negative control);

or teflubenzuron at a rate of 0.01 mg a.i./g diet. Mortality in the mCry3A-0102, negative control, and positive control treatments were 18, 23, and 98%, respectively. Average development time for all treatments was not significantly different. Classification: Acceptable 462656-

09

462656-

10Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

47 Guideline Study Results MRID

885.4380 Honey Bee Testing, Tier 1 ( Apis mellifera) Honeybees were exposed to sucrose solution containing 50 μg mCry3A protein/g sucrose solution, or a positive or negative control. Results suggest that incidental ingestion of mCry3A proteins would not adversely affect the hive condition, survival of larvae in brood cells, or exposed adult worker bees. Classification: Acceptable 461556-

18

885.4240 Aquatic

Invertebrate

Acute Toxicity Test,

Tier 1 The only plausible potentia l route of exposure of freshwater invertebrates to insecticidal proteins produced by transgenic corn plants is co rn pollen drift into aquatic habitats. However, since the pollen of Event MIR604 corn plants has no detectable mC ry3A protein, exposure of freshwater aquatic invertebrates to mCry3A protein will be negligible. Classification: Acceptable Waiver justificati

on

850.620 Earthworm

Subchronic Toxicity Study ( Eisenia fetida) Earthworms were exposed to soil containing mCry3A0102 at a nominal concentration of 370 μg/g dry soil for 14 days, or one of two contro l treatments. At test end, a mortality rate of 5% and a mean weight loss of 5.8% were recorded for mCry3A- 0102 treated worms. For the negative control, mortality wa s 0%, and mean weight loss was 11.4%. Mortality was 100% for the positive control. Classification: Acceptable 462656-

11

462656-

12

N/A Insecticidal Activity Spectrum Study The mCry3A protein has a similar spectrum of activity to native Cry3A, but with enhan ced toxicity to NCRW and

WCRW. Modified Cry3A produced in E. coli and maize were found to be active against WCRM with 144 hour

LC 50 values of 0.43 μ g mCry3A-0102/mL diet and 0.20

μ g mCry3A/mL diet surface, respectively.. Classification: Acceptable 461556-

01

461556-

03

885.5200 Expression in

a Terrestrial

Environment (Soil Fate) A simple first-order kinetic model, based on CPB larvae feeding data, determined that the DT 50 for mCry3A in this silty clay loam soil was 7.6 days. This finding suggests that soil incorporated mCry3A protein degrades over time. Classification: Acceptable 462656-

14

NA Environment

al Fate

Assessment MIR604 corn plants have been shown to express mCry3A protein in leaves, kern els, roots, and silks, but the protein was not detected in corn pollen. Due to corn’s

lack of invasive characteris tics and the low probability

that the mCry3A gene from Event MIR604 would transfer to a wild relative of corn, it is unlikely that mCry3A will 462656-

1348 Guideline Study Results MRID

spread beyond cultivated s ites and persist in weedy populations. It is also unlik ely that genes present in MIR604 corn would be subj ect to horizontal gene transfer at a frequency that ex ceeds the rate of transfer in other plants. Classification: AcceptableModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

49 Guideline Study Results MRID

NA Endangered Species

Assessment The primary route of exposure to mCry3A protein in corn is through ingestion of corn tissue. There are no reports of threatened or endanger ed species feeding on corn plants, therefore such species would not be exposed to corn tissue containing Cry protein. Since mCry3A protein

has not been shown to have toxicity effects on mammals, birds, plants, aquatic species, insects and other invertebrate species at the EEC, a "may affect" situation for endangered land and aquatic species is not anticipated. In addition, EPA does not expect that any threatened or endangered plant species will be affected by outcrossing to wild relatives or by competition with such entities. Hybrid corn doe s not exist in the wild, nor are there wild plants that can interbreed with corn in the

United States.

Because of the selectivit y of mCry3A protein for coleopteran species, endangered species concerns are mainly restricted to the order Coleoptera. Examination of an overlay map showing the county level distribution of the 16 endangered/threatened coleopteran species (currently listed by the U.S. Fish and Wildlife Service) relative to corn production counties in the United States clearly indicated that any potential concern regarding range overlap with corn produc tion was mainly restricted to the American burying beetle ( Nicrophorus

americanus ). The American burying beetle is the largest carrion beetle in North America and is only found in

limited areas of Rhode Island and portions of the Great Plains, including Arkansas and Georgia. Adults are nocturnal and feed on carrion and sometimes prey on other arthropods. Larvae feed exclusively on buried carrion provided by their pare nts. The American burying

beetle’s habitat is variable and often includes deciduous forest, grassland and agricultu ral areas. Considering that

both larvae and adult insects feed exclusively on carrion, it appears that even if Am erican burying beetles did occur in proximity to Bt corn fields, there would be little chance of exposure to Bt protein due to their feeding

habits. After careful review of available data, the EPA determined that exposure of American burying beetle to 462656-

0150 Guideline Study Results MRID

harmful levels of MIR604 corn tissue is not expected. Likewise, a review of the pr eferred habitats of other coleopteran species listed as endangered by the U.S. Fish

and Wildlife Service indicated that exposure to harmful levels of mCry3A protein w ould not take place. The main

reasons for the lack of e xposure are geographical and habitat limitations. These species are located in non-corn production areas and/or their habitat does not encompass agricultural areas. Likewise, other insect species in the orders Diptera, Hemiptera, Lepidoptera, Odona ta and Orthoptera that are listed as endangered/threaten ed species are found in dune, meadow/prairie or open fo rest habitats and are not closely associated with ro w crop production, often times due to the specificity of the habitat of their host plants. The reviewed toxicological data shows the relative insensitivity of a range of insects in non-Coleopteran orders to the mCry3A prot eins, indicating that MIR604 maize hybrids are not likely to have detrimental effects on non-Coleopteran insects included on the endangered/threatened species list.

Further, several of the federally listed insect species are aquatic and consequently, are unlikely to come in contact with MIR604 maize plant material. Many of the endangered and threatened beetles occur in cave or aquatic habitats. Since moveme nt into water bodies of soil containing mCry3A is e xpected to be negligible, pollen drift was considered the primary source of potential hazard to endange red aquatic Coleoptera. According to estimates based on published studies, if 100% of the pollen grains leaving a corn field were deposited in a 1 ha pond with 2 m depth and located ≥ 1 m from the edge of a corn field, <0.0001 μg mCry3A/mL of water would be expected. This is a few orders of magnitude below the toxic level to any insect.Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

51 Guideline Study Results MRID

NA Long-term

field degradation study on the

accumulation

and persistence of

active ingredient Soil microorganism exposure to Bt proteins can occur through contact with corn plan t roots (by direct feeding), corn plant exudates, incor poration of above-ground plant tissues into soil following harvest, or by soil-deposited pollen. Scientists have generated data on Bt proteins in crop residues and their persistence in soil for long periods of time following exposure to a wide range of

environmental field conditions.

The results of these studies show that there is no detectable Cry protein accumu lation in agricultural soils during commercial planting of currently registered Cry protein-producing crops (Icoz and Stotzky, 2007; Sanvido, et. al , 2007). Likewise, no unexpected accumulation of Cry proteins has been seen in numerous studies submitted directly to EPA for currently registered Cry proteins. There are vari ous published studies that

measured Bt protein degradation in a laboratory setting relating the dissipation time of the protein to bioactivity (DT 50 ); data predict that Cry pr oteins in the field degrade rapidly and do not persist or ac cumulate in the soil to any

extent that would affect nontarget organisms (NTOs).

The DT 50 (based on a simple first-order kinetic model) for mCry3A in an artificial soil environment was 7.6 days (MRID No. 462656-14). The results of this study demonstrate a similar rate of degradation to other Cry proteins in artificial soil sy stems (U.S. EPA, 2001) and, along with the literature c ited below, indicate that mCry3A is likely to have a similar field degradation profile to other Bt proteins. In addition, data already exist to support the lack of effects of the mCry3A protein on NTOs (U.S. EPA, 2006; Raybould et al., 2007), which is the primary focus when considering soil fate data.

RECOMMENDATION: The data and rationales in support of the request to waive the need for a soil persistence field study are ACCEPTABLE for mCry3A protein, as expressed in MIR604 corn. 478477-

01

1. Non-target Wildlife Te sting and Hazard Assessment52 a. Mammalian Wildlife

Mammalian wildlife exposure to mCry3A protei n is considered likely; however, mammalian toxicology information gathered to date on Bt Cry proteins does not show a hazard to wild mammals. And an acute oral toxicity test , submitted to EPA in support of the MIR604 registration (see Human Health Risk Assessment), indicated that no significant toxicity was s een when rodents were exposed to mCry3A at the maximum hazard dose level. Therefor e, no hazard to mammalian wildlife is anticipated and data on wild mammal testing is not required for this registration.

b. Avian hazard assessment

Published data and studies on file at EPA show that consumption of Bt corn has no measurable deleterious effects on avian species. However, to comply with published data requirements, the following studies were submitted to EPA in support of the MIR604 product registration. These studies were GLP compliant and, when considered together , meet EPA data requirements for avian species.

i. Northern Bobwhite Quail This study meets current EPA Guideline requirements fo r acute toxicity testing of incidental exposures of plant incorporated Cry proteins to non-target birds in the wild.

Young adult (25 week old) northern bobwhite quail ( Colinus virginianus ) were administered a single nominal oral dose of 722 mg mCry3A-0102/kg body wt and observed for 14 days. There were no

adverse treatment-related clinical signs or mortality. Body weight a nd feed consumption of the test birds were comparable to those of th e negative control. The acute oral LD 50 of mCry3A-0102 was shown to be greater than a nominal concentra tion of 722 mg mCry3A-01 02/kg body wt (approximately 652 mg mCry3A protein/kg body wt). Th ese data show that there will be no adverse effects on avian wildlife from incidental field exposure to mCry3A corn.

ii. Broiler study The submitted study was not EPA GLP compliant, but wa s conducted according to accepted scientific

methods.

In a 49-day avian feeding study, one da y-old commercial broiler chickens ( Gallus domesticus , Ross 344 males and feather-sexable Ross 308 females) were fed formulated diets containing one of the following ingredients: MIR604 corn (contained mCry3A protein); an MIR604 isoline (no mCry3A protein), or a non-transgenic commer cial corn hybrid (no mCry3A pr otein). Starter, grower (days 16- 31), and finisher diets (days 31-49) contai ned 57.5, 63.0 and 67.5% corn, respectively. The concentration of plant produced mCry3A in the tr ansgenic diets was reported to be 0.04, 0.06, and 0.08 μg/g dry weight of the starter, grower, and finish er diets, respectively. Chic ks were separated by sex and placed into single sex pens containing 25 birds each. Each treatment group contained 6 cages each of males and females (12 cages x 25 birds/cage = 300 bi rds). Pen weights (25 bird s/pen) were recorded at days 1 (hatch), 16, 31, and 49. On the later three dates, feed conversion ratios were determined. Feeding was terminated approximately16 hours be fore slaughter on day 51. Body weight, feedModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

53 conversion, and survival data were recorded. Results indicate that sex had a significant effect on body weight and survival, with males weighing more and having higher mortality (which is normal for this species). No adverse clinical signs were noted, and carcass yield and mortality were not significantly different among treatment groups.

Table 5. Mean body weight of broiler chickens fed mCry3A positive or mCry3A negative corn grain.

Body Weight (g) Treatment

Day 1 (hatch) Day 16 Day 31 Day 49

MIR604 (mCry3A positive) 44.60 ± 0.19

a* 547.8 ± 9.6

a 1684.1 ± 50.6

a 3468.4 ± 129.5

a

MIR604 (mCry3A negative) 44.66 ± 0.18 a 563.0 ± 5.4

a 1690.5 ± 40.2

a 3469.4 ±113.1

a

Commercial hybrid (mCry3A negative) 44.70 ± 0.19 a 551.1 ± 9.6

a 1634.0 ± 44.3

b 3365.2 ± 117.6

b
* Within sampling days, means followed by different letters differ significantly (p ≤ 0.05).

Table 6. Carcass and parts yield at day 51 for bro iler chickens fed mCry3A positive or mCry3A negative corn grain.

Males

Dressed

carcass* Thighs Pectoralis major Pectoralis

minor Treatment

------------------ ------------------ ----- g -------

--------------- ---------------
MIR604 (mCry3A positive) 2947.2 ± 57.44

a** 526.3 ± 14.05

a 638.8 ± 19.18

a 150.8 ± 4.63 a

MIR604 (mCry3A negative) 2936.7 ± 56.88 a 484.3 ± 15.10

ab 609.6 ± 16.73

a 145.7 ± 4.57 a

Commercial hybrid (mCry3A negative) 2812.9 ± 61.49 a 454.6 ± 20.30

b 605.1 ± 23.09

a 151.3 ± 4.97 a

Females

MIR604 (mCry3A positive) 2248.3 ± 45.53 a 380.8 ± 7.6 a 515.4 ± 18.9 a 126.2 ± 3.3 a

MIR604 (mCry3A negative) 2291.0 ± 41.4 a 361.9 ± 9.2 a 511.8 ± 13.8 a 128.0 ± 3.5 a54 Commercial hybrid (mCry3A negative) 2204.2 ± 54.8 a 366.2 ± 11.3 a 487.1 ± 22.0 a 125.9 ± 3.4 a

* Fresh carcass without head, neck, feet, feathers, viscera, and blood

** Within carcass parts categories, means followe d by different letters differ significantly (p ≤ 0.05)

c. Aquatic species testing

There is no evidence for sensitivity of aquatic (i ncluding endangered) specie s to anti-coleopteran Bt Cry proteins. Furthermore, aquatic exposure to mC ry3A is extremely small or non-existent since mCry3A was not detectable in MIR604 pollen.

i. Freshwater Fish The Harmonized Testing Guidelines requirement for a static renewal fr eshwater fish toxicity study is usually waived for Bt corn PIPs due to the low potential for exposure to Cry protein produced in this crop. Nonetheless, a 28 day flow-t hrough study was performed and submitt ed for review. This study is scientifically sound.

In this 28-day toxicity study, juvenile rainbow trout ( Onchorhynchus mykiss ) were fed fish feed containing 50% w/w Event MIR604 (0.09 μg mCry3A/g te st diet) or non-transgenic (negative control) corn grain. Prior to test initiation, 40 fish were placed in each of tw o test vessels, the exposure tank and the control tank. Mortality and symptoms of toxic ity were assessed on a daily basis and detailed observations of symptoms and feeding respons es were made on days 4, 7, 10, 15, and 22. No significant differences were detected in the weight of the control or test fish at 0, 14, or 28 days. No significant difference in length was seen at 14 or 28 days. In the MIR604 test group, transient discoloration, sounding, and surfacing were seen in one to three fish after day 15, and one fish (2.5% of test group) was found dead on day 21. No mortalit y was seen in the control group. Due to the lack of demonstrated toxicity of the mCry3A protein to juvenile rainbow trout a nd the low probability that aquatic systems will be exposed to th e protein, no fresh water fish haza rd is expected from commercial

cultivation of Event MIR604 corn.

ii. Aquatic invertebrates

2010 Update. In light of recently published laboratory studies showing reduced growth in shredding caddis flies exposed to anti-lepidopteran Cry1A pr otein corn litter (Ros i-Marshall, et al. 2007), additional aquatic invertebrate da ta are required. A 7 to 14 day Daphnia magna study as per the 885 series OPPTS Guidelines needs to be performe d. The study may be submitted as a condition of registration. Alternatively, a diet ary study of the effects on an aqua tic invertebrate, representing the functional group of a leaf shredder in headwater streams, can be perfor med and submitted in lieu of the Daphnia study.

iii. Estuarine and Marine Animals Estuarine and marine animal studies were not requir ed for this product, because of the low probability that aquatic systems will be exposed to the mCr y3A protein produced in MI R604 corn plant tissues.Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

55 iv. Terrestrial and Aquatic Plants Plant toxicity studies were not required for this produc t because the active ingredie nt is an insect toxin ( Bt endotoxin) that has never s hown any toxicity to plants.

d. Non-Target Insect Testing

The mCry3A protein specifically targ ets corn rootworm species, whic h are within the order Coleoptera (beetles). Since Bt toxins are known to have a limited host ra nge, EPA requires that test species used for non-target insect evaluations should include seve ral species that are rela ted to the target pests (coleopteran species), since it is expected that these species will be most susceptible to the Bt toxin.

i. Ladybird Beetle This study complies with the testing requirements outlined in OPPTS Series 885.4340 (Nontarget Insect Testing, Tier 1), the Organization for Ec onomic Development (OECD) Principles of Good Laboratory Practice, and the UK Good Laboratory Practice Regulations.

Four–day old lady beetle larvae ( Coccinella septempunctata ) were fed live pea aphids that were dipped in one of three solutions for a period of 14 days. For each treatment, aphids were immersed for 30 seconds in a solution containing: mCry3A dissolved in a solution of Agral 90 (a non-ionic surfactant) at a concentration of 50 μg mCry3A protein/mL solution; Agral 90 so lution only (negative control); or 0.5 mL of Nemolt (teflubenzuron)/L Agral 90 solution (positive control) . Freshly-treated live aphids were provided to lady beetle larvae daily until pupati on, and the number of pea aphids provided at each feeding time increased with larval age. Beetle la rvae were assessed daily for developmental stage and mortality. Following adult emergence from pupae, beet les were fed 50 treated aphids 3 times a week for 14 days. Adults were assessed for mortality at each feeding.

There was no significant difference in the rate of pupal development between the negative control and mCry3A-0102 treatment. However, the mean number of days to adult emergence was significantly lower in the mCry3A treatment. There was no signifi cant difference in pre-imaginal or adult survival among the negative control and mCry3A treatments. A ll larvae in the positive control died in the preimaginal stage. Since lady beetles are known to feed on corn pollen or insect prey, rather than corn leaves, and since mCry3A concentra tion in pollen (undetected) and prey is much lower than in corn leaves, it is not expected that lady beetles will be adversely aff ected by MIR604 corn in a field

environment.

Table 7. Mean developmental time for lady beetles exposed to mCry3A-0102 or a control

treatment. Larvae to Pupae Larvae to Adult

Treatment Concentration

Mean Standard

Deviation Mean Standard

Deviation Negative Agral 90 5.48 0.50 9.8 0.6856 controlmCry3A0102 50 μg mCry3A/mL Agral solution 5.33 0.47 9.48* 0.59

*Significantly different from control (p<0.05)

Table 8. Mortality assessment for lady beetles ex posed to mCry3A-0102 or a control treatment. Pre-Imaginal Mortality Adult Mortality Mortality Corrected Mortality Mortality Corrected Mortality Treatment Concentration

--------------- --------------- ---- % -------------

------------ ---------

Negative

control Agral 90 0 - 7.5 -

mCry3A0102 50 μg mCry3A/mL Agral solution 0 0 15.0 8.1

Nemolt 0.5 mL/L Agral

solution 100* 100 - -

* Treatment differed significantly fr om the negative control (p<0.001)

It is noted that the act ual amount of mCry3A protein consumed by larvae and adult ladybird beetles was 9 μg mCry3A/g aphid, which is below the targ eted concentration of 50μ g/g aphid (10 x mCry3A concentration in corn leaves). However, since lady beetles are known to feed on corn pollen or insect prey, rather than corn leaves, a nd since mCry3A concentration in po llen (undetected) and prey is much lower than in corn leaves, it is not expected th at lady beetles will be ad versely affected by MIR604

corn in a field environment.

ii. Carabid Beetle

This study complies with the testing requirements outlined in OPPTS Series 885.4340 (Nontarget Insect Testing, Tier 1), the Organization for Ec onomic Development (OECD) Principles of Good Laboratory Practice, and the UK G ood Laboratory Practice Regulations.

Twenty-four to 48 hour-old Carabid beetle larvae ( Poecilus cupreus ) were fed daily with blowfly pupae until pupation. For each treatment, blow fly pupae were injected with: mC ry3A-0102 at a rate of 50μg mCry3A/g pupa; deionized water only (negative control); or teflubenzur on at a rate of 0.664 ng a.i./g fly pupae (positive control). Fly pupae were replaced dail y with freshly defrosted pupae until beetle larvae entered pupation. Be etle larvae were assessed three times per week for the first two weeks, and two times per week thereafter until day 32 of the study, at which time test containers were checked daily for emerging adult beetles. Adults were sexed, and overall wei ght and mortality were statistically analyzed. Results s howed no significant difference, in the percent of pre-imaginal mortality or mean weight of emerged adults, betw een the mCry3A treatment and the negative control group. The positive treatment resulted in 100% pre-imaginal mortality (Table 9).Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

57 Table 9. Larval mortality and adult weight of Ca rabid beetles fed blowfly pupae treated with or without mCry3A-0102 protein.

Treatment Percent Larval Mortality Percent Corrected Larval Mortality Mean Adult Weight mCry3A-0102 10 0 82.9 Negative control 20 - 81.5

Positive control 100 100 -

Analysis of the test diet showed that the actual concentration of mCry3A in blowfly pupae was 12 μg/g pupae. This amount is less than 10X (50 μg/g) the e xpressed concentration in maize leaves. However, the most likely route of carabid exposure to mCry3A protein is through consumption of prey that has eaten MIR604 plant tissue and studies indicate that the concentration of mCry3A in prey is at least 1.4X lower than the protein c oncentration in plant tissue.

iii. Rove Beetle This study complies with the testing requirements outlined in OPPTS Series 885.4340 (Nontarget Insect Testing, Tier 1), the Organization for Ec onomic Development (OECD) Principles of Good Laboratory Practice, and the UK G ood Laboratory Practice Regulations.

Rove beetles ( Aleochara bilineata ) were obtained from parasitized onion fly ( Delia antique ) pupae and adults were four days old (physiolo gically) at study initiation. During this 35 day feeding trial, beetles were provided one of three treatments of cooked min ced beef. For each treatment, beetles were given approximately 0.2 g of minced beef treated with : 50 μg mCry3A protein/g meat; 10 mL deionized water/90 g meat (negative control); or teflubenzuron at a rate of 0.01 mg a.i./g meat (positive control). For the first 7 days, beetles (10 female and 10 male) were kept in round plastic pots. From days 7 to 35, test arenas were comprised of polystyrene boxes filled with at least 4 cm of quartz sand. On days 1, 7 and 35 living, moribund, dead, and missing beetles were noted. To assess beetle fecundity, approximately 500 onion fly pupae were incorporat ed beneath the sand surface in each test box on days 14, 21, and 28. After approximately seven da ys, fly pupae were removed from the sand and placed in plastic pots. F 1 beetles emerging from onion fly pupae were recorded every 1 to 4 days through day 76. The study was concluded when the m ean number of beetles emerging per replicate declined to less than two per day in the control treatment. The mean number of F 1 progeny was 647 for the negative control and 663 beetles for the mCry3A treatment and thes e results were not significantly different. At day 35, mortality in the mCry3A, nega tive treatment, and positive treatments were 31, 34, and 35%, respectively and did not differ significantly (Table 10). Results also indicate that the reproductive capacity of beetles feeding on the mC ry3A test diet was no t adversely affected.

Table 10. Mortality and number of progeny of r ove beetles supplied mCry3A-0102 or a control treatment for 35 days.

Treatment % Mortality at 35 days * % Corrected Mortality Mean no. of

F 1 Progeny % Effect on Reproduction58 mCry3A-0102 (50 μg mCry3A/g diet) 31 0 663 ± 219 -2.5

Negative control 34 - 647 ± 169 -

Teflubenzuron (10 μg/g diet) 35 2 3 ± 5** 99.5

* Treatments did not differ significantly from the negative control (p>0.05) ** Treatment differed significantl y from the negative control (p < 0.001)

iv. Flower Bug

This study complies with the testing requirements outlined in OPPTS Series 885.4340 (Nontarget Insect Testing, Tier 1), the Organization for Ec onomic Development (OECD) Principles of Good Laboratory Practice and the UK Good Laboratory Practice Regulations.

Nymphal Orius insidiosis were fed, on a daily basis, diet consis ting of cooked beef, liver, yeast, honey, egg, sugar, water, and Nipagin (che mical preservative) for 21 days. Th e diet was treated with one of three treatments: 50 μg mCry3A/g di et; deionized water (negative cont rol); or teflubenz uron at a rate of 0.01 mg a.i./g diet. For each treatment, approxima tely 0.2 g of diet was placed into a small plastic cup covered with parafilm; insects pierced the parafilm to reach th e diet. Nymphs were assessed for mortality and vitality daily until adulthood, or until 21 days after test ini tiation. Bugs that were missing, squashed, or injured during the study were excluded from data analysis. Mortality in the mCry3A, negative control, and positive control tr eatments were 18, 23, 98%, respectively (Table 11). Average development time for all treatments was not significantly different (p >0.5). Analysis of the test diet showed that protei n expression was 47.8 μg mCry3A/g diet, or 95.6% of the nominal concentration (50 μg/g diet). In addition, a bi oassay in which Colora do potato beetle, CPB ( Leptinotarsa decemlineata ) larvae were fed a diet containing 10 or 20% mCry3A-treated diet resulted in mortality of 83 and 90%, respectively. Although control mortality was 23% (test guidelines state that negative control mortality should not exceed 20 %), it is unlikely that study conclusions were affected by this high rate of mortality.

Table 11. Mortality of flower bugs supplied with di et containing mCry3A-0102 or a control diet.

Treatment % Pre-imaginal Mortality % Corrected Mortality

mCry3A-0102 18 0

Negative control 23 -

Teflubenzuron (10 μg/g diet) 98* 97

* Significantly different from negative control (p < 0.001)

v. Honey Bee LarvaeModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

59 An acceptable study was conducted based on OPPTS Se ries 885.4380 (Honey Bee Testing, Tier I), in accordance with Good Laboratory Practice Standard s as published by the U.S. Environmental Protection Agency in 40 CFR Part 160.

Honeybees ( Apis mellifera ) were exposed via in-hive feeders to sucrose solution containing 50 μg/g sucrose solution, a negative control of 50% w/v sucrose solution, or a pos itive control of 480 g/L diflubenzuron insect growth regulator (Dimilin Flo TM ) in sucrose solution. Fresh treatment solutions were provided to each hive daily for five days. Egg cell mortality in the negative control, test, and positive control groups was 28.5, 27.3, and 100%, respec tively. Larval cell mortality was 6.0, 6.8, and 100%, respectively. There was no significant differen ce in mortality between the test and negative control groups for cells with eggs or larvae. There was also no signi ficant difference in pre- and posttest hive condition between the test and negative control treatments. Results for the positive control treatment were significantly different from the ot her treatments for both mort ality and hive condition (Table 12). Adult bees were not affected by any of the treatments. These study results suggest that incidental ingestion of mCry3A pr oteins would not adversely affect the hive condition, survival of larvae in brood cells, or exposed adult worker bees.

Table 12. Honey bee egg and larval cell developmen t and brood count per frame when exposed to mCry3A-0102 or a control treatment. Brood Development Assessments Mean Brood per

Frame Egg Cells Larvae Cells Mortality Correcte

d

Mortalit

y Mortality Corrected Mortality Before

Treatme

nt After

Treatmen

t Treatment

------------------ ------------------ ---------------%-

------------------ -----------------

Negative

control 28.5 - 6.0 - 36.4 49.6

mCry3A0102 27.3 0 6.8 0.9 34.2 40.7

Dimilin Flo 100.0* 100.0 100.0 100.0* 34.5 27.7* * Significantly different from negative control (p<0.01)

vi. Freshwater Aquatic Inverteb rate (waiver justification) The only plausible potential route of exposure of freshwater inverteb rates to insecticidal proteins produced by transgenic corn plants is corn pollen drift into aquatic habitats. However, since the pollen of Event MIR604 corn plants has no detectable mCry3A protein, exposure of freshwater aquatic invertebrates to mCry3A pr otein will be negligible.

vii. Earthworm Toxicity Testing60 This study complies with the testing requirements outlined in OPPTS Series 850.6200 (Earthworm Subchronic Toxicity Study), Good Laboratory Practice Standards as published by the EPA in 40 CFR Parts 160 and 792, and the Organization for Econom ic Development (OECD) Principles of Good Laboratory Practice.

In a laboratory test, earthworms ( Eisenia fetida ) were exposed to test substance mCry3A-0102, incorporated into artificial soil , at a nominal concentration of 370 μg/g dry soil (334 μg mCry3A protein/g dry soil) for 14 days. A negative control of deionized water and artifi cial soil and a positive control of 10, 20, 30 40, or 50 mg 2-chloroacetamide/kg dr y soil were also used in the test. At test end, a mortality rate of 5% and a mean weight loss of 5.8% were recorded for mCry3A treated worms (Table 13). Worms included in the negative control ha d a mortality rate of 0% and a mean weight loss of 11.4%. Positive control worms exposed to ≥ 30 mg 2-chloroacetamide/kg of dry soil had a mortality

rate of 100% and the LC 50 value for earthworms exposed to 2- chloroacetamide was approximately 18 mg active ingredient/kg dry soil. The 14-day LC 50 for earthworms exposed to mCry3A-0102 in an artificial soil substrate was determined to be greate r than 370 μg test substance/g dry soil (equivalent to 334 μg/g mCry3A active ingredient/g dry soil, the highest concentrati on tested), or 67 times greater than the expected field concentration of 5.5 μg/g soil (based on mCry3A concentration in senescent MIR604 plant roots). These study findings, which are consistent with historical Bt cry protein feeding results, indicate that earthworms should not be adversely affected by MIR604 corn plants.

Although some public comments have questioned whet her earthworm test organisms actually ingested the soil incorporated Bt Cry proteins, recently published data show that earthworms do ingest and excrete soil incorporated Bt Cry proteins.

Table 13. Mortality and weight loss of earthw orms exposed to mCry3A-0102 or a control

treatment.

Cumulative mortality (%) Treatment

Day 7 Day 14 14-Day Weight loss (%)

mCry3A-0102 2.5 5.0 5.8

Negative control 0 0 11.4

viii. Insecticidal Activity Spectrum Study Insect susceptibility studies show ed that native Cry3A is primar ily active against Colorado potato beetle and has minimal activity against northern co rn rootworm (NCRW); both species are members of the Chrysomelidae family of beetle s. The mCry3A protein has a sim ilar spectrum of activity to native Cry3A, but with enhanced toxicity to NC RW and western corn rootworm (WCRW).

The bioactivity of the recombinant E. coli and maize event MIR604 mCry3A proteins were compared using a diet incorporation bioa ssay with first instar WCRW. Modified Cry3A from both E. coli and maize were found to be active against WCRW with 144 hour LC 50 values of 0.43 μ g mCry3A/mL diet

and 0.20 μ g mCry3A/mL diet surface, respectively (Table 14).Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

61 Table 14

. Toxicity of mCry3A protei n derived from recombinant E. coli (mCry3A-0102) or from

corn event MIR604 on WCRW larvae.

LC50 Sample μg mCry3A protein/mL

diet 95% Confidence

Interval LPMIR604-0103 (corn-derived protein) 0.20 (0.09 - 0.41)

mCry3A-0102 (bacterial-derived protein) 0.43 (0.14 - 0.94)

2. Soil Fate

Soil organisms may be exposed to mCry3A protein through contact with corn plant roots (by direct feeding), corn plant root exudate s, incorporation of above-ground plant tissues into soil following harvest, or by soil-deposited pollen. Some eviden ce suggests that acidic soils (pH 5.6), and those which are high in clays and humic acids, are more lik ely to bind Cry protein, and thus decrease the rate of protein degradation by soil mi croorganisms. It is noted, however , that the pH factor should not contribute to protein binding in co rn fields, since maize is generall y grown on neutral soils (above pH 5.6). And despite evidence that soils high in clay and humic acids may bind cry proteins, and thus interfere with the microbial degradation processes, the weight of evidence s uggests that Cry proteins do not accumulate in soil to arthropod-toxic levels. Nonetheless, the Agency requires soil fate evaluations for each new insect protected crop.

This study was conducted in accordance with Good Labor atory Practice Standards as published by the U.S. Environmental Protection Agency, Offi ce of Pesticide Programs in 40 CFR Part 160.

The test soil was a silty clay lo am collected from a corn-growing re gion of Iowa. Treatments were the following: mCry3A test mixture ( Bt cry protein and water) mixed w ith soil at a nominal dose of 230 μg mCry3A/g d/w soil, and sampled after 0, 1, 3, 7, 12, and 30 days of incubation; a negative control consisting of soil dosed with deionized water; and a positive control consisti ng of mCry3A mixed with test diet (no soil), at a concentr ation of 230 μg mCry3A/g diet (equ ivalent test dose level at Day 0).

For the CPB bioassay, mCry3A-treated soil collecte d at each incubation time point was incorporated into a stock diet at a concentration of 10% w/w, and the resulting suspension was poured into Petri dishes. Negative and positive contro ls were prepared in the same manner. Ten freshly-hatched CPB larvae were placed in each Petri dish, which we re then covered and maintained under ambient laboratory conditions. Test material and negative control treatments were replicated 12 times (120 larvae/treatment) and the positive control was replic ated four times (40 larvae). Larval mortality was

assessed at 72 hours.62 Mean CPB larval mortality in mCry3A- treated soil ranged from 48-54% during the first week, then declined rapidly to 9% on Day 30 (Table 15). The DT 50 value (time for 50% of initial bioactivity to dissipate) for degradation of mCry3A in this soil was estimated to be 7.6 days. Biomass determinations of soil at study start and end showed that mi crobial activity was maintained during the study.

Table 15. mCry3A protein bioactivity in treated soil as measured by CPB larvae mortality. Treatment % CPB Mortality Negative control 18

Positive control 53

Test Treatment Day 0 53 Day 1 54 Day 3 48 Day 7 51 Day 12 11 Day 30 9

Based on these results, it may be concluded that purified mCry3A-0102 insecticidal proteins degrade rapidly in silty clay loam soil. However, silty clay loam soil is just one of many soil classes used for corn production in the United States. A more us eful study would evaluate protein degradation, accumulation, and/or persistence in a range of soil types, including those with high clay and humic acid content, due to their known binding affinity for proteins.

In addition, this study utilized field soil spiked with purified insectic idal protein. This approach is useful because dose responses can be easily quantif ied. However, the degradation and accumulation of Cry proteins found within decaying plant tissue may be have differently than proteins in artificially spiked soil. Thus, the relevance of these study results is unclear other th an to show that degradation in soil does take place.

To account for the above concerns, it is recommende d that additional studies should be conducted to evaluate insecticidal protein degradation, accumula tion, and persistence in a variety of soil types, including those high in clay and hum ic acids, into which all non-harvested corn plant material is incorporated. Sampling should be conducted each year for three years in a fiel d sown with continuous

MIR604 corn. Soil should be monitored for a mini mum of one growing season after harvest and monitoring should continue until mCry3A protein can no longer be detected. As noted in Table 16 below, the Agency has requested th at the applicant submit this study as a condition of registration.

These recommendations are based in part on th e August 27, 2002 SAP and public comments provided

to EPA at that time. This Panel concluded that several different soils should be examined and monitored for a minimum of one growing season after corn harvest and that monitoring should continue until the Cry protein can no longer be detected. According to the Panel, at least three soil types should be evaluated for Cry pr otein persistence and these soils should be high in organic matter and clay, since the potential of pe rsistence is highest in these soil types. The Panel also recommendedModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

63 that soil degradation studies may be conducted u nder less than optimum environmental conditions, such as high or low temperatures, since protein pe rsistence may be affected by varying environmental conditions. The Panel further suggest ed that since corn roots grow into deep soil with reduced microbial activity, degradation rates in these zones should also be examined. With regards to protein source, the Panel recommended that st udies should utilize plant material that is representative of actual field conditions. For example, soil samples should be collected from field sites where whole plant tissue has been incorporated into the field. And fu rther, plant tissue should not be ground prior to incorporation, because grinding artificially incr eases the plant tissue surface area exposed to microorganisms, and increased surface area may lead to an increase in the rate of protein degradation. Finally, the SAP stated that “(r)eal lif e or true persistence (or Cry protei n) is likely to be equal to or

less than that measured with ELISA.” So if an ELISA is conducted, results should be compared to a bioassay that uses a sensitive species such as the Colorado potato beetle.

3. Effects on Soil Microorganisms

2010 Update: Numerous published studies indicate that exposure to Cry protein produced in Bt PIP crop plants does not adversely a ffect 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 -corn leaves for the meso- and macrofauna soil community (Hönemann et al

. 2008). Although a minimal transient increase and shift in microbial populations may resu lt from the presence of tr ansgenic plant tissue in soil, no adverse effects have been attributed to the Cry protein.

In addition, there are several ongoing U.S. Department of Agriculture and EPA O ffice of Research and Development funded research projec ts evaluating the effects of Bt crops on soil microbial flora. If

adverse effects are seen from this or any other research, the Agency will take appropriate action to mitigate potential risks.

With regard to the impact of genetically 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 variat ions in seasons and weather, plant growth stage, and plant varieties, independent of being genetically en gineered, are also responsible for significant shifts in soil microbial communities. To date, most studies with genetically engineered crops have shown minor or no effects on soil microbes beyond the variation cau sed by the factors listed above.

4. Horizontal Transfer of Transgenes from Bt Crops to Soil Organisms

2010 Update: EPA has evaluated the potential for horizontal gene transfer (HGT) from Bt crops to soil organisms and has considered po ssible risk implications if such a transfer were to occur. Genes that have been engineered into Bt crops are mostly found in, or have their origin in, soil-inhabiting

bacteria. Soil is also the habita t of anthrax, tetanus, and botulinum toxin-producing bacteria. Transfer64 of these genes and/or toxins to other microorganisms or plants has not been detected. Furthermore, several experiments (published in sc ientific journals), that were c onducted to assess the likelihood of HGT, have been unable to detect gene transfer under typical environmenta l conditions. Horizontal gene transfer to soil organisms has only been detected with very promiscuous microbes under laboratory conditions designe d to favor transfer.

As a result of these findings, which s uggest that HGT is at most an artif icial event, and th e fact that the

Bt toxins engineered into mCry3A corn are derive d from soil-inhabiting bacteria, EPA has concluded that there is a low probability of risk from HGT of transgenes found in mCry3A-producing corn.

5. Gene Flow and Weediness Potential

Conclusions gathered from this review process are as follows:

• The potential for pollen-directed gene flow fr om corn to Eastern gamagrass is extremely remote (DeWald et al . 1999). This is evidenced by the difficulty with which Tripsacum dactyloides x Z

. mays hybrids are produced in structured breeding programs. Additionally, the

genus Zea does not represent any species considered as serious or pernicious weeds in the U.S., its possessions, and/or its territories (Holm et al . 1979). Any introgression of genes into this species as a result of cross fert ilization with genetically modified corn is not expected to result in a species that is weedy or difficult to control. In many instances where hybridization has been directed between these two species, the resu ltant genome is lacking in most or all of the corn chromosomal complement in subsequent generations (DeWald, personal communication, 1999).

• Many of the Zea species loosely referred to as “teosi ntes” will produce viable offspring when

crossed with Zea mays ssp. mays . However, none of these plants are known to harbor weedy characteristics and none of the native teosinte spec ies, subspecies, or races are considered to be aggressive weeds in their native or introduced habitats (Schoper, personal communication, 1999). In fact, many are on the brink of extinction where they are indigenous and will be lost without human intervention (i.e., conservation m easures). Further, none of the landraces or

cultivated lines of Z

. mays are considered to have weedy pot ential and are generally considered to be incapable of survival in the wild as a result of breeding practices (i.e., selection) during domestication of the crop.

The October 2000 Scientific Advisory Panel agreed th at the potential for gene transfer between corn and any receptive plants within the U.S., its possessions , and/or its territories wa s of limited probability and nearly risk free. Based on these findings, the EP A has determined that there is no significant risk of gene capture and expression of the mCry3A protei n by wild or weedy relatives of corn in the U.S., its possessions, or its territories.

2010 Update: Movement of transgenes from crop plants into weeds is a signi ficant concern, due to uncertainty regarding the effect th at a new pest resistance gene may have on plant populations in the wild. Under FIFRA, EPA has reviewed the pot ential for gene capture and expression of Bt endotoxinsModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

65 by wild or weedy relatives of corn, cotton, and po tatoes in the U.S., its possessions, and/or its territories. To date, Bt plant-incorporated protectants have been registered for use in agronomic plant species that do not have a reasonable possibility of passing th eir traits to wild native plants. However, due to concern over the possibility that species related to corn ( Zea mays ssp. mays ), such as Tripsacum species and the teosintes, coul d be recipients of gene flow from genetically modified Z

. mays , EPA conducted a thorough review of the scientific literature on what is known about the gene flow potential of Z

. mays (U.S. EPA 2001).

6. Impacts on Endangered Species

The primary route of exposure to mCry3A protein in corn is through ingestion of corn tissue. There are no reports of threatened or endangered species feedi ng on corn plants, therefore such species would not be exposed to corn tissue contai ning Cry protein. Since mC ry3A protein has not been shown to have toxicity effects on mammals, birds, plants, aquatic sp ecies, insects and other invertebrate species at the EEC, a "may affect" situation for endangered land a nd aquatic species is not anticipated. In addition, EPA does not expect that any threatened or endangere d plant species will be affected by outcrossing to wild relatives or by competition with such entities. Hybrid corn does not exist in the wild, nor are there wild plants that can interbreed with corn in the United States.

Because of the selectivit y of mCry3A protein for coleopteran sp ecies, endangered species concerns are mainly restricted to the order Coleoptera. Examin ation of an overlay map showing the county level distribution of the 16 endangered/thr eatened coleopteran species (curre ntly listed by the U.S. Fish and Wildlife Service) relative to corn production countie s in the United States cl early indicated that any potential concern regarding range overlap with corn production was mainly restricted to the American burying beetle ( Nicrophorus americanus ). The American burying beetle is the largest carrion beetle in North America and is only found in limited areas of Rhode Island and portions of the Great Plains, including Arkansas and Georgia. Adults are nocturnal and feed on carrion and sometimes prey on other arthropods. Larvae feed exclusivel y on buried carrion provided by their parents. The American burying

beetle’s habitat is variable and often includes deciduous forest, grassla nd and agricultural areas. Considering that both larvae and adult insects feed exclusively on carrion, it appears that even if American burying beetles did occur in proximity to Bt corn fields, there woul d be little chance of

exposure to Bt protein due to their feeding habits. After careful review of available data, the EPA determined that exposure of American burying beetle to harmful levels of MIR604 corn tissue is not expected. Likewise, a review of the preferred habita ts of other coleopteran species listed as endangered by the U.S. Fish and Wildlife Service indicated that exposure to harmful levels of mCry3A protein would not take place. The main reasons for the lack of exposure are ge ographical and habitat limitations. These species are loca ted in non-corn production areas and/or their habitat does not encompass agricultural areas.

Likewise, other insect species in the orders Diptera, Hemiptera, Lepidoptera, Odonata and Orthoptera that are listed as endangered/threatened species are found in dune, meadow/p rairie or open forest habitats and are not closely associ ated with row crop production, often times due to the specificity of66 the habitat of their host plants. The reviewed toxi cological data shows the relative insensitivity of a range of insects in non-Coleopteran orders to the mCry3A protei ns, indicating that MIR604 maize hybrids are not likely to have detrimental e ffects on non-Coleopteran insects included on the endangered/threatened species list.

Further, several of the federally listed insect spec ies are aquatic and conseque ntly, are unlikely to come in contact with MIR604 maize plant material. Many of the endangered and threatened beetles occur in cave or aquatic habitats. Since move ment into water bodies of soil containing mCry3A is expected to be negligible, pollen drift was considered the prim ary source of potential hazard to endangered aquatic Coleoptera. According to estimates based on published studies, if 100% of the pollen grains leaving a corn field were deposited in a 1 ha pond with 2 m depth and located ≥ 1 m from the edge of a corn field, <0.0001 μg mCry3A/mL of wate r would be expected. This is a few orders of magnitude below the toxic level to any insect.

2010 Update: Current ecological effects data and EPA revi ews of mCry3A protein, as expressed in

MIR 604 and in the stacked event MIR 604 x Bt11, expressing mCry3A x Cry1Ab, support the Agency’s determination that adverse effects w ill not occur to nontarget organisms. Due to a demonstrated lack of toxicity a nd/or exposure, no effects from mC ry3A protein, as expressed in MIR 604 and in the stacked events expressing Bt11 x mC ry3A x Cry1Ab, are anticipated for any nontarget species, including federally-listed th reatened and endangered (“listed ”) lepidopteran and coleopteran species and their designated critical habitats. EPA has also determined that there are no indirect effects on endangered and threatened plant species, such as impacts on lepidopteran pollinators that are important and/or essential to an en dangered or threatened plant. Th e Agency is therefore upholding its determination that the registered uses of mCry3A protein as expre ssed in MIR 604 and in the stacked events expressing Bt11 x mCry3A x Cry1Ab will have “No Effect,” direct or indirect, on endangered or threatened terrestrial or aquati c species as listed by the U.S. Fish and Wildlife Service (USFWS) and the National Marine Fisheries Services (NMFS).

B. Environmental Risk Assessment

The EPA uses a Maximum Hazard Dose Tiered syst em for biopesticide non-target wildlife hazard

assessment. When no adverse effects at the maximu m hazard dose are observed, the Agency concludes

that there are no unreasonable adverse eff ects from the use of the pesticide.

At present, the Agency is aware of no identified si gnificant adverse effects of mCry3A proteins on the abundance of non-target beneficial organisms in any population in the field environment, whether they are pest parasites, pest predators, or pollinators. Further, the EPA believes th at cultivation of mCry3A corn may have fewer adverse impacts on non-target orga nisms than use of chemical pesticides for corn production, because under normal circumstances, mCry3A corn requires substantially fewer applications of chemical pestic ides, compared to production of non-Bt corn. And fewer chemical insecticide applications ge nerally result in increased populations of beneficial organisms that control secondary pests, such as aphids and leafhoppers, in corn fields. In addition, no adverse effect on endangered and threatened species listed by the US Fish and Wildlife Service is expected from theModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

67 proposed MIR604 CRW resistant corn registration (see Section A.II.6 above). Further, the EPA has determined that there is no significa nt risk of gene capture and expres sion of mCry3A protein by wild or weedy relatives of corn in the U.S., its posse ssions, or territories (see Section A.II.5 above),

available data do not indicate that Cry proteins have any measurab le adverse effect on microbial populations in the soil (see Section A.II.3 above), nor has horizontal transfer of genes from transgenic plants to soil bacteria been demonstrated (see Sect ion A.II.4 above). In conclu sion, this risk assessment finds no hazard to the environment at the present time from cultivation of mCry3A protein expressing MIR604 corn for a time-limited registration.

C. Supplemental Studies Needed for Long T erm MCry3A Non-Target Hazard Assessment

The Agency has sufficient information to believe th at there is no risk from the proposed uses of mCry3A corn to non-target wildlife, aquatic, and soil organisms. Howe ver, in response to the August 2002 SAP recommendations, the Agency is requesting s upplementary studies that will evaluate the persistence of mCry3A in the soil and the l ong range effects of cultiv ation of mCry3A on the invertebrate community structure in corn fields. This will facilitate identification of potential adverse effects which may result from long-term use of this product.

Table 16. Supplemental data requirements for Event MIR604 corn.

Testing Category Type of Data

Ecosystem effects Long range field studies s hould be conducted based on recommendations of the August, 2002 SAP.

Soil fate studies Long range soil degradation fiel d studies should be conducted. Studies should follow guidelines outlined by the August 2002 SAP, which are presented in summary fo rm in the conclusion section of the soil fate review (s ee Section A.II.2 above).68 References DeWald, Chester. (1999). Pers onal communication. Plant Breeder and Geneticist, USDA-ARS, Woodward, OK, December, (580_256_7449).

DeWald, C.L., P. Sims, Y. Li, and V.A. S okolov. (1999). A novel cytoplasm for maize. Maize

Genetics Conference Abstracts. 41:P114.

Holm, L, J.V. Pancho, J.P. Herberger, and D.L. Plucknett. (1979). In: A geographical atlas of world

weeds . (pp. 391). John Wiley and Sons, New York.

Hönemann L, Zurbrügg C, Nentwig W. 2008. Effects of Bt -corn decomposition on the composition of

the soil meso- and macrofauna. Applied Soil Ecology 40:203–209.

Oliveira AP, Pampulha ME, Bennett JP. 2008. A two-year field study with transgenic Bacillus thuringiensis maize: Effects on soil microorganisms. Science of the Total Environment

405:351–357.

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 transgen ic crop byproducts may affect headwater stream ecosy stems. PNAS , vol. 104, no. 41, 16204–16208.

Sanvido O, Romeis J, Bigler F. 2007. Ecological imp acts of genetically modifi ed crops: ten years of

field research and co mmercial cultivation. Advances in Biochemical Engineering and Biotechnology 107:235–278.

Saxena D, Stotzky G. 2001. Bacillus thuringiensis ( Bt ) toxin released from r oot exudates and biomass

of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in

soil. Soil Biology and Biochemistry 33:1225–1230.

Schoper, J. (1999). Personal communication. Geneticis t, Pioneer Hi-Bred Inte rnational, Johnston, IA, (515-270-3544).

U.S. EPA (2000). SAP report No 99-06. Sets of scientific issues be ing considered by the Environmental Protection Agency regarding: Section I - Characteriz ation and Nontarget Organism Data Requirements for Protein Plant Pesticides. Dated February 4, 2000. Available at the EPA website: http://www.epa.go v/scipoly/sap/1999/index.htm#december

U.S. EPA. (2001). Biopesticides Registration Action Document – Bacillus thuringiensis Plant- Incorporated Protectants. Available from: http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm

.

U.S. EPA (2001). SAP Report No. 2000-07. Sets of scientific issues being considered by the Environmental Protection Agency re garding: Bt plant-pesticides risk and benefit assessments. Dated March 12, 2001. Web site:Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

69 http://www.epa.gov/scipoly/sap/200 0/october/octoberfinal.pdf

U.S. EPA (2002). SAP Report No. 2002-05. A set of scientific issues being considered by the Environmental Protection Agency regarding: Co rn rootworm plant-inco rporated protectant nontarget insect and insect re sistance management issues. Dated November 6, 2002. Web site: http://www.epa.gov/scipoly/sa p/2002/august/august2002final.pdf

U.S. EPA (2010). Review of Syngenta’s Request to Waive a Soil Persistence Field study, required as a Condition for the Sec. 3 Registration of Ev ent MIR604 corn [EPA File Symbol 67979-5] submitted by Syngenta Seeds, Inc.; MRID N o. 478477-01; Decision No. 420150; DP Barcode

372882. Memorandum from A. Gross and Z. Vaituzis to M. Mendelsohn dated July 7, 2010.

Vaituzis, Z. and R.I. Rose. (2000). Reassessment of Bt crop effects on nontarget wildlife. Biopesticides

and Pollution Prevention Division. U.S. Envi ronmental Protection Agency. Washington, D.C.

II. D. Insect Resistance Management

1. BACKGROUND

Syngenta submitted an amended insect resistance management strategy in support of their FIFRA Section 3 Registration for Event MIR604-derived tr ansgenic maize that expresses modified Cry3A (mCry3A) protein (MRID# 465296-01, McCaffery et al., 2005). Modified Cry3A in MIR604 is expressed at a non-high dose against infestat ion by the Western corn rootworm (WCRW; Diabrotica virgifera virgifera Le Conte) and the Northe rn corn rootworm (NCRW; D. longicornis barberi Smith and Lawrence). A behavioral avoidance mechanism is also present. BPPD reviewed Syngenta’s previously submitted IRM strategy and noted several deficiencies in the simulation modeling and evaluation of cross-resistance pot ential (BPPD, 2005). These defici encies were addressed in the amended IRM strategy submission (McCaffery et al., 2005, MRID# 465296-01) and are discussed in this chapter.

2. SUMMARY OF SYNGENTA’S SUBMISSION (2004 - 2005)

Syngenta’s submission is divided into three main pa rts: 1) Sustainable de ployment of Event MIR604 maize through effective insect resistance manage ment (IRM), 2) IRM plan for MIR 604, and 3) Justification for the proposed IRM plan for MIR604. The first two sections of the revised Syngenta’s submission were originally submitted by Syngenta (McCaffery and Stein, 2004) and reviewed by BPPD (BPPD, 2005). There were significant defi ciencies identified in the IRM modeling and evaluation of cross-resistance poten tial. Syngenta has addressed thes e deficiencies in the section entitled, “Justification for the pr oposed IRM plan for MIR604.”

Syngenta’s IRM plan for MIR604 maize co ntains the following elements:70 1. 20% structured refuge and its deployment and management;

2. IRM as a component of an integrated pest management program; and

3. Product stewardship program.

The product stewardship program consis ts of the following aspects:

1. Grower education plans;

2. Grower obligations;

3. Grower compliance;

4. Annual resistance monitoring;

5. Action plan in the event of unexp ected levels of CRW damage;

6. Remedial action plans; and

7. Annual IRM plan review.

Syngenta has provided the following areas of sup port for its proposed IRM plan for MIR604 maize:

1. Dose of mCry3A expressed in MIR604 (root expression assays, labor atory feeding assays using excised roots, field efficac y, and adult emergence studies);

2. Simulation models for development of resistance;

3. Impact on MIR604 maize on biology of CRW;

4. Cross-resistance potential.

3. BPPD REVIEW (BPPD, 2005; BPPD, 2006a)

a. Nature and Derivation of Syngenta’s MIR604 CRW-Control Maize

A cry3A gene from Bacillus thuringiensis subsp. tenebrionis ( B.t.t. ) (Sekar et al., 1987) was synthesized by Syngenta scientists for optimal expression in maize. The gene was then modified (hereinafter referred to as “m Cry3A” or “modified Cry3A”) to enhance activity against the Diabrotica virgifera virgifera (Western corn rootworm, WCRW) and Diabrotica longicornis barberi Smith and Lawrence (Northern corn rootworm, NCRW). The introduction of a particular serine protease recognition site (both chymotrypsin and cathepsin G are serine protease s) into the native Cry3A results in a much more rapid and complete processing of the 67 kDa protein to 55 kDa and thus increased activity against WCRW and NCRW. After binding to the receptor, the mCry3A protein inserts rapidly and irreversibly into the plasma membrane of the ce ll to form ion permeable pores in a similar fashion as other Cry toxins (Van Rie et al., 1989; Garcia-Alonso and Vlachos, 2003). These ion channels cause a loss in membrane potential and the formati on of these lesions leads to death resulting from septicaemia and/or starvation (Schnepf et al. , 1998).

b. Corn Rootworm Species Controlled

Native Cry3A is primarily active against Colorado potato beetle ( Leptinotarsa decemlineata ) and has minimal activity against NCRW, both members of th e Chrysomelidae family of beetles. The mCry3A protein has a similar spectrum of activity to the native Cry3A, but with enhanced toxicity to NCRWModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

71 and WCRW, both major coleopteran pests of maize in the USA. Modified Cry3A has some activity against Diabrotica balteata (Banded cucumber beetle). Trials were conducted in 2005 (data not available for this review) to determine the level of control of Diabrotica virgifera zea (Mexican corn rootworm; MCRW) by mCry3A expressing maize plan ts (Event MIR604). Modified Cry3A has no activity against non-cole opteran pests including various lepidopter an pests of maize. MIR604 maize is most effective against larvae, esp ecially the first instar. Syngenta’ s IRM strategy focu ses entirely on

rootworms.

c. Pest Biology

A clear understanding of pest biol ogy and ecology is essential to the development of a sound IRM plan. MIR604 Bt corn was developed to control two primary corn rootworm species: western corn rootworm ( Diabrotica virgifera virgifera LeConte, WCRW) and northern corn rootworm ( D. Barberi Smith & Lawrence, NCRW). WCRW is the most pr evalent target pest in the United States and throughout most of the Corn Belt 1 . NCRW, also found throughout the Co rn Belt, is considered the second most prevalent rootworm pest in the United St ates, and is the primary target pest of the northcentral region 2

.

Key factors believed to influence CRW adaptation to MIR604 corn include distribution, univoltinism, adult dispersal among fields, adult dispersal within fi elds, larval dispersal across rows, larval mortality due to density-dependent processes, insecticide us e, egg mortality, fecundity, and adult and larval population density. Syngenta has provided a suffi cient summary of the biology and ecology of the corn rootworm target pest(s) bot h in the current IRM strategy submission (McCaffery et al., 2005, MRID# 465296-01) and the original IRM strategy submission (McCaffery and Stein, 2004, MRID# 462656-17). See BPPD (2005) for the review of Syngenta’s original IRM strategy submission and pest biology information.

d. IRM Plan for MIR604

Syngenta’s IRM plan for MIR604 ma ize contains three major compone nts: 1) a 20% structured refuge; 2) IRM is part of an integrated pest mana gement program; and 3) product stewardship. Each of these components will be discussed below.

1) 20% structured refuge and its deployment and management

1 WCRW is the primary rootworm pest in Colo rado, Kansas, Nebraska, Ohio, Indiana, Illinois, Iowa, Missouri, and Michigan.

2 NCRW is the primary rootworm pest in Wi sconsin, Minnesota, South Dakota and northern

Iowa.72 There are two ways a grower can implement the re fuge requirement: a non-Bt corn refuge can be planted as a continuous block adjace nt to the MIR604 maize fields or as non-transgenic strips planted within transgenic field. Considering the limited movement of CRW larvae, planting refuges close to transgenic fields in large bloc ks is preferred to narrow strips (Gray 1999, Meinke et al. 2001). Syngenta will require growers to plant a minimum structured refuge of non-corn rootworm-control (CRW) maize on at least 20% of their maize acres. Refuge fields must not be planted with other transgenic corn used to control rootworm because neit her acts as a refuge for the other. Use of a 20% refuge with MIR604 maize complements other tec hnologies and provides a degree of uniformity for CRW control as well as with pr oducts with both CRW and lepidopter an control. This uniformity provides growers with a straightfo rward and understandable message about refuge requirements and promotes compliance. Justification of the 20% ref uge requirement is discusse d later in this review.

Syngenta will require growers to plant the structured refuge as blocks, stri ps, perimeter borders or pivot corners. Encouragement will be given to growers to plant these non-CRW control corn acres within their CRW-control acres. Syngenta has proposed that in-field strips be at least 6 to 12 nonrootworm protected Bt corn rows, although this row width size may be larger than necessary based on the current understanding of root worm biology. BPPD has reviewed larval movement data published by Hibbard et al. (2003). This st udy indicated that betw een 0.75% and 6% of larvae moved across corn rows. These results represent a relatively hi gh-end estimate of the numb er of larvae that cross

rows. This means that much narrower in-field strips should be sufficien t to provide adequate protection from sub-lethal select ion caused by CRW larval movement across rows and maintain low functional recessiveness. Any increas e in sublethal selection would be offset by a greater probability that potentially resistant adults emerging from the Bt corn rows w ould be mated by susceptible adults from the refuge row. Single-row strips would likely be too narrow and allow too much larval

movement across rows to sufficientl y maintain low functional recessive ness. Therefore, seed mixes would not be a good refuge strategy for rootworms. In-field strips of ≥ 4 rows would provide the advantage of being more compatible with the curren t in-field strip width requirement for lepidopteranprotected Bt corn hybrids ( ≥ 4 row strips, with ≥ 6 row strips preferred) . In-field strips of ≥ 4 rows would also be more practical and fl exible for the grower b ecause of the compatibility with split-planter designs. Because mCry3A will be stacked with Cry1Ab (submission pending), a recommendation of

≥ 4 row strips will provide the grower a more easily understandable and consistent message regarding the width of in-field strips. Overall, BPPD’s recommendation is that Syng enta require growers to plant in-field strip re fuges with widths of ≥ 4 rows ( ≥ 6 rows preferred) for rootworm-protected Bt corn hybrids. This will simplify refuge deployment and pot entially increase grower compliance with refuge requirements. Use of an in-field strip refuge is no t intended for fields planted to increase inbred seed

since these fields need to be isolat ed from external corn pollen sources. An in-f ield or adjacent non-Bt corn refuge would be inconsistent w ith inbred seed production practices.

Management of the structured refuge must confor m to the rotational and management practices to achieve synchronous CRW development in both refuge and MIR604 maize fields . Growers will have the option of treating the re fuge with conventional insecticides (s eed treatments, soil applications and foliar applications) to control severe damage by CRW and to c ontrol secondary pests. These treatments will impact the overall number of beetles that emerge from the refuge fields even thoughModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

73 their efficacy is less than 100%. Syngenta’s m odeling study examines one scenario in which a nominal 50% of refuge insects are removed by in secticide control that impacted the potential development of resistance (see modeling section belo w). Syngenta indicates that there is no evidence for any cross-resistance, syner gy, or antagonism between the mCr y3A in MIR604 maize and Cry1Ab and other Bt toxins in Lepidoptera-control maize varieties. There are al so no known interactions between mCry3A and insecticides.

Syngenta notes that the use of continuous corn acres for refuge fields is always to be encouraged. It is also permissible for growers to plant the refuge acr es on first-year corn acres, but only where the CRW-control corn is also planted on first-year corn acres. This dis tinction is made for areas in which WCRW has adapted to crop rotation and sizable populations of these in sects will emerge from firstyear corn. There is also a vari ant of NCRW in which there is extended diapause. Both rotationresistant CRW variants impact the efficacy of crop rotation.

2) IRM is Part of an Integrated Program

Syngenta stressed that the IRM strate gy for MIR604 maize is part of an overall package of integrated crop management techniques. These tactics include:

1. Crop rotation. Growers will be encouraged to ma intain crop rotation as a vital part of CRW management. The presence of rotation-resist ant phenotypes, however, may render such an approach ineffective in some areas.

2. Refuge quality. Growers will be educated on management practices to maximize the effectiveness of their refuges.

3. Insecticide use. When moderate to high CRW population pressures in the refuge, growers will be encouraged to treat the refuge with a soil in secticide or use seed treatments in a manner to

maintain overall survival of CRW to func tion effectively as re fuge mating partners.

3) Product Stewardship

Growers are crucial to the success of any IRM plan because they are responsible its implementation on the practical level. Syngenta has developed a product stewardship progr am that stresses the importance of implementation of the IRM plan fo r MIR604 maize. Syngenta’s product stewardship program consists of the following aspects:

1. Grower education plans (e.g., Syngenta guide);

2. Grower obligations (grower agreements);

3. Grower compliance;

4. Annual resistance monitoring;

5. Action plan in the event of unexp ected levels of CRW damage;

6. Remedial action plans; and

7. Annual IRM plan review.74 Compliance programs are important in that they enc ourage growers to comply with IRM requirements, while providing mechanisms by which registrants can be held accountable fo r noncompliant growers. The compliance program presented in this s ubmission mirrors those developed for existing lepidopteran-protected Bt maize registrations . The specific compliance assurance program components are: 1) Annual IRM Survey, 2) Mech anism for Handling Tips and Complaints, 3) OnFarm Visits, and 4) Phased Compliance Approac h. Syngenta will respond to instances of noncompliance through the Phased Compliance Approach and will also use grower agreements and an annual affirmation scheme to reinforce grower u nderstanding and compliance. Grower education programs give growers a clear understanding of the importance of IRM and its implementation. The IRM program will be communicated to growers by S yngenta through its authorized sales agents using both print and other media includi ng workshops, educational pamphlet s/brochures, and an assortment of public relation activities. BPPD finds Syngenta’s compliance assurance program to be “acceptable” with the following caveats. The EP A-approved changes (granted on June 16, 2006) to the annual IRM reporting requirements and the Co mpliance Assurance Program for the lepidopteranprotected Bt maize registrations should be incorporated into S yngenta’s IRM Compliance Assurance Program for MIR604 Corn Products.

The need for proactive resistance detection and monitoring is critical to the survival of Bt technology. Consequently, the Agency mandates that a resistan ce monitoring plan must be implemented for all registered Bt maize products. Resistance can evolve regionally or as a local in crease in resistance ( r -) allele frequency. The resistance monitoring plan de signed for MIR604 maize is an adaptation of the ABSTC program developed for lepidopteran-protected Bt maize. This program will attempt to detect either local or regional resistance early enough to initiate effective re medial action. As for all other Btmaize products, Syngenta’s resistance monitoring program will be implemented through a twopronged approach, including field reports of unexpect ed damage and population testing and sampling. The initial emphasis of their monitoring plan will be on establishing the baseline susceptibility for

WCRW. Growers will be asked to report to Syngenta any unexpect ed field damage. Diagnostic bioassays of larvae from eggs laid by field-co llected WCRW adults will be used to survey susceptibility of the insects to mC ry3A. The monitoring efforts will focus on regions of the Corn Belt where WCRW populations are known to regularly r each high numbers and where the highest MIR604 adoption is expected. Detecting shif ts in the frequency of resistance genes (i.e., susceptibility changes) through resistance monitoring can be an aggressive me thod for detecting the onset of resistance prior to widespread crop failure. As such, the utilization of sensitive and effective resistance monitoring techniques is critical to the success of an IRM pl an. BPPD finds that Syngenta’s basic resistance monitoring plan and remedial action plan for th e mCry3A protein expre ssed in MIR604 maize are “acceptable,” although certain issues need to be addressed. These are discussed below.

1. The monitoring plan only focuses on WCRW. CRW species are challenging to rear in the laboratory. Of the identified target pest specie s, scientists have had the most success rearing WCRW, and have had little suc cess with NCRW. Consequentl y, BPPD agrees with Syngenta that initial monitoring thr ough population sampling will focus on WCRW, but should include NCRW populations when availabl e. No information exists on MCRW. These data should be provided to BPPD when they become available.

2. A diagnostic method cannot be de veloped until the dose-response relationships for WCRW toModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

75 establish the baseline sensitivity to mCry3A. Wh ile BPPD agrees that a sensitive and reliable bioassay should be developed, these efforts are cu rrently under development. Once this work is completed, the registrants must develop guidelines as to what level of root damage will be expected under various conditions , and what level of rootworm control is normally achieved. Growers will have to report a ny “unexpected damage.” Without such guidelines, i.e., what is “acceptable” rootworm damage, then it will not be possible to determine what the “unexpected” rootworm damage is. Once these guidelines are es tablished it will be possible to define what is “suspected resistance” as described under the “R emedial Action Plans” to mitigate the spread of putative resistant populations . Because of the importance of these guidelines, it should be required that the registrant develops an inte rim rootworm damage guidelines by 2008 and final guidelines by 2010 and submits these to the Agency for review.

It is recommended that MIR604 ma ize be given the following resi stance monitoring requirements:

1. The registrants should monitor for resistance and/ or trends in increased tolerance for corn rootworm. Sampling should be focused in those areas in which there is the highest risk of resistance development. The registrants should submit to EPA an appropriate sampling protocol as part of its monitoring plan.

2. The registrants should provide EPA a description of its resistance monitoring plan by January 31, 2007. The description would include: samp ling (number of locations and samples per locations), sampling methodology, bioassay methodology, standardization procedures (including QA/AC provisions), detection technique and sensitivity, and the statistical analysis of the probability of detecting resistance. A final resistance monitoring plan is required by January 31, 2008.

3. The registrants should develop an appropriate discriminating or di agnostic dose assay by January 31, 2008.

4. The registrants should follow-up on grower, exte nsion specialist or c onsultant reports of unexpected damage or control failures for corn rootworm.

5. The registrants should provide EPA with an annual resistance monitoring report.

The remedial action plan is designed as a tiered approach for mitigating potential WCRW, NCRW, and MCRW resistance development to the Cry34/35Ab1 prot ein. BPPD agrees with the general framework for the Remedial Action Plan; however, because the “b aseline sensitivity” has not been calibrated, this plan cannot be implemented. The submission states that mitigation measures will be initiated when unexpected levels of CRW damage occur. But, leve ls of “expected” damage cannot be identified until baseline sensitivity is determined (see discussion above “Resistance monitoring”). Consequently, it should be required that baselin e sensitivity be established within two years of product commercialization, so that expected levels of cr op damage and target pest resistance can be established, and a remedial action plan initiated when needed.76 The following program summary describes, in order of events, the steps that should be taken to implement a remedial action plan if resistance to target pests is c onfirmed (this general process has been implemented for other lepidopte ran and CRW Bt corn products).

1. Definition of Suspected Resistance: Resistance will be suspected if investigations of unexpected damage reports show that:

a. Implicated maize plant roots were expressing the mCry3A protein at the expected level;

b. Alternative causes of damage or lodging, such as non-target pe st insect species, weather, physical damage, larval movement from a lternate hosts, planti ng errors, and other reasonable causes for the observations, have been ruled out;

c. The level of damage exceeds gui delines for expected damage.

If resistance is “suspected”, the registrants will inst ruct affected growers to use alternate pest control measures such as adulticide treatment, crop rota tion the following year, or use of soil or seed insecticides the following year. These measures ar e intended to reduce the po ssibility of potentially resistant insects contributing to th e following year’s pest population.

2. Confirmation of Resistance : Resistance will be confirmed if all of the following criteria are met by progeny from the target pest species sample d from the area of “suspected resistance”:

a. The proportion of larvae that can feed and surv ive on mCry3A roots from neonate to adult is significantly high er than the baseline proportion (currently being established);

b. The LC 50 of the test population exceeds the upper lim it of the 95% confidence interval for

the LC 50 of a standard unselected population and/ or survival in the diagnostic assay is significantly greater th an that of a standard unselecte d population, as established by the ongoing baseline monitoring program;

c. The ability to survive is heritable;

d. mCry3A plant assays determine that damage caused by surviving insects would exceed economic thresholds;

e. The identified frequency of field resistance could lead to widespr ead product failure if subsequent collections in the affected field area(s) demonstrated similar bioassay results.

3. Response to Confirmed Resistance: When resistance is “confirme d”, the following steps will be

taken:

a. EPA will receive notification within 30 days of resistance confirmation;

b. Affected customers and extension agents will be notified about confirmed resistance;

c. Affected customers and extension agents will be encouraged to employ alternative CRW control measures;

d. Sale and distribution of mC ry3A maize in the affected area will cease immediately;

e. A long-term resistance management action plan will be devised according to the

characteristics of the resistance event and loca l agronomic needs. [The details of such a plan should be approved by EPA and all appropria te stakeholders. This process may takeModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

77 longer than 30 days following confirmation of resistance.]

e. Support for Syngenta’s IRM Plan for MIR604 Maize

1) Dose

Identifying the level of dose, as related to selec tion intensity, is crucial wh en determining size and structure of a refuge needed to delay CRW resistance to MIR604 ma ize. Syngenta conducted a series of field efficacy trials across the Corn Belt in 2003 and 2004 to assess dose. This information was used in the modeling study detailed later in this review. Additiona l data were obtained from root expression assays and in vitro feeding assays in the laboratory. Based on the review of Syngenta’s data, it can be concluded that MIR604 maize doe s not provide a high dose for CRW control.

a) Root expression assays

Levels of expression in leaves, ro ots, and whole plants were determ ined at two developmental stages for two MIR604 hybrids. Levels of expression were num erically higher in leaves than in roots at both the whorl stage and at anthesis a nd somewhat higher in roots at the whorl stage than at anthesis, but there were few, if any statistica lly significant differences at p<0.05. Root expression was found to be uniform throughout the root architect ure implying that there are no mi crohabitats. Therefore, larvae feeding on MIR604 roots should be uniformly expos ed to the mCry3A toxin. Average values of mCry3A in roots range from 1.8 to 3.0 μg mCry3A/g fresh weight.

b) WCRW laboratory feeding assays

In the first laboratory feeding study, nine replicate groups of ten larvae were placed in plastic dishes containing 3 cm root sections exci sed from MIR604 hybrids. Five of nine replicates exhibited only minimal root damage ( ≤ 1 feeding hole) with an average of 22% mortality; while, three of nine replicates exhibited two to three feeding holes with an average of 13.3% mortality. Only one replicate had more than four or more feeding holes.

In the second laboratory feeding stud y, nine replicate groups of ten larv ae were placed in plastic dishes were given a choice of two 3 cm root sections excised from MIR604 and non-transgenic (control) hybrids. In this second experiment, larvae preferre d the non-transgenic hybrid to the MIR604 hybrid. Approximately 75% of non-transgenic hybrid roots were heavily damaged with more than four feeding holes per root section. As in the fi rst experiment, the mortality levels on transgenic root sections were low, only 17%. A third experiment, using a similar choice protocol, produced similar results to the second experiment, but there was considerably more variation.

Following investigations by Syngenta and its co-operato rs, there is evidence for both a direct mortality

mechanism of first instar CRW larvae as well as evidence that MIR604 hybrid s have properties that might deter feeding. Thus, in terms of root prot ection, MIR604 plants exhib it a much higher dose than78 would otherwise be assumed from the consideration of the mortality data alone. The behavioral

deterrence mechanism needs to be studied further by Syngenta.

c) Field efficacy

In 2003 and 2004 Syngenta conducted field-based studi es of MIR604’s efficacy against WCRW and NCRW and study results were used to develop models of MIR604. Trials included naturally and artificially infested sites a nd study endpoints were survival un til adult stage, timing of adult emergence, sex ratio, and adult weight. Both artifici al and natural infestation trials were conducted. A brief summary of the results is provided below.

Efficacy trials compared root damage among MIR604 and non-MIR604-derived corn hybrids. Experimental plots were artificially infested (400 to 1450 eggs/trial), located in fields with naturally occurring populations of corn r ootworm, or both approaches to in festation were combined. When artificially infestation was used, at the V2 to V3 leaf stage rootworm eggs were mechanically placed 3 inches deep in soil, within 2 to 3 inches of the corn stalk base.

i. Artificial Infestation with WCRW: The percentage of artificially infested CRW eggs known to survive to the larval stage is low, and even lower for adult survival. Consequent ly, at each trial location the ratio of viable eggs to larvae was assessed, and comparative estimates of mortality made.

Results indicate that MIR604 provi ded better control of WCRW than untreated non-transgenic controls. Emergence data from 2003 show that MIR 604 controlled an average of 89.9% of CRW, with a low of 74% at Walcott, IA and a high of 97% at Bloomington, IL (Table 1). On an emergence per hectare basis, non-transgenic control plots aver aged 740,410 beetles per hectare, while MIR604 plots averaged 72,019 beetles.

Table 1. Summary of artificial infestation tria ls that evaluate control of WCRW by MIR604 corn. Product efficacy is estimated by the re duction in adult emergence from MIR604 plants relative to untreated, non-transgenic control plants.

Location 2003

Walcott

IA 2003

Stanton

MN 2003 Bloomington

IL 2003

Stanton

MN Artificial

infestation

totals Artificial

infestation

averages

Plants/ha 61,507 61,507 61,507 61,507 61,507

Viable eggs/plant 350 428 405 224 352

Initial density eggs/ha 21.6 M 26.4 M 25.0 M 13.8 M 21.7 M

Emergence in

MIR604 total (F+M) 38 48 14 18 118 30

Emergence in negative control

total (F+M) 145 314 406 299 1164 291Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

79 Area evaluated per entry (ha) 0.00047904 0.00038323 0.00038323 0.00038323 0.0016287 0.00040718

MIR604 total

(F+M)/ha 79,325 125,250 36,531 46,969 288,075 72,019

Negative cont. total (F+M)/ha 302,688 819,344 1,059,407 780,203 2,961,642 740,410

% control 73.8% 84.7% 96.6% 94.0% 90.3% 90.3%

ii. Natural Infestation with WCRW: Field trials using natural infest ation were implemented at six locations in 2004. Three of these locations (Clay Center, NE, Ames, IA, and Higginsvill e, MO) were lightly infested – characterized by few beetles and minimal plant root damage. The re maining three locations (Bloomington, IL, Urbana, IL, and Mead, NE) had a high level of infestation – characterized by high b eetle population densities and a high level of plant root damage.

Percent control was determined by comparing emer gence in MIR604 plots against the non-transgenic

control. This method assumed that the natural in festation of WCRW was ev enly distributed across treatments (an unlikely scenario), although the procedure may provide a rough estimate of the efficacy of MIR604. At the lightly infested locations, MIR604 provided greater c ontrol of WCRW than nontransgenic controls, with a mean of 92.2% beetle control compared to the controls (Table 2). At heavily infested locations MIR604 pr ovided an average of 11.1% beetle control, with a low of -13.5% and a high of 44.5%, compared to non-transg enic control treatments (Table 3).

Table 2. Summary of light pressure natural infest ation trials that evaluate control of WCRW by MIR604 corn

. Product efficacy is estimated by th e reduction in adult emergence from MIR604 plants relative to untreated, non-transgenic control plants.

Location 2004 Clay Center

NE 2004

Ames

IA 2004 Higginsville

MO Light

pressure
natural

infestation

totals Light pressure

natural

infestation

averages

Plants/ha 61,507 61,507 61,507 61,507

Emergence in

MIR604 total (F+M) 23 35 16 74 25

Emergence in negative control total (F+M) 263 276 412 951 317

Area evaluated
per entry (ha) 0.00038323 0.00038323 0.00038323 0.0011497 0.00038323

MIR604 total

(F+M)/ha 60,016 91,328 41,750 193,094 64,36580 Negative cont. total (F+M)/ha 686,266 720,188 1,075,063 2,481,517 827,172

% control 91.3% 87.3% 96.1% 92.2% 92.2%

Table 3. Summary of heavy pressure natural infestat ion trials that evaluate control of WCRW by MIR604 corn

. Product efficacy is estimated by th e reduction in adult emergence from MIR604 plants relative to untreated, non-transgenic control plants.

Location 2004 Bloomington

IL 2004

Urbana

IL 2004

Mead

NE Heavy pressure

natural

infestation

totals Heavy pressure

natural

infestation

averages

Plants/ha 61,507 61,507 61,507 61,507

Emergence in MIR604 Total (F+M) 126 386 345 857 286

Emergence in negative

control total (F+M) 227 340 397 964 321

Area evaluated per entry (ha) 0.00038323 0.00038323 0.00038323 0.0011497 0.00038323

MIR604 total (F+M)/ha 328,781 1,007,219 900,235 2,236,235 745,412

Negative cont. total (F+M)/ha 592,328 887,188 1,035,922 2,515,438 838,479

% control 44.5% -13.5% 13.1% 11.1%

iii. Natural Infestation with NCRW: High natural infestations of N CRW were seen at one location in 2003 and six locations in 2004. Infestations were light at St anton, MN, Cavour, SD, Bloomington, IL, and Mead, NE, while pressure was heavier in Walcott, IA, Ames, IA, and Higgins ville, MO. Percent control was determined by comparing emergence in MIR604 plots against the non- transgenic control. This method assumed that the natural infestation of NCRW was evenly distri buted across treatments (an unlikely scenario), although the procedure may provide a rough estimate of the efficacy of MIR604. At the Ames and Higginsville locations NCRW emergence reached one third of total CRW adult emergence. For these locations, control of NCRW ranged from 45 to 95% . Control ranged from 34.7 to 100% in areas with lower NCRW pressure (Table 4).

Table 4. Summary of natural infestation trials that evaluate control of NCRW by MIR604 corn

. Product efficacy is estimated by the reduction in adult emergence from MIR604 plants relative to untreated, non-transgenic control plants.

Location 2003

Walcott

IA 2004

Stanton

MN 2004

Cavour

SD 2004 Bloomington

IL 2004

Ames

IA 2004 Higginsville

MO 2004

Mead

NE

Plants/ha 61,507 61,507 61,507 61, 507 61,507 61,507 127,859Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

81 Emergence in MIR604 Total (F+M) 28 2 0 5 63 10 32

Emergence in negative control total (F+M) 73 22 53 12 115 218 49

Area evaluated per entry (ha) 0.00047904 0.00038323 0.00038323 0.00038323 .00038323 0.00038323 0.00038323

MIR604 total (F+M)/ha 58,450 5,219 0 13,047 164,391 26,094 83,500

Negative cont. total (F+M)/ha 152,388 57,406 138,297 31,313 300,078 568,844 127,859

% control 61.6% 90.9% 100.0% 58.3% 45.2% 95.4% 34.7%

d) Adult emergence To make realistic judgments re garding the likelihood that a resi stant CRW beetle will pass on resistance alleles to the next generation, adult fitn ess and timing of emergence must be understood. Both the fitness of the surviving beetles and the ti ming of their emergence from the crops have a major impact on their ability to mate with other WCRW a dults that will have an im pact on their ability to contribute genetically to the subsequent generati on. Based on all MIR604 replicate treatments, there was a delay in the emergence of adults from the cr op when compared to the untreated negative isoline

control treatments.

Over all locations, a mean delay of 7.7 days was seen for adult emergence in all MIR604 treatments when compared to non-transgenic controls. No di scernable differences were seen between treatments with light or heavy pressure and between male and female beetles.

i) Artificial infestation with WCRW:

In trials that used artificial infe station, delays of 6 to 10 days compar ed to non-transgenic controls were commonly seen among male and female beetles (Table 5).

Table 5. Emergence delay times (days) for adult male and female WCRW in emerging from artificially infested MIR604 corn fields in 2003 and 2004.

Location 2003

Walcott IA 2003

Stanton MN 2003 Bloomington

IL 2004

Stanton

MN Artificial

infestation

averages
Emergence parameter Emergence (days)

5% male emergence 6.1 4.2 0.1 18.4 7.2

50% male emergence 4.6 9.8 0.9 11.0 6.6

80% male emergence 2.6 14.3 1.6 10.2 7.1

5% female emergence 14.8 3.5 1.3 20.4 10.0

50% female emergence 6.9 8.4 -0.3 9.0 6.082 80% female emergence 5.0 8.1 6.6 8.5 7.0

Mean emergence delay for M+F 6.7 8.0 1.7 12.9 7.3

ii) Natural infestation with WCRW: Emergence delays of over 10 days were seen am ong female CRW beetles in locations with light natural infestation (Table 6). Few males were identif ied in these plots, so emergence time could not be reliably measured. Emergence delays in locations with heavy natural infestation were shorter than delays seen in sites with light emergence (Table 7) . This finding was heavily influenced by the Urbana IL results, which were atypical and for efficacy pur poses, considered an outlier. Removing the Urbana results from the heavily infested data set brings the mean value for emergence to 6.5 days, which is

more similar to results seen in the artificial infestation trial.

Overall, statistical analysis s howed no significant difference in rates of emergence among sexes,

locations or treatments. The most reliable data were collected from three trials in 2004 that recorded over 100 adults in emergence cages. For these sites, a mean delay of 5.8 days was calculated for males and females. Consequently, the modeling study (descr ibed in modeling section) uses a standard delay of 6 days for males and females emerging from MIR604 corn fields.

Table 6. Emergence delay times (days) for adul t male and female WCRW in emerging from light pressure naturally infested MIR604 corn fields in 2003 and 2004.

Location 2004 Clay Center NE 2004 Ames

IA 2004 Higginsville MO Light pressure

natural infestation averages

Emergence parameter Emergence (days)

5% male emergence N/A* N/A* N/A* N/A*

50% male emergence N/A* N/A* N/A* N/A*

80% male emergence N/A* N/A* N/A* N/A*

5% female emergence 8.6 12.3 11.5 10.8

50% female emergence 15.4 12.1 7.0 11.5

80% female emergence 19.4 12.5 5.8 12.6

Mean emergence delay for

M+F 14.4 12.3 8.1 11.6

N/A* - < / = 3 beetles emerged from this entry at this loca tion, and therefore data are ex cluded from delay calculations

Table 7. Emergence delay times (days) for adul t male and female WCRW in emerging from heavy pressure naturally infested MIR604 corn fields in 2003 and 2004.

Location 2004 Bloomington IL 2004

Urbana IL 2004

Mead NE Heavy pressure natural infestation averages

Emergence parameter Emergence (days)

5% male emergence 1.1 6.9 9.5 5.8Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

83 50% male emergence 3.4 4.8 5.0 4.4

80% male emergence 11.2 -3.2 5.9 4.6

5% female emergence 2.4 4.5 10.6 5.8

50% female emergence 7.0 -4.9 9.4 3.8

80% female emergence 8.7 -6.5 4.1 2.1

Mean emergence delay for M+F 5.6 0.3 7.4 4.4

iii) Artificial infestation with NCRW: In 2003 and 2004 data on NCRW emergence was coll ected from four loca tions. Although there was some variability among locations, emergence delays ranged from 5.5 to 11 days. As noted with WCRW emergence data, beetle sex was not shown to affect rate of emergence.

Table 8. Emergence delay times (days) for adult male and female NCRW in emerging from naturally infested MIR604 corn fields in 2003 and 2004.

Location 2003

Walcott IA 2004

Ames IA 2004 Higginsville

MO 2004

Mead NE NCRW natural

infestation

averages

Emergence parameter Emergence (days)

5% male emergence 7.5 0.0 N/A* 12.9 6.8

50% male emergence 2.9 6.1 N/A* 9.5 6.2

80% male emergence 1.4 3.9 N/A* 19.6 8.3

5% female emergence 18.4 6.4 13.1 14.4 13.1

50% female emergence 9.7 10.5 5.6 6.3 8.0

80% female emergence 6.3 6.1 -0.5 3.2 3.8

Mean emergence delay

for M+F 7.7 5.5 6.1 11.0 7.6

N/A* - < / = 3 beetles emerged from this entry at this loca tion, and therefore data are ex cluded from delay calculations

iv) Adult body weight The body weights of adult WCRW beet les were recorded at the artificially infest ed trial in Stanton, MN. Results show that the mean body weight of adult beetles emerging from MIR604 corn fields was significantly greater than the weight of those emerging from non-transgenic plots.84 Table 9. Mean dry weight of adult WCRW emergin g from artificial infe station field trial at Stanton, MN in 2003.

Treatment % Control w.r.t. untreated

control Mean adult weight (g x 10-5)

MIR604-3-11 87.0 206.5 bc

MIR604-3-12 90.3 183.4 bc

Untreated, non-transgenic,

control 0.0 155.4 c

Non-transgenic + FORCE 3G 85.9 222.8 ab

2) Simulation Models for Development of Resistance

Simulation modeling was used by Syngenta to pred ict the evolution of WC RW resistance to the mCry3A toxin expressed in MIR604 maize. Models ha ve been useful tools in the development of IRM strategies. Previously, Monsanto modifi ed a Caprio model for cotton bollworm ( Helicoverpa zea ) to predict the risk of CRW deve loping resistance to MON 863 corn (U.S. EPA, 2002). Andow and Alstad (2002), Onstad et al . (2001), and Onstad et al. (2003) used deterministic models to predict the

evolution of WCRW resistance usin g a variety of management stra tegies. The Andow and Alstad model predicted that, under cert ain conditions, a 20% refuge woul d probably delay resistance for greater than 15 generatio ns when the dose was low, but w ould be ineffective for resistance management of a high dose (U.S. EPA, 2003). In On stad et al. (2001), the model showed that when resistance was dominant, the resistance allele frequenc y exceeded 3% within 2-5 years as refuge size ranged from 5 to 30% for all doses of toxin, but th e resistance allele frequency never exceeded 3%

when resistance was recessive. This model was furt her modified to examine the impact of landscape on WCRW resistance evolution (Ons tad et al., 2003). Both of th e Onstad models were further expanded to evaluate the risk of resistance by WCRW to both transg enic crops and crop rotation in

areas with or without rotation-resi stant phenotypes (Crowder et al., 2005). Storer (2003) developed a stochastic, spatially explicit model to simulate adaptation (resistance) of WCRW in much of the US Corn Belt. In this model, the relative rate of adaptation was affected by the refuge size and the manner in which the non-transgenic refuge maize was deployed. Specifically, the adapta tion rate was lowest if the non-transgenic refuge maize was planted in the sa me fields each year (Storer, 2003). This model was further modified to predict the evolution of resistance to Cry34Ab1/Cry35Ab1 and considered the

influence of rotation-resistan t phenotypes (U.S. EPA, 2005).

3) Syngenta’s Specific Modeling for MIR604 Maize

a) Methods Syngenta worked with Dr. David Onstad of the Univer sity of Illinois to customize the Crowder et al. (2005) model using the value for efficacy (i.e., do se) generated by Syngenta (see section on “Dose” above). The model includes patches of crops without explic it spatial structure. Each patch had a basic spatial unit of a 100 ha farm and was in a homogeneous region consisting of similar farms. The typical model landscape had four crops and MIR604 transgen ic maize planted to both continuous and rotatedModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

85 cornfields. The crops include con tinuous corn, rotated corn, rotated s oybean, and extra vegetation. Six

scenarios were studied. Scenario I consisted only of 100% continuous maize fields in the landscape

with no rotation-resistant WCFW in this area. Scenarios II-V had la ndscapes with 5% extra vegetation, 10% continuous corn, 42 .5% soybean, and 42.5% rotated corn based on the standard set in the work of Crowder et al. (2005). In these four scenarios the area was inhabited by rotation-resistant WCRW. Scenario VI was similar to scenario I, but in this case a soil insecticide was used in the refuges, giving 50% survival of refuge insects. In scenarios IV-V, transgenic corn was planted only in

rotated maize fields. Crowder et al. (2005) demonstrated that pl anting transgenic maize only in

continuous maize fields in these ki nds of landscapes was an inferior strategy so this scenario was not evaluated. In areas with rotation-resistant WCRW (s cenarios II-V), there were two kinds of landscapes

evaluated: one with an initial resistance allele frequency of 0.0001 of the rota tion-resistance allele (Y) in the rootworm population (scenario s II and IV) and the second had an initial Y-allele frequency of 0.1 (scenarios III and V). In both cases, the expression of the rotation resistance allele was dominant. Previously, Crowder et al . (2005) and Crowder and Onstad (2005) demonstrated that the simulation of realistic evolution of rotation resistance requ ired either additive or dominant expression.

Three refuge levels were evaluate d: 10%, 20%, and 30%. The refuge fields and MIR604 fields were assumed to be planted together in either continu ous maize or in rotated maize. The refuge was assumed to be planted in the MIR604 field or adjacen t to it. If the refuge was continuous maize, the refuge had to remain in the same place every year; ot herwise, the refuge in rotation maize fields could change location from year to year.

The time horizon used was 15 years af ter introduction of MI R604 transgenic maize. This model had a daily time step during July-September to simulate adult activity.

Resistance to MIR604 transgenic maize and rotation wa s modeled as dominant, recessive, or additive. Precise additive expression for resistance to transgen ic maize was used. Resistance to crop rotation was modeled as dominant (Y allele).

The initial adult density was assume d to be 50,000/ha of maize for all types. The sex ratio of adults was 50:50. The initial allele frequency for resi stance to MIR604 transgenic maize was 0.0001. The initial allele frequency for resi stance to rotation was 0, 0.0001, or 0.1.

A seed insecticide treatment was not included in most scenarios except for Scenario VI, which

included soil insecticide treatment in refuge in continuous corn. Larval survival was 50% before density-dependent survival.

The model used a range of MIR604 transgenic mai ze mCry3A doses derived from the adult emergence data provided by Syngenta (see discussion above) a nd calculated using two larval survival functions described by Onstad et al. (2001, 2003). These calcul ations are not shown in this review, but can be found in McCaffery et al. (2005, MRID# 465296-01). Homozygous resistant i ndividuals (RR) were assumed to always have 100% survival to MIR604 maize. Heterozygous indi viduals with R dominant86 (sR) were assumed to always have a 100% surviv al to the transgenic crop. The survival of homozygous susceptible individual s (SS) and heterozygote individua ls with R recessive (Sr) were assumed to have a survival of 0, 0.001, 0.05, and 0.20 to represent a theoretical high, practical high, medium, or low toxin dose, respectively.

The standard density-dependent survival of larv ae per state is 0.21 X exp (-0.058 EGG), where EGG is the density of eggs in millions per ha. The maximu m larval survival based on this function was 21%. It was assumed that density-dependent mortality o ccurred after mortality due to overwintering and toxin exposure. In a second approach, it was assu med that mortality is de nsity-independent based on the field data collected by Hibbard et al. (2004); thus, 5% of larvae surviv ed after overwintering and toxin exposure. The Urbana, IL field data collect ed in 2004 was omitted from the analyses because it

was considered an outlier. The data from 2003 and 2004 were combined. Assuming that densitydependent survival occurs, the overall mean propor tion of survival by sus ceptibles to the mCry3A

toxin in the nine trials was 0.093. Fo r the five trials with natural in festations, the mean survival was

0.14. For the four trials with artificial infestations, the meansurvival was 0.038. For densitydependent survival, a range of 0.038, 0.093, to 0.14 for the survival rate of susceptible neonates encountering MIR604 transgenic maize was simulated.

The mean larval survival based on the assumption of density-independent mo rtality after surviving toxin was calculated based on proportional differences between emergence of a dults in control cages and treated cages. The overall mean survival by sus ceptibles to the toxin in the nine trials was 0.24.

For the five trials with natural in festations, the mean survival was 0.34. For the five trials with natural infestations, the mean survival was 0.34. For the f our trials with artificia l infestations, the mean

survival was 0.125. Larval WCRW survival on MIR604 maize was simulated as 0.125, 0.24, and 0.34.

Delays in the emergence of adults were determined from data supplied by Syngenta (see discussion on adult emergence above). In the modeling done by Crowder et al. (2005), there was no significant effect of early mortality and delays of 10 and 14 da ys. For all cases in 2003-2004 in which there were at least 38 adults emerging in the treatment cages, there observed delays ranged from no delay to a delay of almost 15 days. A mean delay of 5.8 days was calculated for both males and females for three

situations in which over 100 adults emerged from the treatment cages (most reliable data). The model used a standard delay of 6 days for both suscepti ble males and females emerging in MIR604 transgenic maize fields. Susceptible geneotypes are SS when R is dominant and SS and rS when R is recessive. For the additive case, simulations were run wi th only SS having early mortality, and then other

simulations were run with both SS and RS having early mortality.

Sensitivity analyses were conducted. In all sensitivity analyses a refuge size of 20% was simulated. In the sensitivity analysis all variables were set to standard conditions (unle ss otherwise noted) except for the function being tested. The e ffect of initial population size, effect of initial R allele frequencies (resistance to transgenic corn) and the effect of lower fecundity by su sceptible adults in transgenic maize were also included in the sensitivity analyses. The sensitivity analysis also used a 12-day delay (in addition to the 6-day delay) to determine how the results were aff ected by a longer delay.

Table 10. Landscape Scenarios Modeled.Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

87 I. No rotation resistance (Y alle le frequency = 0); only continuous maize

II. Rotated maize with Y allele frequency = 0.0001

5% extra vegetation, 10% continuous ma ize, 42.5% soybean, 42.5% rotated maize Transgenic MIR604 maize in both ro tated and continuous maize fields

III. Rotated maize with Y allele frequency = 0.1

5% extra vegetation, 10% continuous ma ize, 42.5% soybean, 42.5% rotated maize Transgenic MIR604 maize in both ro tated and continuous maize fields

IV. Rotated maize with Y allele frequency = 0.0001

5% extra vegetation, 10% continuous ma ize, 42.5% soybean, 42.5% rotated maize Transgenic MIR604 maize only in rotated maize fields

V. Rotated maize with Y allele frequency = 0.1

5% extra vegetation, 10% continuous ma ize, 42.5% soybean, 42.5% rotated maize Transgenic MIR604 maize only in rotated maize fields

VI. No rotation resistance (Y allele frequency = 0); only continuous maize

Annual soil insecticide us e in refuge with larval survival of 50%

b) Results from Syngenta’s Sp ecific Modeling for MIR604 Maize

Syngenta’s modeling results are found in Appendix 1 of their submission (McCaffery et al., 2005, MRID# 465296-01).

1. Recessive resistance allele. If resistance to transgenic MIR6 04 maize is recessive, then WCRW never became resistant within 15 year s in all simulations for all scenarios.

2. Dominant resistance allele. For scenarios I-III, and VI, either wh en larval survival was densitydependent or density-independent , resistance to transgenic MIR 604 maize evolved in less than 13 years with a 20% refuge (all three toxin scen arios). For scenarios IV and V, either when larval survival was density-de pendent or dens ity-independent, resistance to transgenic MIR604 maize did not evolve within the 15 years of th e simulation. Resistan ce evolution using the 10% refuge was worse for scenarios I-III, and VI than using the 20% refuge. The 30% refuge delayed evolution of resistance several years be yond 15 in Scenario III, IV, V when larval survival was density-dependent and density-inde pendent, but resistance still evolved in less than 15 years in Scenarios I, II, and VI.

3. Additive resistance expression. Two simulations re presenting two possible effects of additive expression on early mortality were run for each co mbination of toxin survival (3 combinations) and refuge level (3 levels) for a total of 18 simu lations. Like the results for dominant resistance88 expression, the results for scenarios I-II were mu ch different from those for scenarios IV-V when expression was additive.

Density-Dependent . For Scenarios I and II, with density-dependent survival, resistance evolved in less than 15 years using both the 10% and 20% refuge opti ons for all but one simula tion that for additive expression case #1 in which the toxin survival was 0.14 using a 20% refuge. No resistance was predicted if a 30% refuge (with density-dependent surv ival) was used in a ny of the six landscape scenarios with additive expressi on (both case #1 and #2) during the 15 years of the simulation. No resistance evolved during the 15 years of the mo del simulations using sc enarios IV and V (MIR604 maize was planted only in rotated maize fi elds and rotation-resi stance existed).

Density –Independent. Resistance did not evolve during the 15 years of th e simulation under scenarios I-V using a 20% and 30% refuge if there was de nsity-independent survival except in additive expression case #2 when toxin survival was 0.125 fo r larvae in scenarios I -III. In these three simulations, resistance evolved in 13 years. A 10 % refuge delayed resistance for at least 10 years in

scenarios I-III. Resistance did evolve in less th an 15 years using all refu ge sizes and all toxin combinations with scenario VI (continuous maize treate d with soil insec ticide). In th is case, the 20% refuge with an annual soil insecticide causing 50% mortality will effectively become a 10% refuge. No resistance evolved during th e 15 years of the model simulati ons using scenarios IV and V.

c) Conclusions from Syngenta’s Specific Modeling for MIR604 Maize

The sensitivity analysis indicated th at changes in the initial allele frequency had the greatest effect on

results. Results were insensitive to recessive expression in MIR604 ma ize. Simulations using scenario V were insensitive to changes in initial adult density, R-allele frequency, and emergence delay. For scenario I and dominant expressi on of resistance to transgenic MIR604 maize, the results were generally not sensitiv e to increases in initial pe st density, lengthening of the early-mortality period, no reduction in fecundity of survivors in transgenic MIR604 maize fields . For scenario I and additive expression or dominant expression, results were se nsitive to changes in in itial allele frequency.

If rootworm resistance to the mCry3A toxin as e xpressed in MIR604 maize is recessive (this is thought to be the case), then the modeli ng study suggests that an IRM plan with a 20% refuge, as proposed by Syngenta, will be adequate for delaying the evoluti on of resistance for at l east 15 years. If MIR604 maize is planted in areas with observable rota tion-resistance in the W CRW population, then the simulations indicate that planting the transgenic maize only in rotate d maize fields is a good IRM plan that will delay resistance evolution to the mCry3A toxin expressed in MIR604 maize by at least 15 years regardless of gene expression. This strate gy will also counteract th e rotation resistance in WCRW (see Crowder et al., 2005).

The most complicated cases are for areas without ro tation-resistance or for fr amers who want to plant

continuous corn. The simulations show that resistance to MIR604 ma ize may occur in 9-12 years with a 20% refuge, a toxin survival of 0.093 (the overall m ean) and with dominant expression of resistance. If expression is additive, the evolution of resistan ce is delayed a bit more than for dominant expression by 1-2 years. If the initial resistance allele freque ncy is higher than assumed then resistance can occurModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

89 a few years earlier. If soil insectic ides are used on an annual basis in the refuge, then a 20% refuge effectively becomes a 10% refuge when resistance expression is dominant or additive and resistance evolves more quickly.

4) Impact of MIR604 on Biology of CRW

In order to develop an effective IRM plan a nd appropriate deployment strategies for MIR604, Syngenta submitted information describing CRW biol ogy, ecology, behavior and toxicology related to MIR604. This information is especially important for this product because some individuals survive after feeding on the event. Not only is it important to understand the relative fitness of adults that emerge after feeding on MIR604, it is also important to understand the behavior of surviving larvae. For the most part Syngenta submitted detailed information to address these issues. Syngenta considers this research ongoing, however, and BPPD anticipates ad ditional information when it comes available.

Evidence and observations made by Syngenta show that corn rootworm males normally start to emerge before females and this emergence period genera lly continues for over a month (Hein at al., 1998; Elliot and Hein, 1991; Meinke, 1995). Experime nts conducted by Syngenta in 2003 and 2004 indicate that WCRW adults emerging from MIR604 emerge an average of 6-7 days la ter than WCRW adults emerging from non-transgenic isolin e corn. A similar delay in emergence was observed with NCRW. Syngenta found no evidence that there is a differen ce in the delay in emergence between males and females (although there was some variability and th e emergence profiles did vary). This last observation is especially important because it implie s that normal mating patterns are not likely to be disrupted by any shift in the sex ratios normally found in the field. Given that the emergence period of WCRW is over a month, the observed delays of 6- 7 days for WCRW adults emerging from MIR604 plots should not impact their availability to mate with adults from refuge plots. Syngenta also investigated the impact of density-dependent and de nsity-independent mortality with respect to delays in emergence. These factors were evaluated for their impact on the evolution of resistance in Sygenta’s model (discussed above).

It is clear from Syngenta’s submi ssion that the development of surv iving larvae is significantly delayed, which is reflected in the delay of emerging adu lts. Therefore, it is al so necessary to establish that adults emerging from MIR604 are not physiologically compromise d in a way that would prevent them from mating with refuge adults. Syngenta is currently working with collaborators to fully

understand the relative fitness of a dults that emerge from MIR604. Currently little is known about

sub-lethal effects of toxicants on specific Diabrotica species. WCRW female s mate soon after they emerge and need to feed for about two weeks be fore they can lay eggs (Hein and Tollefson, 1985; Hein et al., 1988). Observations have shown early emerging adults survived longer and were more fecund than later emerging adults, conferring reduced fitness to the later emerging results (Boetel and Fuller, 1997). In most cases, the fitn ess of insects that are exposed to sub-lethal doses of an insecticide is decreased and the number of offspring is reduced (Haynes, 1988). The reduc ed relative fitness of insecticide-resistant genotypes is also co mmon among insects (Crow, 1957), which has been

documented for strains of insects resistant to s ynthetic insecticides (e .g., Ferrari and Geoghiou, 1981;90 Roush and Plapp, 1982) and strains resistant to Bacillus thuringiensis (Groeters et al., 1994; Alyokhin and Ferro, 1999). Syngenta’s contin ued research regarding the biol ogical impact of MIR604 on CRW adults is important.

It is evident from submitted information that the prevention of damage to MIR604 corn roots is not necessarily accompanied by high leve ls of larval mortality. Syngenta and collaborators conducted

studies to better understa nd the interactions between CRW larv ae and MIR604 roots and the means by which roots are protected. To inve stigate these interactions, both MI R604 and negative isoline plants were from seed and infested with newly hatched W CRW. At several interval s (1, 2, 3, 7 and 14 days)

after infestation estimates of root weight, larva number, larval wet and dry weights and larval feeding activity were recorded. Results sh owed that the weight of MIR604 roots increased si gnificantly after day 3 when compared with the control roots, show ing that MIR604 roots were protected from WCRW damage. With regard to larval numbers, the num bers of WCRW larvae on both negative isoline and MIR604 roots declined after infestation, but on MIR 604 this decline occurred more quickly and by day

7 no larvae were alive. This findi ng contrasts with the negative isol ine roots where significant survival was observed. Larval feeding was recorded on ea ch assessment day and significantly fewer larvae were observed on the MIR604 roots compared to the c ontrol. For example, on days 1 and 2 less than 10% of the larvae were present on MIR604 roots a nd none were feeding on day 3, whereas about 50%

of larvae on the control roots were observed feeding at any one time. As expected, this difference in feeding resulted in si gnificant differences in larval weight between the two groups. Both wet and dry weights of larvae feeding on MIR604 roots were si gnificantly lower than t hose feeding on negative isoline roots. Possibly the most importing informa tion from Syngenta’s resear ch were the behavioral findings. It was observed that the larvae feeding on MIR604 roots became sick and either died within the first day or survived until day 2 but did not con tinue to feed on root tissue. Larvae on the MIR604 roots appeared to have one of two behaviors: 1) feeding without inhibition followed by death a short time later, or 2) movement through the root zone, not feeding on the root tissue, but sampling root hairs and continuing to search for food. Applying these finding to a field setting, it is possible that

larvae can survive to the 2 nd instar by feeding on root hairs of MIR604 plants, taking in small amounts of root tissue, and supplementing their diet with surrounding grassy weeds a nd plant roots (Wilson and Hibbard, 2004; Clark and Hibbard, 2004). By surviving to the 2 nd instar stage, which is less susceptible to mCry3A than the 1 st instar stage, WCRW could re-e stablish on MIR604. Although a reestablishment of the pest is pos sible, it is documented that na tural loss of WRCW between root penetration and adult emergence is high (Elliot et al., 1989).

5) Cross-Resistance

It is important to consider the impact of cross-re sistance on the evolution of corn rootworm resistance to MIR604 maize. Cross-resistance occur when a pest becomes resistant to one Bt toxin, for example, and by virtue of this resistance, it confers resistance to another distin ct Bt toxin. The degree to which this cross-resistance might occur depends largely on the mechanism of resistance characterizing the original resistance and the degree to which the tw o (or more) toxins are independently compromised by those mechanisms. For example, Cry1Ac and Cry1F , two Bt Cry toxins th at target Lepidoptera, have a least one midgut receptor in common and if targ et site modification is responsible for resistance then a degree of cross-resistance is probably (G ranero et al., 1996; Balle ster et al., 1999).Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

91 Syngenta discussed the potential fo r cross-resistance involving the mC ry3A toxin in MIR604 maize in the IRM submission. To date, no receptor for Cry3 toxins has been definitely isol ated or characterized. Recently, a cadherin-link protein has been identified in the midgut of WCRW (Siegfried et al., 2005).

Syngenta’s event MIR604 CRW-control maize will be deployed into a landscape consisting of

conventional corn and other CRW-control varieties. At present there are two commercially available

CRW-control varieties that must be included in the evaluation of cross-resistance: Monsanto’s YieldGard® Rootworm (MON863) maize that expr esses the Cry3Bb1 toxin and Dow AgroSciences Herculex® RW maize that expresses the Cry34 Ab1/Cry35Ab1 toxins. The IRM plan for MIR604

must therefore consider the like lihood and possible consequences of the evolution of resistance in CRW that confers cross-resistance to multiple transgenic varieties.

The mode of action of mCry3A expressed in MIR 604 CRW-control maize is similar to that of all known Cry toxins (see discussion earlier in this review). The degree of sequence homology may influence the potential for cross-resistance, but th is depends on the specific ity of the mechanism of resistance that might arise. For Bt Cry toxins, only two modes of resi stance have been observed: 1) altered detoxification by protease enzymes in the midgut lume n that cleave the toxin and 2) modification of the target site receptor on the brus h border membrane of the midgut epithelium so that binding of the toxin is prevented or hindered (Ferré and Van Rie, 2002). The latter mechanism was observed in diamondback moth ( Plutella xylostella ) that were selected with formulated microbial Bt products that resulted in field resistance as described in Tabashnik et al. (1997). Other non-target site

mechanisms of Bt resistance have also been describe d: reduced or impaired gut proteolytic activity in Plodia interpunctella (Indian meal moth) (Oppert et al., 1994, 1997; Herrero et al., 2001) and alternation of toxin processing or some other me tabolic step in laboratory-selected strains of Heliothis

virescens (tobacco budworm) (Jurat-Fuentes et al. , 2003). A non-target site mechanism of resistance would be detrimental to IRM strategies based ar ound reduced selection resulting from stacked Cry toxin genes or mosaic plantings of varieties expressing different to xins. The degree to which such non-target site mechanisms of resistance might imp act resistance to Cry toxins in CRW is currently

unknown.

As described in Ballester et al. ( 1999), modifications of the target s ite binding protein resulting from selection are likely to confer a very specific spectrum of resistances to closely related Cry proteins that also have some affinity with the binding site in question. Between mCry3A and native Cry3Bb1 there is a 61.7% identity and 69.1% similarity in amino aci d sequences. Taking into consideration the size difference between the native and modified Cry3A proteins (mCry3A starts at residue #48), the identity is 74.3% and similarity is 66.5%. This level of amino acid homology is not considered to be particularly high and such differences indicate that there may be a differential recognition of these two proteins in the insect midgut. Galitsky et al. (2001) note that there are di fferences between Cry3A and Cry3Bb1 in certain oligomeric structures in domain II and domain III that affect pore formation in the midgut membrane, regulation of ch annel function, and specificity to wards target pests. These92 differences may affect the behavior of each of these proteins and re duce the likelihood of target-site cross-resistance, although this is unknown.

The Cry34Ab1 and Cry35Ab1 toxins expressed in th e Dow AgroScience’s CRW product, Herculex® RW represent a new family of insecticidal crystal pr oteins (Ellis et al., 2002). Although the target site for these CRW-active proteins is completely unknow n, it can be argued that cross-resistance at the target-site between them and the Cry3 toxins is extremely unlikely give the marked divergence in structure of these proteins from the Cry3 group. Therefore, cross-resi stance between MIR604 and

Herculex RW is considered to be unlikel y and need not be considered further.

By in large, the mechanisms of resistance that mi ght evolve to Bt toxins in Coleoptera like the CRW are largely unknown. Based on the revi ew of the literature, the only genui ne cases of resistance to Bt toxins suggest that target site resistance is the most common, a nd probably is responsible for the majority of resistance cases. Whether this woul d actually be the case for CRW is unknown. Work by Siegfried et al. (2005), however, suggested that there is similarity of the target sites in Coleoptera and Lepidoptera so that if resistance to Cry3 toxins evolves in CRW, it might involve a target site modification. For a brief period when there was a commercially available Cry3A-expressing potato in the US, potential resistance mechanisms were studie d in the laboratory. Base d on these experiments, Colorado potato beetle was shown to survive for a s hort period of time on transgenic plants (Rahardja and Whalon, 1997). No further characterization of th is resistance was ever undertaken as NatureMark stopped marketing these Bt potatoes in 2001.

More recent studies by Rausell et al. (2004) compared the toxin-binding capacities of proteolyticallyprocessed Cry3A, Cry3B and Cry3C toxins to mi dgut brush border membranes of Colorado potato

beetle. In vitro heterologous competition binding experiments showed that the three proteoloyticallyactivated Cry3 toxins all shared a common binding site, but Cry3Aa and Cry3Ca have an extra binding site that is not shared with the Cry3Ba toxin. This means that there could be some differences in the binding of mCry3A in MIR604 and that of Cry3 Bb1 in YieldGard RW. The mCry3A has been modified to promote processing so th at the availability of active toxi n for interaction with the binding site may be different for different Cry3 toxi ns in the natural situ ation in CRW midguts.

Receptors for Cry3 toxins have never been isol ated and characterized. To date, cadherin and aminopeptidase-N are most frequently associated with Cry toxin binding in Lepidoptera (e.g., Gahan et al., 2001; Luo et al., 1996), al though actin (McNall and Adang, 2003) , alkaline phosphatase (McNall and Adang, 2003), and glycolipids (Griffith et al., 2005) have also been identified more recently in binding. Only insect cadherins have been proven to mutate to give resistance to Cry toxins in Lepidoptera (Gahan et al., 2001; Mori n et al., 2003). Siegfried et al. (2005) used an expressed sequence tag to identify the first Co leopteran cadherin gene in CRW. Cadherin could be a receptor for Bt proteins in CRW, but further studies are neces sary to confirm this possibility. Genetic and biochemical studies with different insect species have shown that resistance mechanisms based on target-site genes such as cadherin are inherited as r ecessive or incompletely re cessive traits (Ferré and Van Rie, 2002). On the other ha nd, non-target site mechanisms, su ch as altered metabolism or processing, are more likely to be inherited as incompletely domina nt traits. One would therefore predict that a recessively-inherited target site mechanism is the most likely to evolve if resistanceModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

93 occurs to mCry3A in CRW. The modeling studie s (discussed above) incl ude varying degrees of

dominance.

Rausell et al. (2004) found that CryBb1 might c onfer some cross-resistance to Cry3A (mCry3A) through modification of the shared receptor. The reve rse, however, is not necessarily true. That is, resistance to mCry3A could occu r through modification of a unique binding site with which Cry3Bb1 does not interact. Thus, CRW deve loping resistance to mCry3A expr essed in MIR604 maize may not confer complete resistance to Cry3B b1 maize. While Rausell et al. (2004) did demonstrate specific binding of processed toxin they did not demonstrat e functional receptor binding. Because there is a lack of real information on the nature of resistance, especially in CRW, it is best to assume complete cross-resistance between Cry3Bb1 and mCry3A. This “worst-cas e” was assumed in the modeling

studies discussed above. It is more likely, however, that if CR W resistance does occur to mCry3A, only partial cross-resistance to Cry3Bb1 is expected.

To study these issues further, it is recommended th at Syngenta conduct cross-resistance studies using

CRW colonies resistant to mCr y3A. Experiments should be conduc ted to investigate the nature, inheritance, and fitness costs of specific mechanisms of resistance to the mCry3A protein expressed in

MIR604 maize.

4. CONDITIONALLY REQUIRED DATA FOR MIR604 CORN (2010 UPDATE)

As part of the terms and conditions of the MIR604 (mCryA) registration, the registrant was required to submit programs for resistance monitoring and ref uge compliance. Annual reports summarizing the data collected under these requirements must also be s ubmitted to EPA. Further, data were required to address aspects of potential resistance to mCry3A . The following terms and conditions were required for resistance monitoring, compliance, and additional research:

• Resistance Monitoring

o Description of the steps to be taken to esta blish corn rootworm baseline sensitivity and damage guidelines (due within 90 days of registration -- January 31, 2007);

o Submission of a detailed resistance mon itoring plan. Description of the program including baseline sensitiv ity, sampling (number of locations and samples per locations), sampling methodology, bioassay methodology, standardization procedures, detection technique, sensitivity, and the sta tistical analysis of the probability of detecting resistance, and an interim descri ption of rootworm damage guidelines (due January 31, 2008);

o Submission of rootworm damage guidelines (f or unexpected pest da mage) (due January 31, 2010);

o Development and validation of an appropria te discriminating or diagnostic dose assay (due January 31, 2010);

o Annual report of the insect resistance monitoring progr am. Results of monitoring and investigations of damage reports (due August 31 st each year, beginning in 2008).94 • Compliance

o Written description of Compliance Assurance Program (due within 90 days of registration -- January 31, 2007);

o Grower Agreements: proposed system to assure growers sign grower agreements and affirmation system to assure annual affirm ation by growers of their IRM obligations (due within 90 days of regi stration -- January 31, 2007);

o Annual report on Compliance Assurance Progr am (CAP) activities and results: thirdparty grower survey, on-farm visitation pr ogram, phased-compliance report, tips and complaints, and grower education programs (due January 31 st each year, beginning in 2008);

o Grower education program for MIR604 cor n. Subsequent changes to the grower education program must be in cluded in the annual compliance assurance program report (due January 31, 2008).

• Additional IRM Data

o Initiate establishment of CRW strains that are resistant to mCry3A and investigate the nature, inheritance, and fitn ess costs of specific mechanis ms of resistance to the mCry3A protein expressed in MIR6 04 maize (due January 31, 2010);

o Study the behavioral deterrence (avoidance) mechanism further and submit appropriate results (due January 31, 2010);

o Continue studies on the biological impact of adults surviving on MIR604 maize and submit these results (due January 31, 2010).

Syngenta has submitted all of the required reports as mandated under the terms of registration. A discussion of the reports and data for the resistance monitoring and compliance programs for MIR604

corn follows in the next sections.

a) Resistance Monitoring

Monitoring Plan for mCry3A Corn (2008)

A resistance monitoring progr am for MIR604 (mCry3A) was submitted to EPA in 2008 (MRID 473401-01; reviewed in BPPD, 2009a) . This submission described the monitoring protocols for mCry3A corn as well as progress to establish ba seline susceptibility data for mCry3A, develop a diagnostic bioassay, create a res ponse protocol for unexpected pest damage, and develop a mCry3Aresistant colony.

Like other Bt corn registrations , the monitoring plan for MIR604 is based on the framework of the Agricultural Biotechnology Stewardship Technical Committee’s (ABSTC) document titled “Updated Monitoring Plan for Bt Corn.” Specifically, Syngenta considered the following elements of monitoring: spatial patterns of re sistance, geographic and species fo cus, baseline susceptibility data, and an annual sampling plan.

The monitoring program for MIR604 relies on two main components: reports of unexpected pest damage and bioassays of field co llected Western corn rootworm ( Diabrotica virgifera virgifera,Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

95 WCRW). With respect to bioassays and insect co llections, WCRW are cons idered the only reliable species for annual monitoring alt hough the Bt toxin is also activ e against other CRW pests. The regions from which Syngenta plans to collect WCRW are listed below. Modifications to the sampling program may occur based on MIR604 corn a doption and changes in pest importance.

• Region 1: Illinois – s oybean variant of WCR

• Region 2: Nebraska – orga nophosphate resistant WCR

• Region 3: Iowa – wild type WCR

After baseline susceptibility has been established for mCry3A, diet bioassays will continue to be conducted for the three regions described above. Any unusually low sensitivity to mCry3A will be investigated as soon as practical and tied to field relevance. ‘Unus ually low’ sensitivity would be investigated by consideri ng the following questions:

• Is low sensitivity heritable?

• Is the proportion of field collected individuals with unusually low sensitivity that can feed and survive on MIR604 corn significantly lower than the baseline proportion?

• Did plant assays with mCry3A determine that damage exceeded economic threshold?

Based on 2007 baseline susceptibility data (MRID 480470-02), Syngenta proposed that only population samples with LC 50 values >8,500 ng/cm 2 would be considered for further investigation. This value would be 10-times higher than the mean value observed in past assays while still below the

maximum concentration used to test field collecti ons. EPA would be informed by Syngenta within one month when bioassay results exceeding 8500 ng/cm 2 would be obtained.

BPPD Analysis of mCry3A Resistance Monitoring Plan

BPPD’s review of the mCry3A monitoring plan (BPPD, 2009a a nd 2010) concurred with Syngenta that it is appropriate to focus the current mon itoring efforts on WCRW only. Because of WCRW’s widespread distribution a nd abundance, but similarity in life cycles compared to NCRW and MCRW, it may be more likely that they e volve resistance first to mCry3A. BPPD, however, also has concerns for NCRW and recommends that Syngenta begin to de velop baseline susceptibility data for this target pest. Past difficulties with rearing of NCRW and arguments of limited geographic distribution should not be the driving factors for neglecting to mon itor for resistance in this important corn pest.

Syngenta proposed that it would focus its annual m onitoring for resistance on 1) using dose response curves, 2) field reports of unexpected pest dama ge, and the 3) development of resistant WCRW colonies in the lab. For CRW monitoring and do se-response curves, Syngenta proposed that only population samples with LC 50 values >8,500 ng/cm 2 would be considered for further investigation. BPPD notes that although this va lue may be 10-times higher than the mean value observed over the years, it still appears somewhat arbitrarily chosen. It is not clear how sensitive and reliable this96 particular value will be; LC values may be mislead ing because larvae can survive on Bt diets for some time without feeding. BPPD recommends that Synge nta pursue a sublethal measure to monitor for shifts in susceptibility to mCry3A and explor e the use of the Sublet hal Seedling Assay (SSA) developed by Nowatz ki et al. (2008).

Unexpected Pest Damage Guidelines (2010)

Syngenta submitted finalized protocols for monitoring unexpected pest damage in 2010 (MRID 480470-02). The following strategy was proposed for MIR604 corn:

In cases of unexpected root damage on MIR604 co rn, Syngenta proposed a threshold level of > 2.0 on the Node Injury Scale (NIS; Oleson et al., 2005) to trigger investigati on. Syngenta set this level based on their experience with MIR604 cor n. When growers and extension specialists, or consultants report

to seed dealers that there was unus ually high CRW damage (>2.0), then:

• Syngenta representatives will test for presence of mCry3A in root tissue of damaged plants and confirm hybrid and lot number. If tests confirm that mCry3A was present, then additional roots will be dug and rate the root damage. At this point it will be determined if other causes were responsible for excessive root damage. If damage is again >2.0 and due to CRW, then Syngenta will be notified. If confirmed, the representative will dig additional plants and determine if the root damage was caused by ot her corn pests not susceptible to the toxin, weather conditions, hybrid genetics, or planting error. If the root node injury in the field is >2 due to CRW feeding, then Syngenta will be notified.

• Syngenta will perform more flow strip assays to assess the % of plants expressing mCry3A. If more than 95% of the plants tested express the toxin, then Syngenta will collect WCRW for analysis.

BPPD Analysis of Unexpected Pest Damage Guidelines

BPPD did not concur with Syngenta that the th reshold level for unexpected damage for MIR604 should be >2.0 (see review in BPPD, 2010). BPPD be lieves that a damage threshold level of 1.0 (NIS) may be more appropriate and cons ervative under moderate insect pressure for single, non-high dose CRW products such as are currentl y registered by Syngenta (MIR604) . When insect pressure is exceedingly high, then both Bt protected and refuge corn will incur greater damage. Under these circumstances, a threshold level of 1.5 may be more suitable. In addition, for any mCry3A pyramided products (i.e., with two toxins e xpressing a functional high dose), the damage threshold should be lower than 1.0 and based on the efficacy against the target pest.

BPPD recommended that the following be consider ed when Syngenta follow s up with incidents of unexpected damage reports (BPPD, 2010):

1) The inherent dose of the toxin to cont rol CRW (high dose vs. non-high dose control); 2) Prior use and crop history in the Bt field where excessi ve damage was observed; 3) Damage on non-Bt plants in the same field or immediately adjacent to the Bt plants;Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

97 4) Insect pressure during that corn growi ng season (low vs. moderate vs. high); and 5) Weather pattern during the corn growing seas on and possible effects on Bt protein expression and pest population dynamics.

When greater node injuries are observed on refuge corn than on Bt corn, then this could be an indication that damage is not due to resistance but some other factors. Likewise, if Bt plants have less damage than refuge plants that were treated with insecticides, then the cause of damage is not likely due to resistance. But, if damage levels on Bt pl ants exceed the level obser ved on non-Bt plants, then field resistance should be suspected.

Syngenta’s response to unexpected damage reports is protracted but could be made more efficient by collecting CRW immediately after unexpected damage re ports are received (if adul ts are still flying). After sampling, Syngenta representatives should still be able to proceed with flow strip assays and root damage analysis, as well as putative causes fo r damage. Furthermore, BPPD recommended that monitoring samples for potentially resistant insects should be taken from within and adjacent fields where the pest damage has occurred. Studies by Nara njo (1990) and Coats et al . (1986) suggest that a significant fraction of the female WCRW population engages in longe r distance and pr e-ovipositional dispersal. Hence, BPPD recommended that collec tions of putative resistan t individuals should also extend passed a 1⁄2 mile radius from the Bt field with unexpected damage (BPPD, 2010).

Baseline Susceptibility and Diagnostic Assays for mCry 3A (Monitoring Results 2006-2008)

Syngenta collected baseline susceptibility data for mCry3A from WCRW populations sampled in 2006 through 2008. DM Crop Research Gr oup, Inc. (a separate company work ing with Bt corn registrants to conduct CRW sampling) collected adult sample s of WCRW populations fr om the three sampling regions (IA, IL, and NE) during the 2006 to 2008 growing seasons. Between 11 and 15 populations were sampled each year.

Custom BioProducts, Inc. (IA) (another independent co mpany that conducts the susceptibility testing)

maintained the WCRW colonies a nd resulting eggs and performed the subsequent bioassays on the populations using neonate larvae (< 24 hours old). Neonates were transferred into 96-well plates containing artificial diet developed for CRW cove red with surface applied Bt dilutions containing mCry3A at eight different concentrations: 0 (0.1% Triton-X 100 control), 25, 100, 500, 1000, 2500, 5000, and 10,000 ng mCry3A/cm 2 . The protein used in the tests c onsisted of mCry3A in a lyophilized powder at a concentration of 900 μg /mg (supplied by Syngenta). Larvae we re allowed to feed for five to seven days depending on when fungal contaminati on appeared. Mortality was recorded when larvae did not respond or move when probed. Six replicat es of dose response bioassays were conducted for every population sample. A replication was c onsidered valid if control mortality was ≤ 25%. Results from the assays were used to determine LC 50 , LC 90 , and LC 99 values for the co llected populations.

Data from the mCry3A susceptibil ity bioassays are summarized (range of mean susceptibility values for the sampled populations) in Table 11 below. Over all, the baseline susceptibility data for mCry3A98 were variable as was evident by the LC 50 and LC 90 results, although the LC 50 ranges were more consistent year-to-year than the LC 90 values. One population (from Mc Lean Co., IL) collected during 2008 showed much higher to lerance to mCry3A (LC 50 : 4822.6 ng/cm 2 ; LC 90 : 37,820,880.0 ng/cm 2 ) than the other sampled populations (LC 50 : 191.9 - 1518.4 ng/cm 2 ; LC 90 : 1430.0 - 225,779.9 ng/cm 2 ). Based on the susceptibility testing, Syngenta pr oposed that only population samples with LC 50 values > 8,500 ng/cm 2 would be considered for furt her investigation. BPPD not ed that although this value may be 10-times higher than the mean value observed in the testing through 2008, it still appears to be somewhat arbitrarily chosen. It is not clear how sens itive and reliable this part icular value will be; LC values can be misleading because larvae can surviv e on Bt diets for some time without feeding (see review in BPPD, 2010).

LC 99 values and ranges were submitted for each year but are not shown in Table 11 because the extrapolated values were exceedingly high for most populations. Chi square testing resulted in high probabilities values that indicated that the extrapolated mean LC 99 values were not a reliable estimate

for the actual LC 99 . This suggests that the use of ‘lethal concentrations’ wi ll not be useful to monitor for changes in CRW susceptibility to mCry3A and an effectiv e diagnostic concentration (LC 99 ) can not be realistically achieved for this toxin. Given th ese difficulties, Syngenta may need to consider sublethal measures (i.e., molting inhibition) to es timate the mCry3A activity against CRW. BPPD (2010) recommended that Syngenta use a sublethal meas ure for detecting shifts in susceptibility to mCry3A and/or consider the use of the Sublethal Seedling Assay (SSA) devel oped by Nowatzki et al. (2008) to monitor for shifts in CRW susceptibility. Nowatzki et al. (2008) found that the SSA, which measured survival and age structure of larval populat ions in three potential instar groups, was able to detect shifts in susceptibility of CRW at a much smaller scale than the diet bioassay.

Table 11. Cumulative Results of mCry3A Sus ceptibility Testing for WCRW (2006 - 2008; Table created from data submitted by Syngenta).

Year LC 50 range (ng/cm 2 ) LC 90 range (ng/cm 2 ) MRID Citation

2006 357.9 - 1923.6 3788.7 - 28,766.6 473401-01

2007 209.8 - 1172.3 2470.5 - 52,515.0 480470-02

2008 191.9 - 4822.6 1430.0 - 37,820,880.0 1 480470-02

1 One population in 2008 exhibited high tolerance to mCry3A. The other sampled populations had susceptibility ranges of 191.9 - 1518.4 (LC 50 ) and 1430.0 - 225,779.9 (LC 90 ).

b) Compliance

The compliance program for MIR604 (mCry3A corn) is aligned with that de veloped by ABSTC (in consultation with the EPA) for corn-borer protecte d Bt corn products. Specific elements of the program and additional reports with annua l survey data are discussed below.

MIR604 Compliance Assurance Program

As a term of the MIR604 (mCry3A ) registration, Syngenta was require d to develop and submit to EPAModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

99 a compliance assurance program (CAP) to ensure gr ower adherence to IRM re quirements. The terms of registration mandated a number of compone nts for the compliance program including:

• Grower Agreements: Contractual arrangement be tween the registrant a nd grower to obligate adherence to IRM requirements.

• Annual IRM survey: A survey (conducted anony mously by an independent research firm) intended to provide a statistically representative sample of gr owers from various corn-growing regions in the U.S. Results from the survey should assess levels of grower compliance with refuges as well as grower motivations, at titudes, and reasons for non-compliance.

• On-farm assessments: The registrant is required to develop an on-site assessment program in which trained personnel from each company make vi sits to farms growing Bt corn. During these visits, compliance with refuge requirements is assessed and growers not in compliance are

identified for corrective action under the Phased Compliance Approach.

• Tips and complaints: The registrant must esta blish a means for the reporting and investigation of incidences of refuge non-compliance.

• Phased Compliance Approach (PCA): A consistent set of procedures (for all Bt corn registrants) to be employed to address non-complian ce among growers and seed dealers.

The Agricultural Biotechnology Stew ardship Technical Committee (ABSTC), a consortium of Bt corn registrants, previously developed and submitted a CAP for lepidopteran Bt corn PIPs in 2002. Subsequently, ABSTC submitted revised versions of the CAP in 2004 and 2005 in response to EPA reviews of annual growing season reports (see discussion in the Cr y1Ab/Cry1F Bt corn Biopesticide Registration Action Document). MIR604 (and other registered products with mCry3A) have been included as part of this existi ng program, though data are tabulate d separately for lepidopteran, rootworm, and stacked (lepidopteran + rootworm) Bt corn PIPs (and not by toxin). EPA reviews of compliance data for rootworm-protected PIPs (some of which predates the re gistration of MIR604) can be found in BPPD (2004), BPPD (2007), and BPPD (2009b).

Annual Grower Surveys

As a condition of each individual Bt corn registration, the registrant must perform an annual third-party survey of a statistically representative sample of Bt corn growers. The grower survey functions to measure compliance adherence to refuge size and distance requirements at a regional level and to identify educational opportunities to increase grow er compliance with IRM requirements. More than 500 growers from four separate regions are a nonymously surveyed annually. The methodology for conducting the grower survey has remained virt ually unchanged since it was first conducted by Marketing Horizons, Inc in 2000 for th e lepidopteran Bt corn registratio ns. But, starting in 2007 due to an increasing complexity of growers’ Bt corn plan ting practices and a need to standardize the grower survey across insect-protected tra its, Marketing Horizons utilized an internet-based survey approach.100 Surveys for the corn rootworm PIPs encompasses all growers planting rootworm-protected traits (Cry3Bb1, mCry3A, and Cry34/35Ab1). Cumulative results of the surveys are summarized in Table 12 below. Results from the stacked (lepidopter an + rootworm) Bt corn surveys are tabulated separately (Table 13) and also includ e all registered rootworm PIP traits.

Table 12. Summary of Telephone (2005-2006) and Online (2007-2008) Survey Results for

Rootworm-Protected Bt Corn Growers

Survey Question 2005 1
% Respondents 2006 2
% Respondents 2007 % Respondents 2008 % Respondents

Adherence to Refuge 3

Size 93 89 80 74

Adherence to Distance Requirements 3 87 82 79 63

Awareness of IRM Requirements 97 93 97 96

Unaided recall of refuge

size 51 57 63 72

Unaided recall of refuge

distance 58 55 33 34

1 Includes only YieldGard RW and YieldGard Plus corn growers

2 Includes only YieldGard RW, Yi eldGard Plus, Herculex RW, and Herculex XTRA corn growers

3 Weighted averages across all four regions surveyed

Table 13. Summary of Telephone and Online Sur vey Results for Stacked (Rootworm + Corn Borer) Bt Corn Growers (2006-2008)

Survey Question 2006 % Respondents 2007 % Respondents 2008 % Respondents

Adherence to Refuge Size 1 78 70 72 3

Adherence to Distance Requirements 1 92 66 66 4

Awareness of IRM Requirements 95 96 97

Unaided recall of refuge size 59 62 and 55 2 81

Unaided recall of refuge distance

CRW 48 39 36

Unaided recall of refuge distance

ECB 32 77 86

1 Weighted average across a ll four regions surveyed

2 First number listed is for ECB and the s econd number for CRW refuge compliance.

3 On a per field basis, adherence was 73%

4 On a per field basis, adherence was 76%Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

101 Overall compliance (per grower) with refuge requir ements for both single trait and stacked rootwormprotected PIPs has declined from 2005 to 2008. Grow er adherence to the necessary refuge size fell to below 75% in 2008 for single trait and stacked rootwo rm PIPs. Compliance with refuge proximity was lower; in 2008, ≤ 66% of rootworm PIP grower s deployed refuges within th e required distance to the Bt field. The percent of growers who were able to recall the correct refuge distance requirements (unaided) for rootworm PIPs drastically declin ed to the below 40% in 2008. Refuge distance requirements for rootworm-protected Bt corn products may be more challenging for growers because the refuge must be deployed either within or i mmediately adjacent to the Bt field. Stacked products present additional challenges due to the need to plan either two ref uges (for lepidoptera and rootworm) or a combined refuge for both pest complexes. Nevertheless, grower awareness of the distance requirements has been poor and likely expl ains much of the reported non-compliance.

On-Farm Assessments

The on-farm assessment program is the portion of th e CAP that identifies i ndividual non-compliant growers (regardless of farm size) for remedial IRM education, follow-up reassessments, and other activities as part of the phased co mpliance approach (PCA). It can al so serve as a tool to enhance the registrant’s understanding of the obstacles grow ers face in implementing IRM requirements. The mandatory on-farm assessment program was fully implemented for the first time in 2003 (for lepidopteran registrations) and has typically encompassed more than 2,000 growers per seasons (for all types of Bt corn). On-farm assessments for r ootworm-protected PIPs (including mCry3A products) began in 2006.

Data from the on-farm assessments (2006 through 2008) of rootworm-protected Bt corn PIPs are

summarized in Table 14 below. These on-farm assessments encompass all growers planting rootworm-protected PIPs including varietie s expressing the Cry3Bb1, mCry3A, and Cry34/35Ab1

toxins. Results for the on-farm assessments of stack ed (lepidopteran + rootworm) PIPs are detailed in

Table 15. The assessments do not have the statistical power associated with th e consistently stratified and randomized telephone/on-line surveys and are not used to measure representative rates of noncompliance. In 2007 and 2008, no information was pr ovided regarding specif ic non-compliance with refuge size and distance. This inform ation should be provided in future reports to be consistent with previously collected data and to illustrate how growers are out of compliance (see EPA review in BPPD, 2009b).

Table 14. Cumulative Results for the On-Farm Assessments of Co leopteran-Protected Bt Corn Growers (2006-2008) 1

2006 2007 2008

Growers assessed 395 247 134102 Refuge distance

deviations 2 13 N/A N/A

Refuge size deviations 7 N/A N/A

Significant deviations 11 (2.8%) 16 (6.5%) 12 (9.0)

Insignificant deviations 10 (4.0%) 8 (3.2%) 7 (5.2%)

Compliant growers 374 (94.7%) 223 (90.3%) 115 (85.8%)

Non-compliant growers 21 (5.3%) 24 (9.7%) 19 (14.2%)

1 Table adapted from page 12 of MRID 470444-01

2 Some growers had compliance deviations other than refuge size or distance; thus, the total of refuge distance and size deviations does not equal the number of non-compliant growers.

Table 15. Cumulative Results of the On-Farm A ssessments of Stacked Bt Corn (Rootworm + Corn Borer) Growers (2006-2008) 1

2006 2007 2008

Growers assessed 600 1069 1799

Refuge distance deviations 51 2 N/A N/A

Refuge size deviations 8 N/A N/A

Significant deviations 45 (7.5%) 77 (7.2%) 86 3

Insignificant deviations 16 33 (3.1%) 36 3

Compliant growers 539 (89.8%) 959 (89.7%) 1546 (85.9%)

Non-compliant growers 61 (10.2%) 110 (10.3%) 253 (14.1%)

1 Table adapted from page 12 of MRID 470444-01

2 Some growers had compliance deviations other than refuge size or distance; thus, the total of refuge distance and size deviations does not equal the number of non-compliant growers.

3 The numbers of deviations do not add up to the 253 non-compliant cases reported.

Tips and Complaints

As required by the terms of registration, Bt corn regi strants must have a “tip s and complaints” system as a mechanism for individuals (e.g. , growers, sales representatives, et c.) to report alle ged instances of IRM noncompliance. The number of tips and complain ts (summarized for all Bt corn registrations including lepidopteran and rootwo rm varieties) receiv ed through 2008 is summarized in Table 16 below. Each of these growers identified through th e tips and complaints mechanism were visited as part of the on-farm assessment program. It is not possible, however, to determine whether any of the non-compliant growers identified via the tips and complaints route were subject to the Phased Compliance Approach.

Table 16. Anonymous Tips and Complaints ab out Non-Compliance with IRM requirements (Data from ABSTC annual reports 2003 through 2008)Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

103 Year Number of Tips and Complaints

2003 0

2004 0

2005 5

2006 3

2007 14

2008 5

Phased Compliance Approach

ABSTC’s CAP for lepidopteran and r ootworm-protected PIPs includes a standard set of procedures (shown in Table 17), known as the Phased Complia nce Approach (PCA), which is to be used by registrants when responding to in stances of grower noncompliance w ith the IRM requirements. The PCA also established a tiered approach for non-comp liance with “significant” deviations and “other” deviations. For a 20% CRW refuge requirement (Corn Belt), a significa nt size deviation is defined as a Bt grower planting less than 15% non-Bt corn refuge. This de finition is also applicable to “combined” refuges planted for both lepidoptera and CRW for stacked Bt corn PIPs. On the other hand, a significant deviation based on refuge proximity has not been clearly defined for CRW refuges and as of the 2008 CAP report it is unclear what standards are being used by ABSTC. For lepidopteran Bt corn, a significant de viation is triggered if fewer than 2/3 of the Bt corn fields are planted within 1⁄2 mile of a non-Bt corn refuge. Th is definition is not comp atible with CRW refuge because the distance requirement mandates that refuge s be placed adjacent to or within the Bt corn

field. A reasonable extension of the lepidopteran definition for CRW could be “less than 2/3 of the non-Bt refuge is deployed adjacent or within the Bt field” and “fewerthan two-thirds (2/3) of the infield strips are at least four ro ws wide” (see discussion in BPPD, 2006b). This formula would also be applicable to combined refuges for stacked PIPs.

Table 17. Phased Compliance Approach (PCA) – Standards for Bt Corn Refuge NonCompliance (submitted with the ABSTC 2002 CAP)

Mandatory Responses Additional Responses104 Significant Deviations • IRM education.

• Warning letter.

• Compliance assistance contact prior to planting.

• Compliance assessment contact for the following growing season.

• Deny access to the Bt corn product for any significant deviation two years in a row.

Other Deviations • IRM education.

• Letter and/or compliance assistance contact prior to planting.

• Compliance assessment contact in the following growing season. • Invoice monitoring.

• Technical assistance.

• Grower IRM training.

• Reaffirmation of IRM obligations.

• Deny access to the Bt corn product for other

deviations that are repeated over a period of

years.

Under the PCA, sales are to be suspended to in dividual growers for one y ear after two years of significant deviations. Following the one-year susp ension, growers will need to be requalified to purchase seeds. Growers identified as non-compliant (significant or other de viations) are required to receive a “compliance assessment contact” the following year under th e PCA. Non-compliant growers are typically identified through the on-farm assessment program (see discussion in the on-farm assessment section above). Table 18 summarizes th e numbers of non-compliant growers reassessed under the PCA and the growers still found to be out of compliance. As of the 2008 growing season, one grower was denied access to Bt corn technology due to a refusal to be reassessed in the following season after significant non-compliance. Complian ce data, including results of on-farm assessments and PCA activities, are detailed in BPPD (2007) and BPPD (2009b).

Table 18. Reassessment of Rootworm-Protected and Stacked Bt Corn Growers Under the Phased Compliance Approach (taken from ABSTC annual CAP reports) 1

Year Reassessments 2 Significant

Deviations 3 Loss of Access to Technology

2006 62 0 1 4

2007 82 0 0

2008 134 0 0

1 The data in this table include s both growers planting single tr ait rootworm PIPs and stacked (lepidopteran + rootworm) PIPs. The data in the table has been summed for both groups.

2 Reassessments of growers identif ied with deviations (significant and other) to refuge requirementsModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

105 the previous growing season.

3 Significant deviations recorded the following seas on. Two successive years of significant deviations results in loss of access to Bt corn technology.

4 One grower refused to be reassessed in 2006 and was denied access to Bt corn

c) Additional IRM Data

As a condition of registration, Syngent a was required to address three ar eas of research related to the nature of potential resistance to mCry3A in CR W. Syngenta addressed these requirements in a submission to EPA (MRID# 480470-01; reviewed in BPPD, 2010). The specific requirements and responses are described below.

i) “Initiate establishment of CRW strains that ar e resistant to mCry3A and investigate the nature, inheritance, and fitness costs of specific mechanisms of resistance to the mCry3A protein expressed in

MIR604 maize.”

Since 2006, Syngenta has worked with Dr. Bruce Hibba rd (USDA-ARS, Columbia, MO) to establish a mCry3A-resistant (non-d iapausing) WCRW strain. Nine generati ons of selection have occurred since the beginning of the bioassay experiment. A co ntrol strain was reared on non-Bt maize as a comparison (see Table 19).

After three generations of selection, the mean LC 50 of the treatment colony was 0.259 μg/cm 2 (95% C.I.: 0.0906 - 0.5380 μg/cm 2 ) and the LC 50 of the unselected control colony was 0.552 (95% C.I.: 0.318 - 0.917 μg/cm 2 ). Since the 95% C.I, did overlap, th e results did not differ significantly.

After six generations of selection, the mean LC 50 of the treatment colony was 5.541 μg/cm 2 (95% C.I.: 2.361 - 26.429 μg/cm 2 ) and the LC 50 of the unselected control colony was 1.301 (95% C.I.: 0.871 - 2.003 μg/cm 2 ). The 95% C.I, did not overlap, he nce the results differed significantly.

After nine generations of selection, the mean LC 50 of the treatment colony was 11.25 μg/cm 2 (95% C.I.: 5.70 - 32.69 μg/cm 2 ) and the LC 50 of the unselected control colony was 0.73 (95% C.I.: 0.51 - 1.05 μg/cm 2 ). The 95% C.I, did not overlap, hence the results differed significantly.

Table 19. LC 50 Values of a mCry3A-Selected Colony and Lab Reference Strain (Table created from data submitted in MRID# 480470-01).106 Generations of Selection Mean LC 50 Value with

95% C.I. ( μg/cm 2 ) Significance between Selected

and Lab Strain based on

overlapping 95% C.I. Three generations on Bt (treatment colony) 0.259 (0.0906-0.5380) Three generations on non-Bt (lab reference strain) 0.552 (0.318-0.917) not significant

Six generations on Bt (treatment colony) 5.541 (2.361-26.429) Six generations on non-Bt (lab reference strain) 1.301 (0.871-2.003) significant

Nine generations on Bt (treatment colony) 11.25 (5.70-32.69) Nine generations on non-Bt (lab reference strain) 0.73 (0.51-1.05) significant

To determine the field relevance of differences in LC 50 s, field evaluations were performed and damage

to MIR604 and non-Bt corn was assessed.

Despite the differences in the LC 50 s between selected and non-sele cted colonies, no statistically significant differences in damage, be etle emergence, or larval weight were observed when larvae were exposed to MIR604. When the se lected colony was placed on Bt a nd non-Bt plants, no difference in emergence, weight, and root damage were observed. At this point, it is unclear whether similar performance is related to increasing fitness of th e selected colony on MIR604 plants, decreased fitness of the selected colony on isoline plants, or a combination of both.

Additional steps to be performed include maintenan ce of the colony and further selection in upcoming years to investigate the nature, inhe ritance, and fitness costs of specif ic mechanisms of resistance to mCry3A.

ii) “Study the behavioral deterrence (avoidance) mechanism further and subm it appropriate studies.”

Syngenta worked with Dr. Hibbard (USDA-ARS, Columbia, MO) to further study any potential avoidance mechanisms in CRW larvae to mCry3A e xpressing corn. The methods by Strnad and Dunn (1990) were employed to evaluate host recognit ion of WCRW and MIR604, non-Bt corn, oat roots, and filter paper (negative control).

Neonates exposed to MIR604 or isoline diet had moved significantly lower distances and exhibited lower velocities than larvae exposed to the negati ve control or oats. La rvae exposed to isoline treatments had significantly higher angular velocities and turn angle as well as lower distances moved than neonates exposed to MIR604. These results were consistent w ith observations of local search behavior described for larvae feeding on host roots.

These results suggest that CRW la rvae recognize MIR604 corn as a hos t and that larvae engage in short range searching behavior when exposed to mCry3A in corn diet. Hence, it is unlikely that there is an avoidance mechanism unique to mCry3A a nd WCRW. Syngenta suggest ed that any avoidanceModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

107 behavior may be due to the intoxication from exposure to Bt.

iii) “Continue studies on the biological impac t of adults surviving on MIR604 maize and submit

results.”

Syngenta worked with Dr. Lance Meinke (University of Nebraska) to meas ure potential effects of adult WCRW sublethal exposure to MIR 604 (field and greenhouse experiments).

The study design was set up as a 2 x 2 factorial experiment of all possi ble larva/adult diet combinations. The impacts of larval and adult diet on fecundity and long evity were measured.

Treatments:

• Larvae reared on MIR604 – adults fed on MIR 604 tissue

• Larvae reared on MIR604 – adults fed on non-Bt tissue

• Larvae reared on non-Bt corn – adults fed on MIR604 tissue

• Larvae reared on non-Bt corn – adults fed on non-Bt tissue

Results of the study showed no difference in longevity and fecundity between adults of the different

treatments. Since adult CRW do not seem to detect the difference between Bt and non-Bt corn (no repellency to adults), Syngenta concludes that diet switching should not negatively impact the effectiveness of any IRM refuge strategy in place.

Syngenta’s report on these three st udies adequately addresses the condition of registration. The selection experiment is still ongoing, and BPPD recognizes that it may take significant additional time to fully address that condition of registration.

5. CONCLUSIONS

The original proposed IRM strategy (submitted in 2005) and data to support it were found to be “acceptable” except that the in -field strip refuge must be at leas t 4 rows wide based on recent larval movement data (see EPA reviews in BPPD, 2005 a nd 2006a). If resistance is recessive, then the proposed IRM plan using a 20% struct ured refuge should be adequate to delay resistance for at least 15 years given the assumptions of Syngenta’s model. If MIR604 maize is plan ted in areas with known rotation-resistant WCRW, then planti ng transgenic corn only in rota ted maize fields should be a sound IRM strategy that will delay the evolution of resistance by at l east 15 years rega rdless of gene expression.

In accordance with the terms and conditions of re gistration, Syngenta has submitted additional data regarding resistance monitoring, the na ture of potential resi stance to mCry3A, and refuge compliance. These data (as discussed in secti on 4 above), support the original IRM determination including the use of 20% non-Bt corn refuge to mitigate re sistance development by CRW to mCry3A.108 6. IRM TERMS AND CONDITION OF REGISTRATION (2006)

The following are the IRM terms and condition s of the 2006 MIR604 registration. Syngenta’s responses to the conditionally required data are discussed in section 4 of this BRAD.

The Insect Resistance Management program has the following elements:

a. Refuge Requirements

These refuge requirements do not apply to seed incr ease/propagation of inbred and hybrid seed corn.

1] Grower agreements (also known as stewardship ag reements) will specify that growers must adhere to the refuge requirements as described in the grow er guide/product use guide a nd/or in supplements to the grower guide/product use guide.

2] Specifically, growers must plant a structured refuge of at least 20% non-corn rootworm protected Bt corn that may be treated with inse cticides as needed to control corn rootworm larvae. Growers will not be permitted to apply CRW labeled insecticides to the refuge for control of insect pests while adult corn rootworms are present unless the mCry3A field is treated in a similar manner.

3] Refuge planting options includ e: refuge acres should be plante d as blocks adjacent to mCry3A MIR604 corn fields, perimeter stri ps, or as in-field strips.

4] External refuges must be planted adjacent (e .g., across the road) to mCry3A MIR604 fields.

5] When planting the refuge in stri ps across the field, refuges must be at least 4 consecutive rows wide.

6] Insecticide treatments for control of corn root worm larvae may be applied. Instructions to growers will specify that insecticid es labeled for control of corn rootworm adults cannot be applied while adults are present in the refuge unless the mCry3A field is treated in a similar manner.

7] If the refuge is planted in a fi eld that is in a crop rotation system, then MIR604 must also be planted in a field that is in a crop rotation system.

8] If the refuge is planted on continuous corn, then the MIR604 field may be planted on either

continuous or in a field that is in a crop rotation system.

b. Grower Agreements

1] Persons purchasing the Bt corn product must sign a grower agreement. The term "grower agreement" refers to any grower purchase contract , license agreement, or similar legal document.

2] The grower agreement and/or specific stewards hip documents referenced in the grower agreement must clearly set forth the terms of the current IRM program. By signing the grower agreement, aModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

109 grower must be contractually bound to comply with the requirements of the IRM program.

3] The registrant must develop a system (equivalent to what is already ap proved for Bt11 field corn, EPA Reg. No. 67979-1), which is reasonably likely to assure that persons purchasing the Bt corn product will affirm annually that they are contractually bound to comply with the requirements of the IRM program. The proposed system will be submitted to EPA within 90 days from the date of registration.

4] The registrant must use grower agreements a nd submit to EPA within 90 days from the date of registration a copy of that agreemen t and any specific stewardship docum ents referenced in the grower agreement. If Syngenta wishes to change any part of the grower agreement or any specific stewardship documents referenced in the grower agreement that would affect either the c ontent of the IRM program or the legal enforceability of the provisions of the agreement relating to the IRM program, thirty days prior to implementing a proposed cha nge, the registrant must submit to EPA the text of such changes

to ensure that it is consistent with th e terms and conditions of the amendment.

5] The registrant must establish a system (equiva lent to what is alrea dy approved for Bt11, EPA Reg. No. 67979-1), that is reasonably like ly to assure that persons purch asing the Bt corn sign grower agreement(s), and must provide within 90 days from th e date of the registration a written description of that system.

6] The registrant shall maintain re cords of all Bt corn grower agreements for a period of three years from December 31st of the year in which the agreement was signed.

7] Beginning on January 31, 2008, and annually thereaft er, the registrant sha ll provide EPA with a report showing the number of units of its Bt MIR604 corn seeds sold or shipped and not returned, and the number of such units that were sold to pers ons who have signed grower agreements. The report

shall cover the time frame of the twelve-month period covering the prio r August through July.

8] Beginning in January 31, 2008, and annually thereaft er, the registrant shall submit annual reports on units sold by State (units sold by county level will be made ava ilable to the Agency upon request), IRM grower agreement results, and the complia nce assurance program, including the education program on or before January 31 st each year.

9] The registrant must allow a review of the grow er agreements and grower agreement records by EPA or by a State pesticide regulatory ag ency if the State agency can demonstrate that confidential business information, including names, personal information, and grower license number , will be protected.

c. IRM Education and IRM Compliance Monitoring Programs

1] Syngenta must design and implement a compre hensive, ongoing IRM education program designed to convey to Bt MIR604 corn users the importance of complying with the IRM program. The program110 shall include information encourag ing Bt MIR604 corn users to pursu e optional elements of the IRM program relating to refuge confi guration and proximity to Bt MI R604 corn fields. The education program shall involve the use of multiple media, e.g. face-to-face meetings, mailing written materials, EPA reviewed language on IRM requirements on th e bag or bag tag, and el ectronic communications such as by Internet, radio, or television commercials. Copies of the materials will be provided to EPA for its records. The program sha ll involve at least one written co mmunication annually to each Bt MIR604 corn user separate from the grower techni cal guide. The communication shall inform the user of the current IRM requirements. Syngenta shall c oordinate its education programs with educational efforts of other registrants and ot her organizations, such as the Nati onal Corn Growers Association and state extension programs.

2] Annually, the registrant shall revise, and expand as necessary, its education program to take into

account the information collected through the compliance survey requ ired under paragraph 6 and from other sources. The changes shall address aspects of grower compliance that are not sufficiently high.

3] On January 31, 2008, the registrant must provide a report to EPA summarizing the activities carried out under the education program for the prior year. Annually thereafter, the registrant must provide EPA any substantive changes to its grower education activities as part of the overall IRM compliance assurance program report. The required features of the compliance assurance program are described in paragraphs 4-15 below.

4] The registrant must design and implement an ongoing IRM compliance assurance program designed

to evaluate the extent to which growers purchasing its MIR604 Bt co rn product are complying with the IRM program and that takes such ac tions as are reasonably needed to assure that growers who have not complied with the program either do so in the futu re or lose their access to the MIR604 Bt corn product. The registrant shall coordi nate with other Bt corn regist rants in designing and implementing its compliance assurance program and integrate th e mCry3A CAP with the CAP already approved for Bt11, EPA Registration Numb er 67979-1. The registrant must prep are and submit within 90 days of the date of registration a written description of their compliance assurance program. Other required features of the program are descri bed in paragraphs 5 - 15 below.

5] The registrant must establis h and publicize a "pha sed compliance approach," i.e., a guidance document that indicates how the registrant will a ddress instances of non-comp liance with the terms of the IRM program and general criteria for choosin g among options for responding to any non-compliant growers. The options shall include withdrawal of the right to purchase MIR604 Bt corn for an individual grower or for all growers in a specific region. An individual grower found to be significantly out of compliance two y ears in a row would be denied sa les of the product the next year. Similarly, seed dealers who are not fulfilling their obligations to inform/edu cate growers of their IRM obligations will lose their opportun ity to sell MIR604 Bt corn.

6] The IRM compliance assurance program sha ll include an annual survey conducted by an independent third party of a statis tically representative sample of growers of Bt corn products who plant the vast majority of all corn in the U.S. and in areas in which the selection intensity is greatest. The survey shall consider only those growers who plan t 200 or more acres of co rn in the Corn-Belt andModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

111 who plant 100 or more acres of corn in corn-co tton areas. The survey shall measure the degree of compliance with the IRM program by growers in diffe rent regions of the c ountry and consider the potential impact of non-response. The sample size and geographical resolution may be adjusted annually, based upon input from the independent marke ting research firm and academic scientists, to allow analysis of compliance beha vior within regions or between regions. The sample size must provide a reasonable sensitivity for comparing results across the U.S.

7] The survey shall be designed to provide an unde rstanding of any difficulti es growers encounter in implementing IRM requirements. An analysis of the survey results must include the reasons, extent, and potential biological significance of any impl ementation deviations.

8] The survey shall be designed to obtain grower feedback on the usefulne ss of specific educational

tools and initiatives.

9] The registrant shall provide a final written su mmary of the results of the prior year's survey (together with a description of the regions, the methodology used, a nd the supporting data) to EPA by January 31 of each year, beginning with 2008. The registra nt shall confer with ot her Bt corn registrants and EPA on the design and content of the survey prior to its implementation.

10] Annually, the registrant shall revise, and expand as necessary, its compliance assurance program to

take into account the information collected through the compliance su rvey required under paragraphs 6 through 8 and from other sources. The changes shall ad dress aspects of grower compliance that are not sufficiently high. The registrant must confer with the Agency prior to adopting any changes to a previously approved CAP.

11] The registrant shall conduct an annual on-farm assessment program. The registrant shall train its representatives who make on-farm vi sits with growers of their Bt co rn products to perform assessments of compliance with IRM requirements. There is no minimum corn acreage size for this program. Therefore, growers will be selected for this program from across all fa rm sizes. In the ev ent that any of

these visits result in the identific ation of a grower who is not in compliance with the IRM program, the registrant shall take appropriate action, consistent with its "phased compliance approach," to promote compliance.

12] The registrant shall carry out a program for inve stigating legitimate "tips and complaints" that its growers are not in compliance with the IRM progra m. Whenever an investigation results in the identification of a grower who is not in compliance with the IRM pr ogram, the registrant shall take appropriate action, consistent with its "phased compliance approach."

13] If a grower, who purchases MIR604 Bt corn for pl anting, was specifically iden tified as not being in compliance during the previous year, the registrant shall visit with the grower and evaluate whether that the grower is in compliance with the IRM program for the current year.112 14] Beginning January 31, 2008 and annually thereafte r, Syngenta shall provide a report to EPA summarizing the activities carried out under their compliance assurance program for the prior year and the plans for the compliance assurance program dur ing the current year. The report will include information regarding grower intera ctions (including, but not limited to, on-farm visits, verified tips and complaints, grower meetings and letters), the extent of non-compliance, corrective measures to address the non-compliance, and any follow-up actions taken.

15] The registrant and the seed corn dealers for th e registrant must allow a review of the compliance records by EPA or by a State pesticide regulatory ag ency if the State agency can demonstrate that confidential business information, including the na mes, personal information, and grower license number of the growers will be protected.

d. Insect Resistance Monitoring

The Agency is imposing the following conditions for this product:

The registrant must monitor for mCry3A resistance and/or trends in increased tolerance for corn rootworm. Sampling should be focused in those areas in which there is the hi ghest risk of resistance development.

1] The registrant must provide EPA its resistance monitoring plan for approval. A revised monitoring plan must be submitted to the Agency with 3 months of the date of registration consisting of a description of the steps to be taken to establish corn rootworm baseline sensitivity and damage guidelines. A detailed resistance monitoring planmust be submitted to the Agency for review by January 31, 2008. This plan must include: baseline sensitivity data, sampling (number of locations, samples per locations), sampling methodology and life-stage sampled, bioassay methodology, standardization procedures (including QA/QC pr ovisions), detection technique and sensitivity, the statistical analysis of the probability of detecting resistance, and an interim description of rootworm damage guidelines.

2] The registrant must develop and validate an ap propriate discriminating or diagnostic dose assay by January 31, 2010.

3] The registrant must finalize rootworm damage guidelines and submit these to BPPD by January 31,

2010.

4] The registrant must follow-up on grower, extensi on specialist or consultant reports of unexpected damage or control failures for corn rootworm.

5] The registrant must provide EPA with an an nual resistance monitoring report by August 31st of each year beginning with 2008, reporting on popu lations collected the previous year.

e. Remedial Action PlansModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

113 The following program summary describes, in order of events, the steps that must be taken to implement a remedial action plan if resistance to target pests is c onfirmed (this general process has been implemented for other lepidopte ran and CRW Bt corn products).

1. Definition of Suspected Resistance: Resistance will be suspec ted if investigations of unexpected damage reports show that:

a. Implicated maize plant roots were expr essing the mCry3A prot ein at the expected level;

b. Alternative causes of damage or lodging, such as non-target pest insect species, weather, physical damage, larval movement fr om alternate hosts, plan ting errors, and other reasonable causes for the observations, have been ruled out;

c. The level of damage exceeds guidelines for expected damage.

If resistance is “suspected”, the registrants will instruct affected growers to use alternate pest

control measures such as adulti cide treatment, crop rotation the fo llowing year, or use of soil or seed insecticides the following year. These measures are intende d to reduce the possibility of potentially resistant insects contributi ng to the following year’s pest population.

2. Confirmation of Resistance : Resistance will be confirmed if all of the following criteria are met by progeny from the target pest spec ies sampled from the area of “suspected

resistance:” a. The proportion of larvae that can feed and survive on mCry3A roots from neonate to adult is significantly higher than the baseli ne proportion (currently being established);

b. The LC 50 of the test population exceeds the upper limit of the 95% confidence interval

for the LC 50 of a standard unselected population and/or survival in the diagnostic assay is significantly greater th an that of a standard unselecte d population, as established by the ongoing baseline monitoring program; c. The ability to survive is heritable; d. mCry3A plant assays de termine that damage caused by surviving insects would exceed economic thresholds; e. The identified frequency of field resist ance could lead to widesp read product failure if subsequent collections in the affected field area(s) demonstrated similar bioassay results.

3. Response to Confirmed Resistance: When resistance is “confirmed”, the following steps

will be taken:

a. EPA will receive notification within 30 days of resistance confirmation;

b. Affected customers and extension agents wi ll be notified about confirmed resistance;

c. Affected customers and extension agents will be encouraged to employ alternative CRW control measures;

d. Sale and distribution of mCry3A maize in the affected area will cease immediately;114 e. A long-term resistance management acti on plan will be devised according to the

characteristics of the resistance event and loca l agronomic needs. [The details of such a plan should be approved by EPA and all appropriate stakeholders.]

f. Annual Reporting Requirements

1) Annual Sales: reported and summed by state (count y level data available by request), January 31st each year;

2) Grower Agreement: number of units of Bt corn seeds shipped or sold and not returned, and the

number of such units that were so ld to persons who have signed gr ower agreements, January 31st each

year;

3) Grower Education: substantive changes to edu cation program completed pr evious year, January 31st each year;

4) Compliance Assurance Plan: Compliance Assuran ce Program activities and results, January 31st each year;

5) Compliance: to include annual survey results and plans for the next year; full report January 31st each year;

6) Insect Resistance Monitoring Results: results of monitoring and investigations of damage reports, August 31st each year, beginning in 2008.

7. REFERENCES

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EPA Reviews

BPPD, 2005. Insect Resistance Management Stra tegy Preliminary Review and Deficiencies for Syngenta’s Event MIR 604 Maize (modified Cry3A). USEPA/O PP/BPPD Memorandum from S. Matten to M. Mendelsohn. Dated February 3, 2005.

BPPD, 2006a. Review of Syngenta’s Revised Ins ect Resistance Management Strategy for Event MIR604 Maize (modified Cry3A). USEPA/OPP/ BPPD Memorandum from S. Matten, T. Milofsky, and M. Hunter to M. Mendelsohn. Dated September 9, 2006.

BPPD, 2006b. Technical Review of Dow AgroScie nces’ Grower Agreements, Grower Affirmation Procedures, and Compliance Assurance Program (C AP) as Required as Part of the Terms and Conditions of the Herculex Rootworm (EPA Reg. No. 68467-5) and Herculex Xtra (EPA Reg. No. 68467-6) Registrations. USEPA/ OPP/BPPD Memorandum from S. Matten to M. Mendelsohn dated November 27, 2006.

BPPD, 2007. Review of IRM Compliance A ssurance Program (CAP) components covered in

ABSTC’s submission on behalf of corn bore r-protected (EPA Reg. Nos. 524-489, 68467-2, 67979-1 and 29964-3), corn rootworm-protected (EPA Reg. Nos. 524-528, 68467-5, and 29964-4), and stacked (corn-borer/corn rootworm-protected ) (EPA Reg. Nos. 524-545, 68467-6, and 29964-5) Bt corn registrations. USEPA/OPP/BPPD Memorandum from T. Milofsky and S. Matten to M. Mendelsohn dated August 30, 2007.

BPPD, 2009a. Review of Syngenta’s Corn Rootworm Monitoring Plan and 2006 Baseline Susceptibility Data for MIR604 Corn (EPA Re g. No. 67979-5 and 67979-8). USEPA/OPP/BPPD Memorandum from J. Martinez to M. Mendelsohn. Dated September 17, 2009.

BPPD, 2009b. EPA Review of ABSTC’s 2007 a nd 2008 Corn Insect Resistance Management Compliance Assurance Program [EPA Regi stration Nos. 524-489, 68467-2, 67979-1, 29964-3, 524- 528, 524-551, 68467-5, 67979-5, 29964-4, 524-545, 524-552, 68467-6, 67979-8, and 29964-5; MRIDs120 473396-01 and 476633-01]. USEPA/OPP/BPPD Memorandu m from J. Martinez to M. Mendelsohn dated April 15, 2009.

BPPD, 2010. Review of Syngenta’s Corn Root worm Monitoring Plan and 2007 and 2008 Baseline Susceptibility Data for MIR604 Corn (EPA Reg. No. 67979-5 and 67979-8). USEPA/OPP/BPPD Memorandum from J. Martinez to M. Mendelsohn. Dated June 30, 2010.

Studies Cited by MRID Number

462656-17 McCaffery, A.; Stein, J. (2004) Insect Resistance Management for Syngenta Event MIR604 Maize (Corn). Project Number : MIR604. Unpublished study prepared by Syngenta Jealotts Hill International. 47 p.

465296-01 McCaffery, A.; Stein, J.; Onstad, D. (2005) Amended Insect Resi stance Management of Syngenta Event MIR604 Maize (Corn). Unpublished study prepared by Syngenta Jealotts Hill International, SYNGENTA SEEDS, INC. - FIELD CROPS - NAFTA and University of Illinois. 83 p.

473396-01 Bailey, L. (2008) 2007 Insect Resistance Management Compliance Assurance Program Report for Corn Borer-Protected Bt Corn, Corn Rootworm-Protected Bt Corn and Corn

Borer/Corn Rootworm-Protected Stacked Bt Corn. Project Number: CAP/2007. Unpublished study prepared by Agricultura l Biotechnology Stewardship Technical Committee, 17 p.

473401-01 Kurtz, R. (2008) Steps Taken to Determin e the Baseline Susceptib ility of Diabroctica virifera virgifera to the mCry3A Protei n and the Interim Damage Guidelines for MIR604 corn: 2006 Summary. Unpublished study prepared by Syngenta Biotechnology, Inc. 18 p.

476633-01 Bailey, L. (2009) 2008 Insect Resistance Management Compliance Assurance Program Report for Corn Borer-Protected Bt Corn, Corn Rootworm-Protected Bt Corn and Corn

Borer/Corn Rootworm-Protected Stacked Bt Corn. Project Number: CAP/2008. Unpublished study prepared by Agricultura l Biotechnology Stewardship Technical Committee, 15 p.

480470-01 Kurz, R. (2010) A Summary of Studies A ddressing the Conditions of Registration for

MIR604 and Bt11 x MIR604 Corn Traits. Project Number: SSB/109/10. Unpublished study prepared by Syngenta Biotechnology, Inc. 21 p.

480470-02 Kurtz, R.; Crespo, A. (2010) Steps Taken to Determine the Baseli ne Susceptibility of Diabrotica virgifera virgifera to the MCry3A Protein and the Final Damage Guidelines for MIR604 Corn. Project Number: SSB /114/10. Unpublished study prepared by Syngenta Biotechnology, Inc. 29 p.Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

121 II. E. Benefits and EPA Public Interest Finding

1. PUBLIC INTEREST FINDING

The criteria for determining whether registration of a pesticide chemical is in the public interest are set forth in a Federal Register Notice dated Ma rch 5, 1986 volume 51, No. 43 (OPP-32500; FRL-2977-2)

titled Conditional Registration of New Pesticides . There is a presumption that registration of a pesticide chemical is in the public interest if one of the following crit eria is met: i) the use is for a minor crop; (ii) the use is a replacement for anothe r pesticide that is of continuing concern to the Agency; (iii) the use is one for which an emergency exemption under FIFRA Section 18 has been granted for lack of an alternative pest control method, or (iv) the use is against a pest of public health significance. Further, EPA may determine that such a registration is in the public interest on the basis of the following criteria: i) there is a need for the new chemical that is not being met by currently registered pesticides; ii) the new pesticide is co mparatively less risky to h ealth 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 alternativ e registered pesticides and other available nonchemical techniques.

Syngenta has provided data to suppo rt its claim that MIR604 CRW-pr otected corn is in the public interest. EPA’s analysis suppor ts the following conclusions:

1. Results of efficacy trials conducted in 2002, 2003, and 2004 indicate that MIR604 corn provides effective control of key rootworm pests of field corn.

2. MIR604 corn has unique biochemical propert ies which may benefit insect resistance management for this and other CRW-protected corn products.

3. If MIR604 corn is registered, it will be the third CRW-protected Bt corn product on the market. The availability of multiple CRW-protected corn products will increase grower choice and price competition, resulting in lower seed prices for consumers and higher adoption rates.

4. Registration of MIR604 corn is expected to resu lt in further reduction of chemical insecticide use by growers. This is of sp ecial importance since many pestic ides registered for CRW-control are highly toxic to humans and the environm ent, while mCry3A-expressing corn poses no

foreseeable human health or environmental risks.

2. BACKGROUND

Corn is the most widely cultivated U.S. crop, in terms of acreage planted and net value. In 2004, U.S. corn acreage totaled 80.9 million, yielding 11.8 billion bushels. Corn rootworm (CRW, Diabrotica122 spp.), one of the most damaging pests of field corn, can cause yield losses in th e range of 8 to 16% if

left uncontrolled.

Prior to the advent of insect pr otected field corn, CRW was controlled through the use of crop rotation and insecticides. Although crop rota tion is regarded as an effectiv e CRW control tool (Levine and Sadeghi, 1991), behavior changes in northern corn rootworm (NCRW, Diabrotica barberi ) (extended diapause) and western corn rootworm (WCRW, D. virgifera virgifera ) (soybean rotation) have reduced the effectiveness of this management option in some corn growing regions. Inse cticidal cont rol, a pest management alternative to crop rotation, employs chem icals that are highly toxi c to fish, birds, and other wildlife species. In addition, re sistance to some CRW insecticides , such as methyl parathion and carbaryl, (Meinke et al., 1998; Scha rf et al., 1999; Zhu et al., 2001), may result in increased chemical

use.

Since 2003, a third method of CR W control, CRW-protected Bt corn, has been available to farmers.

The first and second Bt field corn products registered fo r CRW control were Monsanto’s event MON863 (expressing the Cry3Bb1 protein) a nd Dow/Pioneer’s DAS 59122-7 (expressing the Cry34Ab1 and Cry35Ab1 proteins) field corn products, respectively. This review concerns the third CRW-protected Bt corn product submitted for registrati on, Syngenta’s MIR604, which produces the mCry3A insecticidal protein. Similar to ev ent MON863 and DAS 59122-7 corn hybrids, event MIR604 Bt corn is targ eted against the WCRW, NCRW, and Mexican corn rootworm (MCRW, D. virgifera zeae ).

3. EFFICACY AND YIELD TRIALS

Early and late maturity MIR604- derived non-commercial, experiment al hybrids were evaluated in northern, Midwest, and southern U.S. corn-belt locations in 20 02, 2003, and 2004. Small plot trials utilizing either randomized complete block or split plot designs were conducted. Most trials included application of chemical insec ticides to assess synergies between MIR604 corn and chemical

insecticides. Chemical insecticide treatments in cluded one of two seed treatments, ProShield ST ®
(tefluthrin) and Cruiser ® (thiamethoxam), plus one of three in-f urrow soil-applied granular treatments,

Force 3G ® (tefluthrin), Aztec 2.1G ® (Tebupirimiphos + cyfluthrin) and Lorsban 15G ® (chlorpyrifos).

a. Efficacy Efficacy trials compared root damage among MIR604 and non-MIR604-derived corn hybrids. Experimental plots were artificially infested (400 to 1450 eggs/trial), located in fields with naturally occurring populations of corn r ootworm, or both approaches to in festation were combined. When artificially infestation was used, at the V2 to V3 leaf stage rootworm eggs were mechanically placed 3 inches deep in soil, within 2 to 3 inches of the corn stalk base. At the VT to R1 stage, corn plants were manually dug from the ground, soil was washed from th e roots and roots were examined and rated for corn rootworm damage according to the Iowa State 0-3 node-injury rating scale.

1) 2002 Field Season In 2002, results from trials located in Minnesota and Illinois showed that MIR604 derived hybrids hadModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

123 significantly less root damage comp ared to non-transgenic controls , with and without insecticide applications. In all but one case, MIR604 seed treate d with Cruiser or ProShield ST (ProShield is an older seed treatment that has been replaced by Cruise r), or plants treated w ith Force 3G soil-applied insecticide, had significantly reduc ed root damage ratings relative to non-transgeni c control hybrids with the same chemical treatment. The exception was in Stanton, MN, trial SYN137 which included a Force 3G insecticide ap plication, where similar reductions we re seen but the difference was not statistically significant.

2) 2003 Field Season Eight event MIR604 hybrids were assessed for efficacy at over a dozen locations in Illinois, Iowa, Kansas, Minnesota, Missouri, Nebraska, South Dakota, Texas, and Wisconsin in 2003. At all locations, MIR604 corn had significantly lower mean root damage , compared to non-transgenic controls with the

same chemical treatment. And for most trials, untreated Even t MIR604 hybrids demonstrated significantly greater efficacy compared to negative is oline controls treated with Cruiser at low or high rates. No significant difference c ould be detected between rates of Cruiser applied to MIR604 hybrids; however, efficacy of Cruiser seed treatment on nega tive control plants did appear to increase with increasing application rates. Over all, Event MIR604 hybrids seem to provide similar protection against CRW, compared to nontransgenic controls treated with Lorsban 15G , Force 3G, or Aztec 2.1 granular soil-applied insecticides. In gene ral, MIR604 hybrids delivered equi valent or better efficacy than chemical controls currently employed to c ontrol root damage from corn rootworm.

Due to difficulties in rearing NCRW, a limited numb er of efficacy trials were conducted with this rootworm species. In greenhouse trials that employe d artificial infestatio n of NCRW, mean root damage ratings show significantly greater effi cacy among MIR604 hybrids compared to negative controls. In these trials, no significant difference was seen am ong MIR604 hybrids. The study author stated that MIR604 hybrids were expos ed to natural populations of NCRW at the majority of locations. However, the Willmar, MN location was populated prim arily with NCRW and re sults from this trial indicate that MIR604 hybrid s provided greater CRW control, comp ared to negative hybrids with and

without chemical treatment.

At two locations in Texas, Event MIR604 corn hybrids showed efficacy toward MCRW, but not to the

same extent as WCRW and NCRW. At both locations, application of Cruiser seed treatment to Event MIR604 hybrids enhanced the efficacy in a rate dependent manner.

3) 2004 Field Season In 2004, root damage ratings were significantl y different among untreated MIR604 hybrids and negative controls. More specifica lly, at all locations, except Champaign, IL, MIR604 hybrids treated

with Cruiser ® seed treatment had significantly less root dama ge than untreated controls. Further, at all locations, except Urbana, IL, root damage ratings were not significantly diffe rent between untreated MIR604 hybrids and MIR604 hybrids treated with Cruiser ® insecticide at 0.125 or 0.75 mg AI/seed. These findings suggest that negative interact ions between mCry3A protein and Cruiser ® insecticide did not result from use of the seed treatment on MIR604 hybrids. The efficacy of MIR604 corn for124 rootworm protection was also compared to Force 3G ® , a granular soil insecticide that is used to control

CRW. Results of these trials show ed that untreated MIR604 corn, MI R604 corn treated with Cruiser ®
insecticide, and a negative contro l hybrid treated with Force 3G ® insecticide provided statistically equivalent root protection. These results suggest th at the efficacy of MIR604 corn, with or without

Cruiser ® insecticide, is similar to that of Force 3G ® , a commercially available corn rootworm

insecticide.

Significant natural populations of northern corn rootworm (NCRW, Diabrotica barberi ) were

identified at three trial locations in 2004. At the Redwood Falls, MN location, adults seen in the field

before and after root evaluations we re said to be primarily NCRW. At university trials in Ames, IA and Higginsville, MO, adult emergence c ounts indicated that the NCRW pop ulation in these sites was 35% and 32%, respectively, of the total rootworm population. Results from the three locations identified

above were similar to results seen for the overall experiment. Mean root damage ratings for MIR604 hybrids were found to be significantly lower than damage ratings for the untreated negative control hybrid and damage ratings for MIR 604 hybrids with or without Cruiser ® insecticide applications were statistically equivalent to the negative control hybrid tr eated with Force 3G ® insecticide.

An MIR604 hybrid, identified as “l ate hybrid 25”, was also shown to be effective at the Texas site, which contained large populations of MCRW. As seen in the trials described above, rootworm damage on the untreated MIR604 corn hybrid was statistically equivalent to the nega tive control hybrid treated

with Force 3G ® insecticide. In addition, root damage rati ngs were not significan tly different between the MIR604 hybrid treated w ith and without Cruiser ® insecticide. Based on limited data collected during the 2003 and 2004 cropping seasons, the regi strant concluded that MIR604 corn hybrids provide moderately high levels of root damage pr otection against MCRW and that, on average, greater control was provided against WCRW than MCRW. Additional data will be collected in 2005 to confirm these findings.

b. Yield Yield trials compared grain production among MIR604 and non-MIR604-derived corn hybrids. Experimental plots were located on sites with or without natural CRW pressure and chemical insecticides were applied to some sites, but not to all.

1) 2002 Field Season In 2002, Event MIR604 derived hybrids were evaluated for yield (bu/acre) at se veral field trials in Minnesota and Illinois. Growing conditions were normal at the Minnesota locations, while some Illinois sites experienced drought and significant CRW feeding pressu re. At the Minnesota locations, the average yield of MIR604-derived hybrids was st atistically equivalent to the negative isogenic controls (with and without insecticide). However, at the Illinois locatio ns, MIR604-derived hybrids produced greater yields than negati ve controls with and without chemical insecticide applications.

2) 2003 Field Season MIR604 yield trails were repeated in 2003 at fi eld sites in Illinois, Indiana, Iowa, Kentucky, Minnesota, Nebraska, South Dakota and Wisconsin. In contrast to 2002, growing conditions were good at most trial sites. Mean yiel ds from untreated (no chemical insecticides) MIR604 hybrids were, onModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

125 average, statistically equivalent to control hybrids, regardless of maturity group and insecticide treatment. This data suggests that where CRW pr essure exists, MIR604 has a positive effect on yield, and this effect may be magnified under adve rse environmental conditions; however, where CRW pressure does not exist, yield among MIR604 a nd non-transgenic plots will be similar.

3) 2004 Field Season Grain yield for MIR604 plots that were untreated or treated with Cruiser ® insecticide produced an average of 27.5 bushels per acre more than the untr eated negative control hybrid. In addition, results at Bloomington-2, IL, which experience d extreme rootworm pressure a nd stressful growing conditions, suggest that MIR604 corn is most beneficial where rootworm pre ssure is greatest. The submission states that although some of these trials lacked the precision to show st atistical differences among treatments, the numerical trends in grai n yield were positive for MIR604 corn.

A multiple location yield trial conducted in the abse nce of CRW feeding pressure (granular insecticide applied to all plots within trial) showed no signi ficant difference in yield between MIR604 hybrids and the negative isogenic control at all trials and locations except SYNAM445, where MIR604 hybrid 22 yielded 10.5 bushels more per acre than the isoge nic negative control hybri d. These results suggest that, under low stress conditions, MIR604 hybrids pe rform as well as negative isogenic hybrids.

c. Conclusions Results from the 2002, 2003, and 2004 efficacy trials suggest that MIR604 hybrids provide greater protection against CRW than negative control h ybrids. Further, the efficacy of MIR604 hybrids appears to be similar to that of negati ve control hybrids treated with Force 3G ® insecticide and the mCry3A protein that is presen t in MIR604 hybrids does not seem to interact with Cruiser ® seed treatment. Conclusions regarding the relative yield from 2002 and 2003 fi eld trials data were obscured by poor experimental design and extreme weather condi tions. Data from 2004 yield trials indicated that under high stress conditions, MIR604 hybrids (±Cruiser ® seed treatment have greater yields than untreated negative controls, althoug h there was no statistical differe nce among treatments. Under low stress conditions, there was no st atistical difference in yield between MIR604 hybrids (±Cruiser ® seed treatment) and the negative contro ls (± insecticide). Yield data suggest that MIR604 hybrids perform about the same as the isoline hybrids.

4. BENEFITS

EPA has completed an independent analysis of th e potential grower benefits associated with BtCRWprotected corn hybrid technologies. Grower demand for CRW control tech nologies is influenced by the level of CRW infestation (acreage and degree of infestation), comparative cost-benefit of competing CRW control technologies, U.S. and global market acceptance and approval of a technology, and other regulatory constraints ( e.g. refuge requirements). In the year 2000, almost 8 million pounds of CRW insecticide, costing $172 million ($ 12.29/acre), were applied to 14 milli on acres, or 17% of U.S. corn acreage. And between the years 2005 to 2013, CRW infested acreage is projected to increase from approximately 31.8 million acres to 39 million acres (Table 36). BPPD anticipates, through evaluation126 of USDA/NASS and DOANE databases on CRW damage and control costs, that the market for transgenic in-plant CRW protection will increase by 2.6% per year, reachi ng 18 to 19 million acres by the year 2013.

Data on product efficacy suggests that Event MIR 604 corn provides greater protection against CRW damage than does unmodified corn (see Efficacy s ection above). However, submitted yield data was inconclusive and should be supporte d by a third year of field tria ls (see Yield section above).

Table 36. Projected acreage with corn rootworm infestation and breakdown of associated CRW control practices for the years 2000 to 2013. Information presented for 2000 and 2002 reflect

actual infestation and insect control tactics.

Acres Infested Acres Treated CRW-Protected Bt Corn Acreage Conventional

Treatments Year

------------------ ------------------ -----acreage x 10 6

--------------- ------------- ------------ 2000 28.0 14.0 0.0 14.0

2001 1

- - - - 2002 29.5 14.7 0.0 14.7

2003 30.2 15.1 1.0 14.1

2004 31.0 15.5 2.5 13.0

2005 31.8 15.9 4.0 11.9

2006 32.6 16.3 6.0 10.3

2007 33.5 16.7 7.2 9.5

2008 34.3 17.2 8.6 8.5

2009 35.2 17.6 10.4 7.2

2010 36.1 18.1 11.9 6.1

2011 37.1 18.5 13.7 4.8

2012 38.0 19.0 15.8 3.2

2013 39.0 19.5 16.8 2.7

Annual Growth

Rate 2.58% 2.58% 16.8% -14.36%

1 EPA does not have data for the 2001 growing season.

a. Human Health Benefits Event MIR604 CRW-protected corn is expected to be safer for handlers, applic ators, farmers, and the public than chemical pesticides in current use. Twenty-five of the 39 registered conventional

insecticides used to control CRW are classified as “Restricted Use”, 12 ha ve the “Danger” label classification, and several are in Agency Special Review ( e.g. , dimethoate, phorate, and terbufos). Further, each year there are confirmed reports of human illness associated with these registered chemical insecticides (See EPA’s Incident Data Base, http://www.opp.gov/pesticides). Adoption of mCry3A corn hybrids has the poten tial to reduce occupational, fa rmer, and public health risks associated with the manufacture, transport, storag e, handling, application, and disposal of conventional insecticides, by providing a safer alternative for CRW control.Modified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

127 Increased adoption of transgenic CRW-protected co rn products is of special importance to EPA, because many of the chemical insecticides register ed for CRW control are highly toxic to humans and the environment, are in Special Review, and have restricted use labels. Th e chemical insecticides (larvicides and adulticides) subj ect to the greatest use reducti on following adoption of transgenic CRW-protected hybrids are: organophosphates (c hlorpyrifos, tebupirimphos, tebufos, methyl parathion, and chlorethoxyf os), pyrethroids (tefluthrin, cyfluthr in, bifenthrin, lam bda cyhalothrin), carbamates (carbofuran), and pyr azoles (fipronil) (Appendix A). Adoption of transgenic CRWprotected corn products is not expe cted to result in reduced use of seed treatments (nicotinoids); however seed treatment products are generally less toxic than at-plant and post-plant products.

In contrast to the registered chemical insectic ide alternatives, EPA has concluded that there is reasonable certainty of no harm fr om aggregate exposure to the mCry3A protein as expressed in corn. See mCry3A Human Health and Pr oduct Characterization reviews for more information on potential human health effects [Fellman, 2005(a,b); 2006(c)].

b. Environmental Benefits Chemicals commonly used for CRW control can cau se adverse environmental effects when used according to label instructions. Of the most comm only used CRW control products, 15 are labeled as “toxic,” six as “highly toxic,” one as “very highly toxic,” and 14 as “extremely toxic” to birds, fish, and other wildlife. Several of the synthetic insecticides (e.g. organophos phates, carbamates, and synthetic pyrethroids), are moderate ly to highly toxic to terrestria l non-target species. Of special concern are methyl parathion and ca rbofuran, both of which are implicated in bird kills (Appendix A, Table 1A). In contrast, the ecological risk assessmen t and characterization of the mCry3A protein, as expressed in corn, suggests that thes e proteins pose no signifi cant ecological risk to non-target species, including endangered and bene ficial species (Milofsky, 2006).

c. Insect Resistance Management Benefits There is concern about the ability of target pest s to evolve resistance to CRW control mechanisms, including crop rotation, chemical insecticides, and CRW-protected Bt corn products. Currently, the favored management practice for CRW control is crop rotation, specifically corn-soybean rotations, complimented by application of chemical insec ticides – pyrethroids, organophosphates, carbamates, pyrazoles, and more recently, neonicotinoids as s eed treatments. CRW has developed resistance to a number of chemical control products used as ad ulticides. The recent development of the NCRW extended diapause and WCRW soybean resistant vari ants have further reduced the efficacy of crop rotation and chemical control options.

Since mCry3A CRW-protected corn appears to ha ve good efficacy against CRW, introduction of this product is expected to extend the durability of other commercially available CRW-protected Bt corn products ( e.g. , Cry3Bb1 PIPs and Cry34/35Ab1 PIPs). To en sure the long-term efficacy of mCry3A CRW-protected corn hybrids, an insect resistance ma nagement plan should be implemented (Matten et al. 2006).128 5. REFERENCES

AgroTrak U.S. Crop Chemical Usage Study. 2003 Doane Marketing Research, Inc.

Chen, E. and Stacy, C. 2003. Modified Cry3A toxi ns and nucleic acid sequences coding. Patent Application WO Patent No. 03/018810

Federal Register. 2004. Phosphomannose Isomerase a nd the Genetic Material Necessary for Its Production in all Plants; Exempti on from the Requirement of a Tole rance. Vol. 69(94); Friday, May 14, 2004; pp. 26770-26775; 40 CFR Part 180.1252

Fellman, A. 2005 (a). Addendum to product characteri zation and expression analysis for modified Cry3A (mCry3A) Bacillus thuringiensis insecticidal protein and th e genetic material necessary for their production (via pZM26) in transgenic maize plants derived from Syngenta Seeds’

transformation Event MIR604. U.S. EPA/BPPD Memorandum from A. Fellman to M. Mendelsohn. Dated Feb 11, 2005.

Fellman, A. 2005 (b). Review of product characteriza tion and expression analys is for modified Cry3A (mCry3A) Bacillus thuringiensis insect control protein and ma ize plants derived from Event MIR604 in support for a permanent exemption fr om tolerances and Section 3 registration, submitted by Syngenta Seeds, Inc. – Field Crops- NAFTA. U.S. EPA/BPPD Memorandum from A. Fellman to M. Mendelsohn. Dated May 31, 2005.

Fellman, A. 2006 (c). Review of product characteriza tion and human health da ta for modified Cry3A (mCry3A) Bacillus thuringiensis insect control protein and mai ze plants derived from Event MIR604 in support for a permanent exemption fr om tolerances and Section 3 registration, submitted by Syngenta Seeds, Inc.-field crops -NAFTA. U.S. EPA/BPPD Memorandum from A. Fellman to M. Mendelsohn. Dated Feb. 15, 2006.

Gallagher, S.P., Mitchell, L.R. and Beavers, J.B. 2003. An acute oral toxicity study of modified Cry3A protein (MCRY3A-0102) in the north ern bobwhite. Syngenta, MRID No. 46155616.

Garcia-Alonso, M. and Vlachos, D. 2003. Charact erization and safety of modified Cry3A Bacillus thuringiensis protein and maize (corn) plants derived from event MIR604. EUP submission for MIR604. Syngenta, MRID No. 46155601

Johnson, I. 2003. Acute oral toxic ity study of modified Cry3A protei n (MCRY3A-0102) in the mouse. Syngenta, MRID No. 46155610.

Matten, S.R., Milofsky, T., and M. Hunter. 2006. Insect resistance management assessment for modified Cry3A (mCry3A) corn rootworm-protect ed corn (Event MIR 604). U.S. EPA/BPPD

Memorandum from S. Matten to M. Mendelsohn. Dated Sept. 06, 2006.

Milofsky, T. 2006. Environmental Risk Assessment for modified Cry3A (mCry3A) BacillusModified Cry3A Corn Biopesticide Registration Action Document (BRAD) July, 2010

129 thuringiensis protein and the genetic material necessary for it s production in Event MIR604

corn. U.S. EPA/BPPD. Memorandum from T. Milofsky to M. Mendelsohn, Dated Feb. 9,

2006.

NCBI. 2003. National Center for Biotechnology Information

Rabe, S. 2003. Molecular characterization of event MIR604 maize (corn) expr essing a modified Cry3A Bacillus thuringiensis protein. Syngenta, MRID No. 46155602

Raybould, A. 2004. Environmental safety assessment of modified Cry3A protein and event MIR604 corn to non-target organisms. Syngenta, MRID No. 46265601

University of Illinois Agricultural Extension Service Bulletins #22 and #25. 2003.

U.S. Department of Agriculture, Economic Res earch Service (USDA-ERS). 2001. Commodity costs

and returns.

U.S. Department of Agriculture, National Agri cultural Statistics Serv ice (USDA-NASS). 2001. Agricultural Statistics 2001.

U.S. Department of Agriculture, National Agri cultural Statistics Serv ice (USDA-NASS). 2002. Agricultural Statistics 2002.

U.S. Environmental Protection Agency. 2001. Biopesticides Registra tion Action Document, Bacillus thuringiensis Plant Incorporated Protectants.

U.S. Environmental Protection Agency. 2003. Biope sticides Registration Action Document, Event MON863 Bt Cry3Bb1

Vlachos, D. and Joseph, R. 2003. Characterization and safety of phosphomannose isomerase (PMI), a selectable marker expressed in event MIR604-derived maize (cor n) plants. Syngenta, MRID

No. 45934402130Appendix ATable 1A. Insecticide end-use products registered by EPAfor use on corn for control of corn rootworm species.ProductActiveIngredientsTypeaUse RatebUseClassificationcAmbush®Insecticide–SyngentaPermethrin –25.6% SP 0.2 lb/acAdultcontrolWARNING. Restricted Use; extremely toxic to fishand aquatic invertebrates, highly toxic to beesAsana®XL Insecticide0.66 EmulsifiableConcentrate- DuPontEsfenvalerate –8.4% SP 0.05 lb/acAdultcontrolWARNING. Restricted Use; extremely toxic to fishand aquatic invertebrates, highly toxic to beesAztec2.1% GranularInsecticide– BayerCorp.Tebupirimfos –2.0%cyfluthrin –0.1%OPSP0.15 lb/ac0.01 lb/acLarvalcontrolWARNING. Restricted Use; toxic to fish andwildlifeBaythroid2Emulsifiable PyrethroidInsecticide- BayerCyfluthrin –25%SP 0.04 lb/acAdultcontrolDANGER. Restricted Use; extremely toxic to fishand aquatic invertebrates, highly toxic to bees, maycause allergic skin reactionsCapture2ECInsecticide/Miticide–FMC Corp.Bifenthrin –25.1% SP 0.3 lb/acLarvalcontrolWARNING. Restricted Use; extremely toxic to fishand aquatic invertebrates, highly toxic to beesChlorfos15GInsecticide Granular–Griffin LLCChlorpyrifos –15% OP 2.02 lb/acLarvalcontrolCAUTION.Toxic to birds and wildlife, extremelytoxic to fish and aquatic organismsChlorfos4EInsecticide– GriffinLLCChlorpyrifos –42% OP 2.52 lb/acAdult &LarvalcontrolWARNING.Toxic to birds and wildlife, extremelytoxic to fish and aquatic organismsModified Cry3A CornBiopesticide Registration Action Document (BRAD) July, 2010131ProductActiveIngredientsTypeaUse RatebUseClassificationcCounterCR SystemicInsecticide-Nematicide– American CyanamidCompany Terbufos – 20% OP 1.30 lb/acLarvalcontrolDANGER. Restricted Use; fatal if swallowed,inhaled or absorbed through skin, extremely toxic tofish and wildlifeD-z-ndiazinon AG500Insecticide- Syngenta Diazinon – 48% OP 0.48 lb/acAdultcontrolCAUTION. Restricted Use; highly toxic to birds, fishand other wildlife, highly toxic to beesD-z-ndiazinon AG600WBC Insecticide-SyngentaDiazinon - 56% OP 0.45 lb/acAdultcontrolCAUTION. Restricted Use;highly toxic to birds, fishand other wildlife, highly toxic to beesDeclareEmulsifiableInsecticide Concentrate– Griffin LLCMethylparathion –45.11% OP 0.22 lb/acAdultcontrolDANGER. Restricted Use: fatal if swallowed,inhaled or absorbed through skin, highly toxic toaquatic invertebrates and wildlife, highly toxic to beesDiazinon 500-AGOrganophosphateInsecticide– UAPDiazinon – 48% OP 0.48 lb/acAdultcontrolCAUTION. Restricted Use;highly toxic to birds, fishand other wildlife, highly toxic to beesDimethoate 4 ECSystemic Insecticide-HelenaDimethoate –44.8% OP 0.45 lb/acAdultcontrolWARNING.Toxic to wildlife and aquaticinvertebrates, highly toxic to beesDimethoate 400Systemic InsecticideMiticide- UAPDimethoate –43.5% OP 0.44 lb/acAdultcontrolWARNING.Toxic to wildlife and aquaticinvertebrates, highly toxic to bees132ProductActiveIngredientsTypeaUse RatebUseClassificationc5 lb. DimethoateSystemic Insecticide-HelenaDimethoate –57% OP 0.46 lb/acAdultcontrolDANGER.Toxic to wildlife and aquatic invertebrates,highly toxic to beesForce3G Insecticide-Syngenta Tefluthrin – 3% SP 0.17 lb/acLarvalcontrolCAUTION. Restricted Use; very highly toxic tofreshwater and estuarine fish and invertebratesFortress2.5Ggranular insecticide-DuPontChlorethoxyfos– 2.5% OP 0.16 lb/acLarvalcontrolDANGER. Restricted Use; toxic to wild mammals,birds, fish and aquatic invertebratesFortress5G granularinsecticide– DuPontChlorethoxyfos– 5% OP 0.16 lb/acLarvalcontrolDANGER. Restricted Use; toxic to wild mammals,birds, fish and aquatic invertebratesFuradan4Finsecticide/ nematicide– FMC Corp.Carbofuran –44% C 0.88 lb/acAdult &larvalcontrolDANGER. Restricted Use; poisonous if swallowed orinhaled, toxic to fish, birds and other wildlife, highlytoxic to bees, can seep or leach through soil and cancontaminate groundwaterLannateLV insecticide– DuPontMethomyl –29% C 0.65 lb/acAdultcontrolDANGER. Restricted Use; fatal if swallowed, toxicto fish, aquatic invertebrates and mammals, highlytoxic to bees, known toleach through soil intogroundwaterLannateSP insecticide- DuPontMethomyl –90% C 0.45 lb/acAdultcontrolDANGER. Restricted Use; fatal if swallowed, maycause blindness, toxic to fish, aquatic invertebrates andmammals, highly toxic to bees, known to leach throughsoil into groundwaterLorsban15G GranularInsecticide– DowAgrosciencesChlorpyrifos –15%OP 2.03 lb/acLarvalcontrolCAUTION.Toxic to birds and wildlife, extremelytoxic to fish and aquatic organismsModified Cry3A CornBiopesticide Registration Action Document (BRAD) July, 2010133ProductActiveIngredientsTypeaUse RatebUseClassificationcLorsban-4EInsecticide– DowAgrosciencesChlorpyrifos –44.9% OP 2.69 lb/acAdult &larvalcontrolWARNING.Toxic to birds and wildlife, extremelytoxic to fish and aquatic organismsMocap10% GranularNematicide Insecticide–Aventis CropScience Ethoprop – 10% OP 3.53 lb/acLarvalcontrolWARNING.Toxic to aquatic organisms and wildlifeMocapECNematicide-Insecticide– Aventis CropScienceEthoprop –69.6%OP 3.34 lb/acLarvalcontrolDANGER. Restricted Use; toxic to aquatic organismsand extremely toxic to birdsPenncap-MMicroencapsulatedInsecticide– ElfAtochemMethylParathion – 22%OP0.44 lb/acAdultcontrolWARNING. Restricted Use; highly toxic to aquaticinvertebrates and wildlifePhorate 20 GOrganophosphateInsecticide- UAP Phorate – 20% OP 1.3 lb/acAdult &larvalcontrolDANGER. Restricted Use; extremely toxic to fishand wildlifePounce®WSBInsecticide– FMCCorporationPermethrin -24.7% SP 0.2 lb/acAdultcontrolWARNING. Restricted Use; extremely toxic to fishand aquatic invertebrates, highly toxic to beesPounce®3.2 ECInsecticide– FMCCorporationPermethrin –38.4% SP 0.2 lb/acAdultcontrolCAUTION. Restricted Use; extremely toxic to fishand aquatic invertebrates, highly toxic to bees134ProductActiveIngredientsTypeaUse RatebUseClassificationcPounce®25 WPInsecticide– FMCCorporationPermethrin –25% SP 0.2 lb/acAdultcontrolWARNING. Restricted Use; extremely toxic to fishand aquatic invertebrates, highly toxic to beesRegent4 SCInsecticide– AventisCropScienceFipronil – 39.4% PP 0.13 lb/acLarvalcontrolWARNING. Restricted Use; toxic to birds, fish andaquatic invertebratesSevinbrand 80SCarbaryl Insecticide–Aventis CropScience Carbaryl – 80% C 2.0 lb/acAdultcontrolWARNING.Extremely toxic to aquatic and estuarineinvertebrates, highly toxic to beesSevinbrand XLRPLUS CarbarylInsecticide– AventisCropScienceCarbaryl –44.1% C 1.76 lb/acAdultcontrolCAUTION.Extremely toxic to aquatic and estuarineinvertebrates, highly toxic to beesThimet20-G Soil andSystemic Insecticide–American CyanamidPhorate – 20% OP 1.3 lb/acLarvalcontrolDANGER. Restricted Use; extremely toxic to fishand wildlifeThimet20-G Soil andSystemic Insecticide–American CyanamidPhorate – 20% OP 1.3 lb/acLarvalcontrolDANGER. Restricted Use; extremely toxic to fishand wildlifeWarrior®Insecticidewith Zeon Technology-SyngentaLambdacyhalothrin –11.4%SP 0.03 lb/acAdultcontrolWARNING. Restricted Use; extremely toxic to fishand aquatic organisms and toxic to wildlife, highlytoxic to beesCruiser®5FS-SyngentaThiamethoxam N0.1 lb/ac(seeds)LarvalcontrolCAUTION. toxic to aquatic organismsPoncho®1250 -Gustafson LLCClothianidin N0.1 lb/ac(seeds)LarvalcontrolCAUTION.This product is toxic to aquaticinvertebratesModified Cry3A CornBiopesticide Registration Action Document (BRAD) July, 2010135ProductActiveIngredientsTypeaUse RatebUseClassificationcPrescribe®-Gustafson LLCImidacloprid N0.11 lb/ac(seeds)LarvalcontrolCAUTION. Highly toxic to birds and aquaticinvertebratesTable reproduced from MON 863 registration and updated with dataprovided by DAS in MRID # 46123921.a - OP: organophosphate; SP: synthetic pyrethroid; C:carbamate; PP: phenyl pyrazole; N: nicotinoidb – maximum labeled use rate expressed in pounds of active ingredient per acre (assumethat 1 liq pt  1 lb)c – precautionary language as stated on label.136 III. Regulatory Position for Modified Cry3A Protei n and the Genetic Material Necessary for its Production (Via Elements of pZM26) in Event MIR604 Corn SYN-IR604-8

A. Initial Regulatory Position

In 2006 EPA conditionally registered Syngenta Seeds Inc.'s new active ingredient, modified Cry3A protein and the genetic material necessary for its production (via elements of pZM26) in event MIR604 corn SYN-IR604-8. The Agency determined that the use of this pesticide was in the public interest and that it would not cause any unreas onable adverse effects on the environment during the time of conditional registration.

Results of efficacy trials conducted in 2002, 2003, and 2004 indicate that MIR604 corn provides effective control of key rootworm pests of field corn. MIR604 corn has unique biochemical properties that may benefit insect resistance management for this and other CRW-protected corn products. MIR604 contains the third CRW-active Bt corn prot ein on the market. The availability of multiple CRW-protected corn products will increase grower choice and price competition, resulting in lower seed prices for growers and higher adoption rates. Registration of MIR 604 corn is expected to result in further reduction of chemical insecticide use by gr owers. This is of special importance since many pesticides registered for CRW- control are highly toxic to huma ns and the environment, while mCry3A-expressing corn poses no foreseeable human health or environmental risks.

The new corn plant-incorporated protectant, Even t MIR604 corn, produces its own insecticide within the corn plant. This protectant, mCry3A protein, is derived from Bacillus thuringiensis (Bt), a naturally occurring soil bacterium. The mCr y3A protein used in this product controls corn rootworm, a highly destructive pest res ponsible for the single largest use of conven tional insecticides in the United States.

In order to reduce the possibility of corn rootworm developing resist ance to Bt, EPA required Syngenta Seeds, Inc. to ensure that 20 percent of the plante d acreage of this product be set aside in which nonCRW-protected corn will be grown to serve as a "refuge." These refuge areas will support populations of corn rootworm not exposed to the CRW-protected corn. The insect populations in the refuges will help prevent resistance development when they cro ss-breed with insects in the CRW-protected fields. This resistance management strategy was develope d as a condition of the registration, and EPA will require routine monitoring and documentation that these measures are followed. The submitted insect resistance management data support a registration until 2010.

A tolerance exemption at 40 CFR Part 174.456 was established for residues of Bacillus thuringiensis modified Cry3A protein and the genetic mate rial necessary for its production in corn.

Pursuant to FIFRA section 3(c)(7)(C), EPA ma y conditionally register a new pesticide active ingredient for a period of time reasonably sufficient for the generation and submission of required data that are lacking because insufficient time has elap sed since the imposition of the data requirement for those data to be developed. EPA may grant such conditional registration only if EPA determines that (1) the use of the pesticide product during the period of the conditional registration will not cause any

unreasonable adverse effect on the en vironment, and (2) the registrati on and use of the pesticide during the conditional registration is in the public interest. EPA determines that all of these criteria have been

fulfilled.137 The first criterion under FIFRA Section 3(c)(7)(C) me ntioned above has been met because insufficient time has elapsed since the imposition of the data requirements for:

1) Field degradation studies evaluating accumulation and persistence of mCry3A in several different

soils in various strata.

2) Three (3) year full-scale fiel d or semi-field studies for evalua tion of mCry3A Event MIR604 corn

exposure on non-target invertebrates.

3) Data to augment the mCry3A amino acid sequence analysis to known toxins and allergens within six months of the date of registrati on: specification of which version of NCBI database was utilized; descriptions of parameters utilized; and dates accessed for the BLAST search.

4) Insect resistant management da ta: a) Specific cross-resistance stud ies. Establish strains of CRW that are resistant to mCry3A and investigate the nature, inheritance, and fitness costs of specific mechanisms of resistance to the mCry3A protein e xpressed in MIR604 maize; b) Study the behavioral deterrence (avoidance) mechanism further and submit a ppropriate results; c) Continue studies on the biological impact of adults surviving on MIR604 maize and submit these results.

The applicants submitted or cited data sufficient fo r EPA to determine that conditional registration of modified Cry3A protein and the gene tic material necessary for its pr oduction (via elements of pZM26) in event MIR604 corn SYN-IR604-8 under FIFRA 3(c )(7)(C) will not result in unreasonable adverse effects to the environment, as discussed above. Th e applicants submitted and/or cited satisfactory data pertaining to the proposed use. The human health e ffects data and non-target or ganism effects data are considered sufficient for the period of the conditio nal registration. These data demonstrate that no foreseeable human health hazards or ecological effect s are likely to arise from the use of the product and that the risks of resistance developing to mCr y3A protein during the conditional registrations are not expected to be significant.

Registration of modified Cry3A pr otein and the genetic material necessary for its production (via elements of pZM26) in event MIR604 corn S YN-IR604-8 is in the public interest because:

1. Results of efficacy trials conducted in 2002, 2003, and 2004 indicate that MIR604 corn provides effective control of key rootworm pests of field corn.

2. MIR604 corn has unique biochemical propert ies which may benefit insect resistance management for this and other CRW-protected corn products.

3. If MIR604 corn is registered, it will be the third CRW-protected Bt corn product on the market. The availability of multiple CRW-protected corn products will increase grower choice and price competition, resulting in lower seed prices for consumers and higher adoption rates.138 4. Registration of MIR604 corn is expected to resu lt in further reduction of chemical insecticide use by growers. This is of sp ecial importance since many pestic ides registered for CRW-control are highly toxic to humans and the environm ent, while mCry3A-expressing corn poses no

foreseeable human health or environmental risks.

In view of these minimal risks and the clear benefits related to modi fied Cry3A protein and the genetic material necessary for its production (via elemen ts of pZM26) in even t MIR604 corn SYN-IR604-8, EPA believes that the use of the product during the limited period of the conditional registration will not cause any unreasonabl e adverse effects.

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

EPA has determined, as explained in section II.E. , that the third criterion for a FIFRA 3(c)(7)(C) conditional registration has been fu lfilled because the use of modifi ed Cry3A protein and the genetic material necessary for its production (via elements of pZM26) in event MIR604 corn SYN-IR604-8 under this registration is in the public interest.

The submitted data in support of this registration unde r section 3(c)(7)(C) of th e Federal Insecticide, Fungicide, and Rodenticide 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 effects 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 Update: Regulatory Position

Section 3(c)(7)(A) of FIFRA provides for the regi stration or amendment of a pesticide when the pesticide and proposed use “...are iden tical or substantially similar to any currently regi stered pesticide and use thereof, or differ only in ways that would not significantly increase the risk of unreasonable adverse effects on the environment, and (ii) appr oving 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 defi ned under section 2(bb) of FIFRA as “... any unreasonable risk to man or th e environment, taking into account the economic, social, and environmental costs and benefits of th e use of any pesticide...” Thus, pursuant to section 3(c)(7)(A), EPA may conditionally re gister a pesticide if (1) the pesticide and its proposed use are identical or substantially similar to a currently regist ered pesticide; or (2) the pesticide and its proposed use differ only in ways that would not significantly in crease the risk of unreasonable adverse effects; and (3) approving the registration w ould not significantly increase the risk of any unreasonable adverse

effect.139 The Agency concludes that the following mCry3A corn product registrati ons, set to expire in September 2010 and described in-depth throughout this BRAD, meet both criteria (1) and (2):

(1) Agrisure RW Rootworm-Pro tected Corn (EPA Reg. No. 67979-5) (2) Agrisure CB/LL/ RW (EPA Reg. No.67979-8)

All of these mCry3A corn products are identical in both composition and use (field corn) to plantincorporated protectants that are currently registered. Thus, criterion (1) has been fulfilled.

With regard to criterion (2), the Agency maintains, as was previously determined for the original registration of these particular products, that cu ltivation of mCry3A-contai ning corn will not cause

unreasonable adverse effects on the environment. The conditional environmental effects data, submitted in response to terms and conditions of re gistration and summarized in section II(C)(2)(b) of this BRAD, strengthen the Agency ’s initial position and also confirm that long-term effects on nontarget organisms are not anticipated. Lastly, the continued use of these products will likely still provide many of the benefits as were evaluated in sectio n II(E) of this BRAD to support the 2005 registration of Event MIR604 corn (e.g., reduction in use of conven tional insecticides that are highly toxic to both humans and the environment).

In conclusion, as the expiring mCry3A products (i.e., Agrisure RW Rootworm-Protected Corn, (MIR604 Corn, EPA Reg. No. 67979-5) and Agrisu re CB/LL/RW (Bt11 x MIR604 Corn, EPA Reg. No.67979-8)) have met the required criteria under s ection 3(c)(7)(A) of FIFRA, the Agency is proposing to amend these registrations to extend their respective expirati on dates as follows:

Product Name (EPA Reg. No.) Proposed Expiration Date
Agrisure RW Rootworm-Protected Corn (EPA Reg. No. 67979-5) September 30, 2015

Agrisure CB/LL/RW (EPA Reg. No. 67979-8) September 30, 2015

Although data provided were satisfac tory to make the determinations required by section 3(c)(7)(A) of FIFRA, they were not sufficient to support an unc onditional registration under FIFRA section 3(c)(5). Additional data, specifically in relation to aqua tic invertebrate testi ng and insect resistance management, are necessary for a finding of registra bility 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 Bt Corn reassessment, EPA determined th at it was appropriate to amend the then-existing registrations to extend the period of registration of those products to an expiration da te of October 15, 2008. All of the products being assesse d at that time were efficacious against lepidopteran pests. EPA based this action on the finding that use of Cry1Ab or Cry1F expressed in corn will not significantly140 increase the risk of unreasonable adverse effects on the environment "for the limited time period of 7 additional years (to October 15, 2008)." These registrations were late r amended to extend the period of registration to an expiration date of Septem ber 30, 2010. EPA subsequently granted time-limited registrations to products efficacious against coleopteran corn rootworm pests. For example, EPA registered Cry3Bb1 on February 24, 2003, to May 1, 2004, and extended that re gistration twice, to February 24, 2008, and September 30, 2010.

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

As discussed in the 2001 Bt PIP BRAD (at IID13), th e earliest Bt corn regi strations 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 th at the limited market penetration of these early crops would be so low as to gua rantee that adequate natural re fuges would be available from neighboring non-Bt corn fiel ds. From 1995 to 1997, Bt corn regist rations included voluntary refuge requirements of 0% to 20% in the corn belt. In 1999, the ABSTC, in conjunction with the National Corn Growers Association, proposed uniform IRM requi rements for Bt corn registrations. With some modifications, this proposal, put in place for the 2000 growing season, formed the baseline IRM requirements for almost all Bt corn registrations for the better part of a decade: farmers were required to plant a 20% refuge that could be treated for insects, or a 50% trea ted refuge in cotton-growing areas; all refuges to be planted within one-half mile of the Bt corn field.

These uniform requirements brought cer tainty and consistency to the ma rket after the initial period where many Bt corn products had different re fuge requirements. Recently, however, as product developers have begun to conceive of products with different combinations of "pyramided" products (i.e., products containing two or more toxins efficacious against the sa me pest) and "stacked" products (i.e., products combining toxins e fficacious against different pests) , the refuge requirements have begun to vary. For example, cert ain products require a 20% external refuge; some products permit a 5% external refuge; one product inco rporates a 10% seed blend refuge; we have applications in process for products that propose to incorpor ate a 5% seed blend refuge; and other permutations are possible.

Given the profusion of various t oxin combinations and refuge opti ons, we can no longer proceed on the basis that, as concerns insect resistance manage ment, all products are equal. It was a relatively simple proposition when the default requirement of a 20% sprayed refuge applied to almost all of the Bt corn crops in the market. Under those circumstan ces, the relative durability of products against the development of resistance was functionally equiva lent, and, as a consequence, imposing functionally equivalent registration peri ods was appropriate. That is now no longer the case.141 As part of our continually e volving regulatory approach to th e continually evolving product mix wrought by developers, we think it a ppropriate to revise our regulator y requirements in scientifically defensible ways to reflect the actual level of risk s posed by the products that we regulate. Here, for example, where we've determined that a particular product, or category of products, likely will pose less risk of insect resistance developing to a particul ar PIP protein, we think it appropriate to grant that particular product, or category of products, a regi stration for a period greater than that granted a corresponding product that poses a gr eater risk of insect resistance developing. This approach is reflective of complementary principles: first, to ensure that we apply our limited resources to the products that pose greater risk of adverse effect s to the environment; and, second, to conserve the resources that registrants and applicants must e xpend 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 re gistration periods of five, eight, and twelve years; a fifteen year registration period will also be available, if adequately supported by our science assessment. In this scheme, (i) a product with a single PIP toxin, and a 20% exte rnal refuge, qualifies for a five year registration; (ii) a product with py ramided PIP toxins (i.e., tw o 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 py ramided PIP toxins (i.e., two or mo re toxins with distinct, non-cross reacting modes of action), that are high-dose, with eith er a seed blend or extern al refuge, qualifies for a twelve year registration; (iv) a product with pyramided PIP toxi ns (i.e., two or more toxins with

distinct non-cross reactin g 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 baselin e single toxin product with a 20% external refuge, woul d qualify for a fifteen year regi stration. Products determined by

EPA's science assessment to be less than 100% as durable as the baseline si ngle toxin product with a 20% external refuge would not qualify for a five y ear registration and the re gistration 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 may arise, or where novel resistance concerns may be present, would also be determined on a case-by-case basis.