**DRAFT BIOPESTICIDES REGISTRATION ACTION DOCUMENT** 

Bacillus thuringiensis eCry 3.1Ab insecticidal protein and the genetic material necessary for its production (via elements of vector PSYN12274) in 5307 Corn ( SYN
-  53  7
- 1 )
PC Code: 016483

**U.S. Environmental Protection Agency Office of Pesticide Programs**  **Biopesticides and Pollution Prevention Division**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) June 2012 DRAFT

2 

### Table of Contents

**I. OVERVIEW**  A. Background .................................................................................................3 B. Use Profile ....................................................................................................4

**II. S**  **CIENCE ASSESSMENT** A. Product Characterization .................................................................................5 B. FQPA Safety Assessment
............... ...............................................................21 C. Environmental Assessment .............................................................................42 D. Bt11xMIR604xTC1507x5307 Environmental Assesment .........................................60 E. Bt11xMIR162xMIR604xTC1507x5307 Environmental Assessment ...........................67 F
. Insect Resistance Management .......................................................................73 G
. Public Interest Finding..................................................................................97 **I II**  **. REGULATORY**  **P OSITION**  **FOR 5307**  **C ORN**  .................................99Bacillus thuringiensis eCry3.!Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

3 Bacillus thuringie nsis eCry 3.1Ab insecticidal protein and the genetic material necessary for its production (via elements of vector PSYN12274) in 5307 Corn ( SYN
-  53  7
- 1 ) Corn Regulatory Action Team **Product Characterization and Human Health** John Kough, Ph.D. Annabel Waggon er **Environmental Fate and Effects** Gail Tomimatsu, Ph.D., Shannon Borges **Insect Resistance Management** Jeannette Martinez Alan Rey nolds **Benefit s**  **Assessment** Alan Rey nolds **Registration Support** Mike Mendelsohn **I. O**  **VERVIEW A.**  **Background** Sy ngenta Seeds, Inc.

– Field Crops

- NAFTA (S yn genta Seeds, Inc.) is seeking a FIFRA Sec. 3 (c)(5) Registration for Event 5307 maize [OECD Unique ID. SYN

-  53  7
- 1] for use as a plant

- incorporated protectant (PIP). S yn genta developed PIP Event 5307 maize (Zea mays ) through Agrobacterium

mediated transformation (via plasmid vector PSYN12274 ) to express eCry3.1Ab insecticidal crys tal protein to provide protection against root feeding damage caused b y the following coleopteran pest species: Diabrotica virgifera (western corn rootworm, WCR), D. longicornis barberi (northern corn rootworm, NCR), and D. virgifera zeae (Mexican corn rootworm, MCR). The eCry 3.1Ab is an engineered chimera protein , composed of portions of modified Cry 3A (mCry 3A) protein derived from the native Cry 3A protein from Bacillus thuringiensis ( Bt) subsp. tenebrionis and Cry 1Ab protein derived from Bt subsp. kurstaki HD
- 1 (Hofte and Whiteley , 1989 ; Walters et al. 2010). Chimeric cry genes can be engineered with novel insecticidal specificities and acti vities by exchanging various domains that are homologous between different cry genes (Hofte and Whiteley , 1989 ). Event 5307 corn also contains the pmi gene, which was introduced along with the eCry3.1Ab protein with the same PSYN12274 transformation vector . The gene represents the manA gene from Escherichia coli and encodes the enzyme phosphomannose isomerase (PMI), which was employed as a selectable marker during the process of regenerating plant material following transformation. The PMIprotein is aBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

4 co mmon enzy

me involved in carbohydrate metabolism to allow for selection of transformants in cell culture, b y only allowing transformed corn cells to utilize mannose as a sole carbon source, while corn cells lacking the pmi gene fail to grow (Negrotto, et al

. 2000). In conjunction with the Sec.3 registration of event 5307 maize, Sy ngenta Seeds, Inc. submitted a petition for an exemption from the requirement of a tolerance pursuant to section 408(d)(1) of the Federal Food, Drug, and Cosmetic Act with respect t o the PIP Bacillus thuringiensis eCry3.1Ab insect control protein and the genetic material necessary for its production in food and feed commodities of field corn, sweet corn, and popcorn. The genetic material necessary for the production of the PIP activ e ingredients are the nucleic acids (DNA, RNA) which comprise genetic material encoding these proteins and their regulatory

regions. The genetic material (nucleic acids) that are part of any PIP are exempt from the requirement of a tolerance [40 CFR § 174. 507]. In addition, an exemption from the requirement of a tolerance has been established for PMI enzy me in all food commodities when used as a plant

incorporated protectant inert ingredient [40 CFR § 174.527], thereby , the PMIenz ym e expressed in event 530 7 corn is covered in this exemption. The Agency

previousl y granted a Sec. 5 Experimental Use Permit (EUP) for event 5307 corn [EPA Reg. No. 67979
- EUP
- 8] to conduct experimental field tests, and in conjunction with the EUP, the Agency established a tempo rary

exemption from the requirement of a tolerance for Bt eCry 3.1Ab protein residues on food and feed corn commodities [40 CFR § 174.532], which is set to expire on December 31, 2013. **B. Use Profile Pesticide Name:**  Bacillus thuringiensis eCry 3.1Ab in secticidal protein and the genetic material

necessary for its production (via elements of vector PSYN12274) in 5307 Corn ( SYN
-  53  7
- 1 ) **Trade and Other**  5307 Corn; Agrisure TM VipteraTM 3222; Agrisure TM NGRW 3122 **Names: OPP Ch**  **emical Code:** 016483 **Basic Manufacturers: S**  yn genta Seeds, Inc.

– Field Crops

– NAFTA

P.O. Box 12257 Research Triangle Park, NC 27709

- 2257
**T ype of Pesticide:**  Plant

- In corporated Protectant **Use:** Field Corn **Ta rget Pests:**  w estern corn rootworm ( Diabrotica virgifera virgifera ), n orthern cornBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

5 r ootworm (

Diabrotica barberi ), and Mexican corn rootworm ( Diabrotica virgifera zeae ) **Products Expressing**  5307 Corn

– EPA File Symbol 67979

- EE **This Pesticide:** Bt11xMIR162xMIR604XTC1507x5307 Corn

– EPA File Symbol 67979

- EG

Bt11xMIR604XTC1507x5307 Corn

– EPA File Sy mbol 67979

- EU
**II. SCIENCE ASSESSMENT**  The classifications that are found for each data submission are assigned b y Environmental Protection Agency ( EPA ) science reviewer s and are an indication of the usefulness of the information contained in the documents for risk assessment . A rating of “ACCEPTABL E” indicates the study is scientifically sound and is useful for risk assessment . A “SUPPLEMENTA L” rating indicates the data provide some information that can be useful for risk assessment. T he studies may have certain aspects determined not to be scientificall y acceptable ( “ SUPPLEMENTAL : UPGRADABLE ” )
. If a study is rated as “SUPPL EMENTAL : UPGRADABLE ,” EPA alway s provides an indication of what is lacking or what can be provided to change the rating to “ACCEPTABLE.” If there is simply a “SUPPL EMENTAL” rating, the reviewer will often state that the study is not required b y the current 40 Code of Federal Regulations (CFR) Part 158. Both “ ACCEPTABLE ” and “ SUPPLEMENTAL ” studies may be used in the risk assessment process as appropriate. An “UNACCEPTABLE” rating indicates that the study is not useful for risk assessment and cannot be upgraded. **A. Product Cha**  **racterization** Sy ngenta Seeds, Incorporated has developed Event 5307 corn [OECD Unique ID. SYN

-  53  7
- 1] to express eCry 3.1Ab protein for use as a plant

- incorporated protectant (PIP) against root feeding damage caused b y the following coleopteran pest speci es: Diabrotica virgifera (western corn rootworm, WCR), D. longicornis barberi (northern corn rootworm, NCR), and D. virgifera zeae (Mexican corn rootworm, MCR). This proposed PIP is a recombinant fusion protein, composed of portions of modified Cry 3A (mC ry 3A) (derived from the native Cry 3A protein from B acillus thuringiensis ( Bt) subsp. tenebrionis ) and Cry 1Ab protein (derived from Bt subsp. kurstaki HD
- 1). Event 5307 maize also contains the manA gene from Escherichia coli , which encodes phosphomannose is omerase (PMI) as a selectable marker in regenerating plant material following transformation (Negrotto et al. 2000). The PMIprotein is a common enzy

me involved in carbohydrate metabolism to allow for selection of transformants in cell culture, b y only all owing transformed corn cells to utilize mannose as a sole carbon source, while corn cells lacking the pmi gene fail to grow (Negrotto, et al. 2000).Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

6 **1 . Transformation System**  Based on amino acid sequence similarit y and crystal structures, known Cry p roteins have a similar three

dimensional structure comprised of three domains, Domain I, II and III (Nakamura et al ., 1990; Li et al ., 1991; Ge et al ., 1991; Honee et al ., 1991). The toxin portions of Cry proteins are characterized b y having five conserved blocks (CB) across their amino acid sequence. These are numbered CB1 to CB5 from the N

- terminus (5’ end) to the C

- terminus (3’end) (Hofte and Whiteley , 1989). The sequences preceding and following these conserved blocks are highl y variable and are design ated as variable regions V1 to V6. Because Cry proteins share structural similarities, chimeric cry genes can be engineered via the exchange of domains that are homologous between different cry genes. eCry 3.1Ab was engineered as a chimera protein b y exchanging the variable regions (V1 to V6) of mCry 3A protein between Cry 1Ab protein for enhanced toxicity against western corn rootworm ( Diabrotica virgifera ) and other Diabrotica spp . The ecry3.1Ab gene (Entrez Accession Number GU327680 [NCBI, 2011]) (Walters et al. 2010) consists of a fusion between the N

- terminus (Domain I , Domain II, and a portion of Domain III) of the mcry3A gene a and the C

- terminus (a portion of Domain III and Variable Region 6) of the cry1Ab gene b (Hofte and Whiteley , 1989) (see Figure 1)
. **Figure 1. Schematic**  **Illustrating the Origin of**  **the Amino A**  **cid Residues Present in**  **eCry3.1 Ab** 

Figure 1 was reproduced from Figure 1(A), page 11 in MRID No. 48442 501 **a Description of the**  **mcry3A gene:**  A maize

- optimized cry3A gene was synthesized to accommodate the preferred codon usage for maize (Murray et al. 1989). The synthetic sequence w as based on the native Cry3A protein sequence from Bac illus thuringiensis subsp. tenebrionis (Sekar et al. 1987). The maize

- optimized gene was then modified to incorporate a consensus cathepsin

- G protease recognition site within the expressed protein. T he mcry3A gene encodes for mCry3A protein , which is exp ressed in Event MIR604 maize . Event MIR604 is currently registered for use as a PIP (EPA Reg. No.67979
- 5) and its risk and benefits assessment is found in the Agency’s BRAD for mCry3A corn (US EPA, 2006). An exemption from the requirement of a tolerance wa s established for mCry3A in maize [40 CFR § 174.505 ]. **b Description of the**  **cry1Ab gene:**  The cry1Ab gene w as originally cloned from Bacillus thuringiensis var. kurstaki strain HD
- 1 (Geiser et al. 1986). Its amino acid sequence has been codon

- optimized (Koziel et al. 1997) to accommodate the preferred codon

- usage for maize (Murray et al. 1989). T he cry1Ab gene encodes for Cry1Ab protein , which is expressed in Event Bt11 maize . Event Bt11 is currently registered for use as a PIP (EPA Reg. No.67979

- 1) and its risk and benefits assessment is found in the BRAD for the Agency’s Reassessment for Bt crops in 2001 (US EPA, 2001). An exemption from th e requirement of a tolerance w as established for Cry1Ab in all crops [40 CFR § 174.511 ].Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

7 T ransformation of Zea mays to produce event 5307 maize was accomplished using immature embryos of a proprietary maize line via Agrobacterium tumefacie ns - mediated transformation (Negrotto et al . 2000). The region between the left and right borders of the transformation plasmid included ecry3.1Ab and pmi gene expression cassettes. This T

- DNA was transferred into the maize genome during transformation wi th plasmid vector pSYN12274. The ecry3.1Ab expression cassette consisted of the ecry3.1Ab coding region regulated b y a cestrum y ellow leaf curling virus promoter (CMP) and a nopaline s ynthase (NOS) poly adenylation termination sequence. The pmi expression cassette consisted of the pmi coding region regulated b y a Z. mays poly ubiquitin promoter (ZmUbiInt) and the NOS poly adenylation termination sequence. The ecry3.1Ab expression cassette consisted of the ecry3.lAb coding region regulated b y a cestrum yellow leaf curling virus promoter (CMP) and a nopaline s ynthase (NOS) poly adenylation sequence. The pmi expression cassette consisted of the pmi coding region regulated b y a Z. mays poly ubiquitin promoter (ZmUbilnt) and the NOS poly adenylation sequence. **2 . Ch**  **aracterization of the DNA Inserted in the Plant and Inheritance and Stability** Characterization of genomic DNA isolated from event 5307 corn leaf tissue using restriction enzy me digests and Southern blot anal ys es indicated that the DNA was inserted in the corn genome at a single locus, and the insert contains one cop y of the ecry3.1Ab and pmi expression cassettes. There were no other detectable genetic elements other than those associated with the respective cassettes. No vector backbone sequences from the transformation plasmid pSYN12274 were found in event 5307 maize
. Segregation patterns over several generations of event 5307 maize confirmed the expected inheritance ratio for both the ecry3.1Ab and pmi genes consistent with Mendelian principles. Sequenci ng data demonstrated that the insert is intact with the exception of one single nucleo tide change when compared to the transformation plasmid pSYN12274. This nucleotide change was located 48 base pairs upstream of the CMP promoter, in a non

- coding region of the T

- DNA, and has no effect on the functionalit y of the T

- DNA insert. Therefore, the overall integrity of the insert and the functional elements within the insert are contiguous, as present in pSYN12274. **3 . Protein Characterization and Equivalence**  Production of microbial

- derived eCry 3.1Ab protein was chosen in order to obtain sufficient material for testing, therefore, its equivalence to the plant

- derived eCry 3.1Ab protein expressed in event 5307 corn was determined. The ECRY3.1AB
- 0208 test substan ce, containing purified microbially

- produced eCry 3.1Ab protein, was prepared from an Escherichia coli ( E. coli ) overexpression sy stem to contain the same ecry3.1Ab gene that was introduced into Event 5307 corn plants
. The eCry 3.1Ab protein expressed in tes t substance ECRY3.1AB

- 0208 is identical to that expressed in transgenic maize Event 5307 except that it contains one additional methionine and six additional histidine residues at the N

terminus, which were added to aid purification purposes in microbial p roduction. Sodium dodecyl sulfate

- poly acrylamide gel electrophoresis (SDS

- PAGE) analy ses showed the eCry 3.1Ab proteins from both sources had the same approximate molecular weight ( ca . 74.8 kDa) based on the predicted molecular weight of ca . 74.8 kDa. De nsitometric anal ys es showed the purit y of eCry 3.1Ab in the test material to be ca. 89.6% by weight. Western blot analy sis showed that both plant

-Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

8 produced and microbially produced eCry 3.1Ab cross

- reacted with the same antibodies, confirming the identity and integrit y of the eCry 3.1Ab protein. The mass of the eCry 3 .1Ab protein contained in ECRY3.1AB
- 0208 measured b y mass spectrometry, was 74832.80 Da (74.8 kDa) confirming the theoretical mass based on amino acid sequence analy sis. In addition, the expected N

- terminal peptide was confirmed for eCry 3.1Ab from both sources. Both proteins produced comparable insecticidal activity in insect feeding bioassay s with first instar Colorado potato beetle ( Leptinotarsa decemlineata ) larvae , based on overlapping ranges of LC 50 values. There was also no evidence of post

- translational gl ycos yl ation of eCry 3.1Ab protein from either source. Collectivel y, the results support the conclusion that eCry 3.1Ab produced in recombinant E. coli (test substance ECRY3.1AB

- 0208) is biochem icall y and functionall y equivalent to the eCry3.1Ab produced in Event 5307 maize, and is a suitable surrogate for eCry 3.1Ab produced in Event 5307 maize. **4 . Protein Expression**  Exp ression level data were submitted to quantify eCry 3.1Ab protein concentrati ons in different plant tissues, sampled at key

developmental growth stages in event 5307 corn using ELISA anal ys es. A summary of the results are provided in Table 1 below.
**Table 1. Protein Expression levels**  **of eCry3.1Ab protein in Event**  **5307 Corn Tissue s** 

**Tissue Type eCry3.1Ab**  **Protein (μg/g dry weight ± SD)**  **a** Leaves <LOQ **b**

- 142.96 ± 53.44 Roots 9.13 ±6.38
- 42.72 ±4.67 Whole plants 8.27 ±2.90
- 111.08 ±38.36 Kernels 4.45 ±0.82
- 6.19 ±1.87 Pollen **c** 0.10 **b**
**a** Range reflects means at different growth stages across field sites **b** LOQ = 0.10 μg/g dry weight **c** Pollen samples were pooled for each location, however, due to insufficient material or samples detected at the LOQ, the mean protein concentration for pollen across the field locations was n ot calculated. How ever, m ost of the samples were detected at the LOQ (0.10 μg/g dry weight ), which is sufficient for reporting the eCry3.1Ab protein in pollen. Table 1 was reproduced from Tables 3

- 7 in MRID No. 48442509
.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

9 **Ta ble 2**  **. Product Char**  **acterization Data Submitted for Event 5307**  **maize STUDY TITLE**  **STUDY SUMMARY MRID NO.**  Molecular characterization of Event 5307 maize **a** Transformation of Zea mays (maize) to produce event 5307 was

described and the presence of the intended transgenic DNA insert, containing the ecry3. 1Ab and pmi genes into the plant genome, was confirmed via PCR anal yses. Segregation patterns over several generations of 5307 maize confirmed the expected Mendelian inheritance ratio for both the ecry3.1Ab and pmi genes were inherited in the predicted manner consistent with Mendelian principles. A single intact insert in 5307 maize and single copies of the ecry3.1Ab and pmi genes were identified. Event 5307 maize is free of an y backbone sequen ces from the transformation pl asmid pSYN12274. Sequence anal ys is of the 5307 maize insert showed one single nucleo tide change when compared to the transformation plasmid pSYN12274. This nucleotide change was located 48 base pairs upstream of the CMP promoter, in a noncoding region o f the T
- DNA, and has no effect on the genes encoded by event 5307 maize. **CLASSIFICATION: ACCEPTABLE; This report is superseded by MRID No. 484425**  **- 01.**  **477347 -**  **02**

Comparison of eCry 3.1Ab protein produced in Event 5307

- derived maize plants and eCry 3.1Ab p rotein produced in recombinant Escherichia coli **a** The plant

- derived eCry 3.1Ab protein (extracted from 5307 maize

tissue) and the bacterial

- derived eCry 3.1Ab protein (produced in a recombin ant E. coli over - expression sy stem) revealed similar molecular wei ghts (73.7 and 74.8 kDa, respectively). Both eCry 3.1Ab plant

- and bacterial

- derived proteins were immunologically

cross - reactive with antibodies capable of detecting the eCry 3.lAb protein. Both eCry 3.1Ab plant

- and bacterial

- derived proteins showed comp arable insecticidal activit y against Colorado potato beetle larvae and no evidence of post

translational gl yc osylation. Peptide mass mapping anal ys is showed that the identified peptides corresponded to regions throughout the sequence of eCry 3.1Ab from both eCry 3.1Ab plant

- and bacterial

derived proteins, except that the microbial

- derived version contains one additional methionine and six histidine residues at the N

terminus. **CLASSIFICATION: ACCEPTABLE 477347**  **- 03** 

Characterization of Test Substance ECRY3.1 AB
ECRY3.1AB
- 0208 test substance (containing bacterial

- derived

eCry 3.1Ab protein) was tested at various buffers and was readily soluble at 10 mg/mL ; ECRY3.1AB
- 0208 was determined to **477347 -**  **04**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

10 **STUDY TITLE STUDY SUMMARY**  **MRID NO.** 0208 and Certificate of Analy sis **a** contain 92.4% protein (estimated by ab sorbance at 280 nm via spectrophotometry) and densitometry anal ys is after SDS

- PAGE determined the test substance to contain 97.0% eCry 3.1Ab protein. Thereb y, the overall purity of test substance ECRY3.1AB

- 0208 was calculated to be 89.6% eCry 3.1Ab (w/w). Western blot anal ys is revealed an immunoreactive band representing a molecular weight of ~74.8 kDa; The total mass of the protein was measured by a quadrupole time

- of

- flight mass spectrometer and confirmed the molecular weight of ECRY3A.1AB

- 028 as 74832.80 Da (74.8 kDa). **CLASSIFICATION: ACCEPTABLE** Bioactivity

of Test Substance ECRY3.1AB
0208 **a** The bioactivity of the bacterial

- produced eCry 3.1Ab protein in test material ECRY3.1AB

- 0208 was assessed in three independent insect feeding bioassay s with first instar Colorado potato beetle ( Leptinotarsa decemlineata ) larvae. The larvae were provided diets containing various dose concentrations of eCry 3.1Ab μg/mL of diet for six day s. The larvae demonstrated a dose response to the test material, with 100% mortality at the highest dose. Mortality in the buffer control and the assay control group was 14% and 17%, respectivel y. The L C 50 for the test was 2.3 μg/mL(95% c.i., 1.4

- 3.3 μg/mL). **CLASSIFICATION: ACCEPTABLE 477347**  **- 05** 

Evaluation of Transgeni c Protein L evels in Multiple Generations of Plants Derived from Transformation Event 5307 Maize **a** The concentration of eCry3.1Ab and PMIprotein expression were

measured via E LI SA in plant tissues collected from five Event 5307 maize plants, representing 4 generations (at plant growth stages VT
- R1) in a greenhouse study . The mean eCry 3.1Ab protein concentration in leaves, roots, and pollen of the four generations ranged from 83.40 to 93.67 μg/g dry weight, 23.88 to 35.39 μg/g dry

weight, and <LOD (0.08 μ g) to 0.15 μg/g dry weight, respectively . The mean PMIprotein concentration in leaves, roots, and pollen of the four generations ranged from 1.77 to 1.95 μg/g dry

weight, 1.05 to 1.19 μg/g dry weight, and 18.96 to 25.58 μg/g dry

weigh, respectively . There fore, the concentrations of eCry 3.1Ab and PMI protein were comparable across four generations of event 5307 maize at VT

- R1 growth stages were comparable, indicating consistency of protein expression across multiple generations. **CLASSIFICATION: SUPPLEMENT AL**  **- Data deficiencies**  **477347 -**  **07**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

11 **STUDY TITLE STUDY SUMMARY**  **MRID NO.**

**were addressed in MRID N**  **o. 484425 -**  **09**

Molecular characterization of Event 5307 maize in support of the EUP application; Amended Report No. 1: Replaces Original Report Dated February 17, 2009; MRI D No. 47734702 Transformation of Zea mays (maize) with plasmid pSYN12274 to

produce event 5307 was described. The presence of the intended transgenic DNA insert, (containing the ecry3. 1Ab and pmi genes) in the plant genome was confirmed via PCR anal yses. Segregation patterns over se veral generations of 5307 maize confirmed the expected inheritance ratio for both the ecry3.1Ab and pmi genes consistent with Mendelian principles. Southern blot analyses demonstrated that the T

- DNA insert in event 5307 maize carries a single cop y of the ecry3.1Ab and pmi genes. In addition, event 5307 maize is free of an y backbone sequen ces from the transformation plasmid pSYN12274. Sequence anal ys is of the 5307 maize insert showed one single nucleo tide change when compared to the transformation plasmid pSYN12274. This nucleotide change was located 48 base pairs upstream of the CMP promoter, in a non

coding region of the T

- DNA, and has no effect on the genes encoded b y event 5307 maize. **CLASSIFICATION: ACCEPTABLE 484425**  **- 01**  **[This report**  **supersedes MRI**  **D No. 47734702]** 

Characterization of Phosphomannose Isomerase Test Substance PMI

0105 and Certificate of Analy sis The purpose of this stud y was to characterize microbially produced

test substance PMI

- 0105 for its solubility , purit y, protein identity , integrit y and enzymatic activity . The PMI

- 0105 test substance was produced b y the manA gene in the recombinant Escherichia coli over - expression s ys tem and encodes the same amino acid sequence as the plant

- expressed PMIprotein present in maize event 5307 as a P IP inert ingredient. The results of the solubility of PMI

- 0105 in various buffers were appropriate. Spectro photometric and densitometric anal ys is showed the overall purity /concentration of the PMIprotein in PMI
- 0105 test substance as 89.5% PMIby **484425 -**  **02**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

12 **STUDY TITLE STUDY SUMMARY**  **MRID NO.**

weight
. SDS
- PAGE analy ses showed a molecular weight of 42.8 kDa. Western blot anal ys is revealed a dominant immunoreactive band corresponding to the expected PMI molecular weight of 42.8 kDa. The predicted PMI molecular weight was also confirmed as 42,850 Da b y qu adrupole time

- of

- flight mass spectrometry . N
terminal sequencing analy ses confirmed that the first 15 amino acids of PMI in test substance PMI

- 0105 corresponded to the predicted N

- terminal sequence of PMI protein encoded by the manA gene. Results of the en zy matic activity

assay showed the PMIprotein in test substance PMI

- 0105 was active, with an average specific activity

of 503.77 U/mg PMI. In conclusion, the results confirmed the solubility , purit y, protein identity , integrit y and enzy matic activity

of PM I protein in the microbial

- expressed PMI
- 0105 test substance, and its suitability as biochemically

and functionally

equivalent to the plant

- expressed PMI protein in event 5307. **CLASSIFICATION: ACCEPTABLE** Comparison of phosphomannose isomerase produced in event 5307
- derived maize plants and phosphomannose isomerase produced in recombinant Escherichia coli. The purpose of this stud y was to demonstrate the biochemical and

functional equivalence between the plant

- expressed phosphomannose isomerase (PMI) protein in event 5307 maize and PMI
- 0105 test substance (the microbiall y - produced PMIprotein prepared from a recombinant E. coli over - expression sy stem). PMIfrom event 5307 plant leaf material (pedigree NP2171 x BC5F3) and from PMI

- 0105 test substance showed the same mobility in the Western blot analy sis and confirmed an apparent molecular weight consistent with the predicted molecular weight of approximately

42.8 kDa. Western blot analy ses showed the PMI protein from both sources were immunolog icall y cross - reactive with anti

- PMIantibodies. Inan enzy matic activit y assay , both the plant

- produced PMI and the microbially

- produced test substance were active. The specific activity for plant produced PMIwas 455.67 Units/mg PMIand the specific activ ity for the microbially

produced PMI was 526.26 Units /mg PMI
. These results support the conclusion that PMIproduced PMI

- 0105 test substance is biochemically

and functionally

equivalent to the PMIproduced in 5307 maize. Therefore, the PMIproduced in rec ombinant E. coli (test substance PMI

- 0105) is suitable surrogate for PMIproduced in event 5307 maize. **CLASSIFICATION: ACCEPTABLE 484425**  **- 03** 

Event 5307 maize: Copy Southern blot analy ses of 5307 maize demo nstrated that (1) 5307

maize contains a single copy of ecry3.1Ab , pmi , the CMP promoter **484425 -**  **04**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

13 **STUDY TITLE STUDY SUMMARY**  **MRID NO.** number functional element southern blot analy sis sequence, the ZmUbiInt promoter sequence and two copies of the

NOS terminator sequence, as expected for a single insertion site; (2) there are no extraneous DNA fragments of the functional elements inserted elsewhere in the maize genome; and (3) 5307 maize is free of backbone sequence from the transformation plasmid pSYN12274. **CLASSIFICATION: ACCEPTABLE** Event 5307 maize: Genetic stability anal ys is Southern b lot analy ses of 5307 maize demonstrated that (1) the

5307 maize carries a single, complete cop y of the T

- DNA insert with no extraneous DNA fragments of plasmid pSYN12274 T

DNA inserted elsewhere in the maize genome, (2) the transgenic locus is stable acros s all the 5307 maize generations analyzed, and (3) all generational samples of 5307 maize examined were negative for the vector backbone sequence from the transformation plasmid pSYN12274. **CLASSIFICATION: ACCEPTABLE 484425**  **- 05** 

Event 5307 maize: Genome to insert junction anal ys is for translated open reading frames with a minimum size of 30 amino acids: Assessment of amino acid sequence similarity to known or putative toxins. Bioinformatic anal ys is of the DNA sequences spanning the

junctions between the mai ze genomic sequence and the 5307 maize insert were used to identify any putative open reading frames (ORFs) with a minimum putative translation size of 30 amino acids. One putative ORF spanning the junction between the maize genomic sequence and the 3' reg ion of the 5307 maize insert was determined. The Basic Local Alignment Search Tool for Proteins (BLASTP) program was used to search the National Center for Biotechnology

Information (NCBI) Entrez® Protein Database (2010) to determine whether the translatio ns of this putative ORF showed significant similarity to known and putative toxins. Results showed the amino acid translation of the putative ORF shares no biologicall y relevant amino acid sequence similarity to an y known or putative protein toxins. **CLASS IFICATION: ACCEPTABLE**  **484425 -**  **06**

Event 5307 maize: Genome to insert junction anal ys is for translated open reading frames with a minimum Bioinformatic anal ys is of th e DNA sequences spanning the

junctions between the maize genomic sequence and the 5307 maize insert were used to identify any putative open reading frames (ORFs) with a minimum putative translation size of 30 amino acids. One putative ORF spanning the junc tion between the maize genomic sequence and the 3' region of the 5307 maize insert was identified. The translation of the putative ORF was sy stematicall y **484425 -**  **07**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

14 **STUDY TITLE STUDY SUMMARY**  **MRID NO.** size of 30 amino acids: Assessment of amino acid sequence to known or putative allergens. compared against the protein sequences in the Food Allergy

Research and Resource Program AllergenOnlin e database (2010; version 10.0) using a FASTA search algorithm (2010, version 3.45). No amino acid sequence similarities were observed after comparing 80 sequential amino acid peptides of this translated putative ORF with an y known allergen in the Allergen Online database spanning either junction of the maize genomic DNA and event 5307 maize insert . Also, no matches of eight or more contiguous amino acids were observed between the translated putative ORF and an y entry in the AllergenOnline database. Therefor e, the amino acid translations of the putative ORF spanning the junction between the maize genomic sequence and the 3' region of the 5307 maize insert have no biologically relevant amino acid sequence similarity to known or putative protein allergens. **CLA SSIFICATION: ACCEPTABLE**  Validation of the ELISA method for quantitative detection of eCry 3.1Ab protein in maize grain, broiler diets, and rat diets. A previously validated enzy

me - linked immunosorbent assay (ELI SA) method (Beacon Analy tical Sy s tems, Portland, ME) was evaluated for its performance as a suitable anal yt ical method for the quantification of eCry 3.1Ab protein in maize grain, broiler chicken diets, and rat diets. The parameters for validation included accuracy , quantitative range, int ermediate and repeatability precision, sensitivity

(minimum dilution factor, limit of detection (LOD), limit of quantitation (LOQ)), short

- term and freeze/thaw stability , specificit y of the assay based on cross

- reactivity with non

target transgenic protein s . Inaddition, the ECRY3.1AB
- 020 test substance ( a microbiall y - derived eCry 3.1Ab recombinant protein prepared from an over

- expression E. coli sy stem ) used in the ELISA method as a positive assay control sample (PACS) for eCry 3.1Ab was anal yz ed to establis h a new specification range for the eCry 3.1Ab
. Results showed the performance parameters evaluated in this study for the ELISA method were within the acceptance criteria defined for each parameter; therefore, the validity of the ELISA method used for de termining eCry 3.1Ab protein concentrations in event 5307 maize grain, broiler diets, and rat diets is confirmed. In addition, the dilution factors, LODs, LOQ, and extraction efficiencies can be used for subsequent studies using ELISA anal ys es for quantifyi ng eCry 3.1Ab in maize grain, broiler chicken **484425 -**  **08**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

15 **STUDY TITLE STUDY SUMMARY**  **MRID NO.**

and rat diets. In a separate report, the accuracy of the values reported in this study were verified based on the review of individual data used in calculating the accuracy , precision, percent recoveries, coeff icient of variations, dilution factors, extraction efficiencies, and assay specificit y (MRID No. 48802604). **CLASSIFICATION: ACCEPTABLE** Quantification of eCry 3.1Ab and phosphomannose isomerase in maize tissues derived from transformation eve nt 5307. Field studies were conducted to quantify eCry 3.1Ab and PMI protein concentrations in event 5307 maize plant tissue matrices (leaves, roots, whole plants, pollen, and kernels) at key developmental growth stages (whorl, anthesis, maturity , and sene scence) via enzy

me - linked immunosorbent assay (ELISA) anal ys es across four different locations in the 2008 growing season. The mean concentrations of eCry 3.1Ab across all locations and plant stages on a dry

- weight basis ranged from <LOQ to 142.96 ± 53.44  g/g in leaves, from 9.13 ±6.38 to 42.72 ±4.67  g/g in roots, and from 8.27 ±2.90 to 111.08 ±38.36  g/g in whole plants. The mean concentrations of eCry 3.1Ab across all locations on a dry

weight basis ranged from 4.45 ±0.82 to 6.19 ±1.87  g/g in kernels at maturity

and senescence stages. The mean concentration of eCry 3.1Ab in pooled pollen samples (collected only at the anthesis stage) was not calculated across locations, because some of the eCry 3.1Ab samples were not quantifiable or unavailable. However, most of the samples were detectable at the LOQ (0.10  g/g); therefore, the 0.10  g/ g can be designated as the eCry 3.1Ab protein concentration in pollen for the purposes of this study
. The mean concentrations of PMIacross all locations and plant developme ntal stages on a dry

- weight basis ranged from <LOQ to 4.83 ±1.47  g/g in leaves, <LOQ to 2.11 ±0.60  g/ g in roots and 0.97 ±0.37  g/g to 4.38 ±2.43  g/g in whole plants. The mean concentrations of PMIacross all locations on a dry

- weight basis ranged from 1 .11 ±0.05  g/ g to 2.08 ±0.49  g/g in kernels at maturity and senescence stages, while the mean concentration of PMIacross locations in pooled pollen samples was 6.51 ±0.72  g/ g. **484425 -**  **09 [This**  **report supersedes**  **MRID No. 47734707]** Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

16 **STUDY TITLE STUDY SUMMARY**  **MRID NO.**

The results show the protein concentrations of eCry3.1Ab and PMI protein extracted from various plant tissue matrices collected from four field sites of event 5307 maize were either detectable or quantifiable in all tissues anal yz ed with the exception of some leaf tissue samples for PMIprotein at the senescence stage. Moreover, the ranges of concentrations for eCry 3.1Ab and PMIproteins expressed in various plant tissue matrices of event 5307 maize were similar for each developmental stage across multiple field sites. These results indicate the stability of the transgenic insertio n of ecry3.1Ab and pmi gene cassette b y encoding consistent levels of eCry 3.1Ab and PMI protein expression in event 5307 maize. **CLASSIFICATION: ACCEPTABLE** Validation of the ELISA method for quantitat ive detection of eCry 3.1Ab. This study was conducted to validate the performance of an enzy me - linked immunosorbent assay (ELISA) method as a suitable anal yt ical method for the quantitative anal ys is of eCry 3.1Ab protein. The ELISA is a double

- antibody sandwich assay

specific to eCry 3.1Ab developed by Beacon Anal yt ical Sy stems, Inc. (BAS). Analy sis of eCry 3.1 Ab reference protein standard curves was performed to evaluate the precision of the assay , evaluate the goodness of fit of a four

- parameter logistic mo del, determine the quantitative range of the assay , evaluate lot

- to - lot variability of the ELISA test kits, and cross

- reactivity with non - target transgenic proteins (AMY797E, Cry 1Ab, PAT, mCry 3A, PMIas expressed in event MIR604 corn, Vip3Aa20, and mEPSPS)
. Results show the performance parameters evaluated in the BAS ELISA method were within the acceptance criteria defined for each performance parameter. T herefore, the validity of the ELI SA method used for determining eCry 3.1Ab protein concentrations in event 5307 maize is confirmed and the BAS eCry3.1Ab ELI SA kit is suitable as a quantitative anal yt ical detection method for eCry 3.1Ab. In addition, the dilution factors, LODs, L OQ, and extraction efficiencies can be used for subsequent studies using ELISA anal ys es for quantifying eCry 3.1Ab in maize. In a separate report, the accuracy of the values reported in this study were verified based on the review of individual data used in calculating the intra

- and inter

- assay precision , goodness

- of

- fit , assay ba ckground , quantitative range , lot

- to - lot variability , and cross reacti v ity with non

- target proteins (MRID No. 48802604). **CLASSIFICATION: ACCEPTABLE 484425**  **- 14** Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

17 **STUDY TITLE STUDY SUMMARY**  **MRID NO.** Validation of a quantitative ELISA method for indication of eCry 3.1Ab protein stability and qu alification of the eCry 3.1Ab positive control for use in the eCry 3.1 Ab quantitative ELISA method. This study was conducted to test the suitability of a previousl y validated enzy

me - linked immunosorbent assay (ELISA) kit (Beacon Anal yt ical Sy stems, Inc. (B AS)) for use as a protein stability indicating method. The BAS ELISA is a double

- antibody sandwich assay

specific for the quantitative anal ys is of eCry 3.lAb protein. The suitability

was evaluated b y the ability of the ELISA method to detect a decrease in the amount of eCry3.lAb protein due to destabilization, which may occur during long term storage. Using accelerated protein destabilization through forced denaturation, ELISA analy ses of the eCry 3.lAb protein treated with sodium dodecyl sulfate (SDS) demons trated decreasing eCry 3.lAb recovery with increasing SDS concentration. Thus, the BAS eCry 3.1Ab ELISA method is suitable for indicating the protein stability of eCry 3.1Ab protein. Additionally , the qualification of ECRY3.1AB

- 0208 test substance (a micro biall y - derived eCry 3.1Ab protein prepared from an Escherichia coli overexpression sy stem) for use as the eCry 3.1Ab positive control sample material was conducted. Results showed the concentration of the eCry 3.lAb positive control sample m aterial is 116.73 ug/ml, within an acceptable range of 72.82 ng/ml to 160.64  g/ml. Therefore, the eCry 3.1Ab positive control sample material is qualified for use in the BAS eCry 3.1Ab ELISA method. **CLASSIFICATION: ACCEPTABLE 484425**  **- 15** 

Effect of temperature on the bioacti vity

of eCry 3.1Ab protein as contained in test substance. The study showed that eCry 3.1Ab (ECRY3.1AB

- 0208) prepared from an E. coli over - expression sy stem pre - incubated at 25oC, 37oC, and 65oC for 30 minutes and tested for activity in a bioassay with L. d ecemlineata (Colorado potato beetle) larvae retained activity relative to the protein pre

- incubated at 4oC. No activity was observed when eCry 3.1Ab was pre

- incubated at 95oC for 30 minutes. Thus, the bacterial

- expressed eCry 3.1 Ab protein becomes denatured and loses its biological activity after heat treatment for 30 minutes at 95°C. **CLASSIFICATION: ACCEPTABLE 484425**  **- 16** 

Validation of an ELISA method for quantification of eCry 3.1Ab protein in maize An enzy me - linked immunoabsorbent assay (ELISA) me thod was re - validated for its use as a suitable anal yt ical method for the quantitation of eCry 3.1Ab protein in maize kernels because minor modifications to the previously validated ELISA method have resulted in increased sensitivity of eCry 3.1Ab protein qu antitation **484425 -**  **17**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

18 **STUDY TITLE STUDY SUMMARY**  **MRID NO.** kernel. in kernel matrix. The method performance characteristics evaluated

included sensitivity

(minimum dilution factor, limit of detection), accuracy , quantitative range (upper and lower limits), linearity , method specificit y (cross

- reactivity), and e xtraction efficiency
. Results show the performance parameters evaluated for the BAS ELISA method were within the acceptance criteria defined for each performance parameter. T herefore, the validity of the ELI SA method used for determining eCry 3.1Ab prote in concentrations in event 5307 maize kernel is confirmed and the BAS eCry 3.1Ab ELISA kit is suitable as a quantitative anal yt ical detection method for eCry 3.1Ab in maize kernels . In addition, the dilution factors, LODs, LOQ, and extraction efficiencies can be used for subsequent studies using ELISA analyses for quantifying eCry3.1Ab in maize kernels
. In a separate report, the accuracy of the values reported in this study were verified based on the review of individual data used in calculating the accuracy , precision, percent recoveries, coefficient of variations, dilution factors, extraction efficiencies, and assay specificit y (MRID No. 48802604). **CLASSIFICATION: ACCEPTABLE** Independent Laboratory Validation/ Kit for eCry 3.1Ab Testing in Leaf

, Seed, and Bulk Grain Corn Two commercial qualitative lateral flow strip test kits were

developed b y EnviroLogixTM to determine the presence or absence of eCry 3.1Ab protein in corn grain, leaf or seed samples. The QuickStixTM Kit for eCry3.1Ab Corn Bulk Grain (Cat. No. AS TBD BG) dete cts eCry 3.1Ab protein at a level of 1% or greater (one kernel in 100). In grains samples prepared from two separate seed lots of event 5307 corn, eCry 3.1Ab protein tested showed 100% accuracy

in its detection and demonstrated its precision based on using t est kits taken from three different manufactured lots. The QuickStixTM Kit for eCry 3.1Ab Leaf and Seed (Cat. No. AS TBD LS) also detects eCry 3.1Ab protein at a level of 1%. Results from the cross

- reactivity test using the bulk grain test kit using samples f

rom event MIR604 transgenic corn (known to contain a protein with similar homology to eCry 3.1Ab protein) were negative. Additional cross reactivity anal ys es were negative for leaf tissue and seed samples from transgenic corn containing other unrelated Cry proteins. Collectively , these r esults demonstrate the test kits are able to detect eCry 3.1Ab protein residues in corn grain, leaf and seed with sufficient accuracy , precision, and **477347 -**  **17**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

19 **STUDY TITLE STUDY SUMMARY**  **MRID NO.**

sensitivity
. Thus, the EnviroL ogixTM QuickStixTM Kit for eCry 3.1Ab Corn Bulk Grain and the QuickStixTM Kit for eCry 3.1Ab for leaf and seed are suitable analy tical methods for the qualitative detection of eCry 3.1Ab protein residues
. EnviroL ogixTM also obtained USDA GIPSA performanc e certification of the QuickStixTM Kit for eCry 3.1Ab Corn Bulk Grain (Certificate of P erformance No. GIPSA 2009
- 011), which is also acceptable as confirmation of the test kit’s performance in lieu of an ILV stud y. **CLASSIFICATION: ACCEPTABLE** V alidation of a quantitative eCry 3.1Ab ELISA for anal ys is of maize leaf, root, kernel, and pollen extracts The study was conducted to validate the use of an enzy me - linked immunosorbent assay

(ELISA) method for the quantitative anal ys is of eCry 3.1Ab protein expression levels in event 5307 maize leaf, root, kernel, and pollen tissue matrices. Recoveries of eCry 3.1Ab from fortified extracts of leaf, root, kernel, and pollen matrices ranged from 85.42% to 113.88%, indicating that the assay is accurate across the range of the eCry 3.1Ab reference protein standard curve. Extraction efficiencies of 77.09% and 74.66% were achieved for leaf and root matrices, respectivel y. Recoveries of eCry 3.1Ab from fortified extracts spiked with mCry 3A and Cry 1Ab proteins ranged fro m 75.81% to 112.87% for the matrices tested, indicating that the assay is specific for the quantitative anal ys is of eCry 3.1Ab protein. These results show the ELI SA method is sufficient for accurately quantifying eCry 3.1Ab protein concentrations in various corn tissue matrices. In addition, the LODs, LOQ, and extraction efficiencies values can be used for subsequent studies using ELISA anal ys es. **CLASSIFICATION: ACCEPTABLE 477539**  **- 02** 

Event 5307 Maize: Insert Sequence Analy sis; Amended Report No. 2 This study was conducted to determine the deox yribonucleic acid

(DNA) sequence of the event 5307 maize insert and to assess the intactness of the insert, the organization of the functional elements, and the presence of an y rearrangements, deletions, and/or base pai r changes within the event 5307 maize insert. Two overlapping fragments that span the 5307 maize insert were amplified from genomic DNA extracted from 5307 maize using pol ym erase chain reaction. These fragments were cloned, and sequences of the clones wer e aligned to create a consensus of the transferred- DNA (T

- DNA) sequence. This sequence was **4880 26**  **- 01** Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

20 **STUDY TITLE STUDY SUMMARY**  **MRID NO.**

compared to plasmid pSYN12274, the transformation plasmid used to create 5307 maize. Therefore, DNA sequence analy sis demonstrated that the 5307 maize insert was intact and the organization of the functional elements within the insert is contiguous, as present in pSYN12274. **CLASSIFICATION: SUPPLEMENTAL; No DER prepared**   The data in this report were used to support the results reported in MRID No: 484425
- 01. Event 5307 Maize: Flanking Sequence Determination: Amended Report No. 1 This study was conducted to determine the maize genomic sequence flanking both sides of the 5307 maize insert. The sequence of each flanking region was amplified using pol ym eras e chain reaction anal ys is. These amplification products were cloned, and multiple clones were sequenced and compared to generate a consensus sequence for each flanking region. Results showed f or each flanking region, 1,000 base pairs were reported. ** CLASSI** **FICATION: SUPPLEMENTAL; No DER prepared**   T he data in this report were used to support the results reported in MRID No: 484425
- 01. ** 488026** **- 02** 

5307 Corn: Supplemental ELISA Validation Data for MRIDs 484425
- 08, 484425
- 10, 484425
- 11, 484425
- 14, and 484425
- 1 7 During the Agency ’s review of S yn genta Seeds, Inc. event 5307

data submission, the Agency identified data deficiencies in the ELISA method validation studies for eCry 3.1Ab (specificall y in MRID No. 484425
- 08 , 484425
- 14 , and 484425

- 17). The Agency reques ted the raw data used to calculate ELISA method sensitivity , accuracy , precision , quantitative range , linearit y , specificit y , and extraction
. Inresponse, Sy ngenta submitted a compilation of raw data for each data deficiency identified from each study repo rt in a single volume. Review of the individual data for each study verified the accuracy

of the values reported for the ELISA validation studies
. **CLASSIFICATION: SUPPLEMENTAL; No DER prepared**   The raw data compiled for each stud y were used to support th e results reported in MRID No. 484425
- 08, 484425
- 14 and 484425

- 17
. ** 488026** **- 04** 

**a Previously Reviewed in US EPA 2010b** Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

21 

### B. **FQPA F**  **OOD** 

### S **AFETY** 

### A **SSESSMENT 1**  **. Background and Statutory Findings** Section 408(c)(2)(A)(i) of the FFDCA allows EPA to establish an exemption from the requirement for a tolerance (the legal limit for a pesticide chemical residue in or on a food) only if EPA determines that the exemption is “safe.” Section 408(c)(2)(A)(ii) of the FFDCA defines “safe” to mean that “there is a reasona ble certainty

that no harm will result from aggregate exposure to the pesticide chemical residue, including all anticipated dietary exposures and all other exposures for which there is reliable information.” This includes exposure through drinking water an d in residential settings, but does not include occupational exposure. Section 408(b)(2)(C) of the FFDCA requires EPA to give special consideration to exposure of infants and children to the pesticide chemical residue in establishing a tolerance and to “en sure that there is a reasonable certaint y that no harm will result to infants and children from aggregate exposure to the pesticide chemical residue”... Additionally , section 408(b)(2)(D) of the FFDCA requires that the Agency consider “available information concerning the cumulative effects of a particular pesticide's residues and other substances that have a common mechanism of toxicity .” EPA performs a number of anal ys es to determine the risks from aggregate exposure to pesticide residues. First, EPA deter mines the toxicity

of pesticides. Second, EPA examines exposure to the pesticide through food, drinking water, and through other exposures that occur as a result of pesticide use in residential settings. **2 . Toxicological Profile**  Consistent with sectio n 408(b) (2) (D) of the FFDCA, EPA has reviewed the available scientific data and other relevant information in support of this action and considered its validity , completeness and reliability and the relationship of this information to human risk. EPA has also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children.
**a ) Product Characterization Overview**  Based on amino acid sequence homology and crystal structures, known Cry proteins have a similar three

- dimensional structure comprised of three domains, Domain I , II, and III (Nakamura et al . 1990; L i et al . 1991; Ge et al . 1991; Honee et al . 1991). The toxin portions of Cry proteins are characterized by hav ing five conserved blocks (CB) across their amino acid sequence. These are numbered CB1 to CB5 from the N

- terminus to the C

- terminus (Hofte and Whiteley 1989). The sequences preceding and following these conserved blocks are highl y variable and are designated as variable regions V1 to V6. Because Cry

proteins share structural similarities, chimeric cry genes can be engineered via the exchange of domains that are homologous between different cry genes. Sy ngenta Seeds, Inc.

– Field Crops

- NAFTA (S yn gent a) h as developed Event 5307 maize (Zea mays ) [OECD Unique ID. SYN
-  53  7
- 1] to express Bacillus thuringiensis ( Bt) eCry 3.1Ab insecticidalBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

22 crys tal protein for use as a plant

- incorporated protectant against root feeding damage from Western corn rootworm (WCR, Dia brotica virgifera ) and related Diabrotica species. This proposed PI P is an engineered chimera protein , composed of portions of modified Cry3A (mCry3A) protein, a protein derived from the native Cry 3A protein from Bt subsp. tenebrionis, and on the Cry 1Ab pr otein from Bt thuringiensis subsp. kurstaki HD
- 1. The ecry3.1Ab gene (Entrez Accession Number GU327680 [NCBI, 2011]) (Walters et al. 2010) consists of a fusion between the N

- terminus (Domain I, Domain II, and a portion of Domain III) of a mcry3A gene and th e C
- terminus (a portion of Domain III and Variable Region 6) of a cry1Ab gene (Hofte and Whiteley , 1989). The eCry 3.1Ab protein is 654 amino acid residues in size and is approximately 74.8 kilodaltons. Event 5307 maize also contains the manA (also known a s pmi ) gene from Escherichia coli , which encodes phosphomannose isomerase (PMI) as a selectable marker in regenerating plant material following transformation (Negrotto et al. 2000) . An exemption from the requirement of a tolerance was established for PMI in all crops when used as a PIP inert ingredient [ 40 CFR § 174.527 , effective April 25, 2007 ]. **b ) Mammalian Toxicity Assessment**  Sy ngenta has submitted acute oral toxicity data demonstrating the lack of mammalian toxicity at high levels of exposure to th e pure eCry 3.1Ab protein. These data demonstrate the safet y of the product at a level well above maximum possible exposure levels that are reasonabl y anticipated in the crop. Basing this conclusion on acute oral toxicity data without requiring further toxi city testing and residue data is similar to the Agency

position regarding toxicity testing and the requirement of residue data for the microbial Bacillus thuringiensis products from which this plant

- incorporated protectant was derived (see 40 CFR 158.2130( d)(1)(i) and 158.2140(d)(7)). For microbial products, further toxicity testing and residue data are triggered by significant adverse acute effects in studies such as the mous e oral toxicity study , to verify

and quantify the observed adverse effects and clarify the source of these effects (Tiers II & III). An acute oral toxicity

study in mice (MRID No. 477539

- 01) indicated that eCry 3.1Ab is non

- toxic
. Two groups of ten male and ten female mice were orally d osed (via gavage) with 2, 000 milligrams/kilograms b ody weight (eCry 3.1Ab protein mg/kg bwt) of the ECRY3.1AB

- 0208 test substance, a biochemically and functionall y equivalent microbially

- produced eCry 3.1Ab protein. All treated animals gained weight and had no test material

- related clinical signs and no test material

- related findings at necrops y. Since there were no significant differences between the test and control groups related to the oral administration of ECRY3.1AB

- 0208 test material; the eCry 3.1Ab protein does not appear to cause any significant advers e effects at an exposure lev el of up to 2000 mg/kg bwt and supports the finding that the eCry 3.1Ab protein would be non

- toxic to mammals. When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad et al. 1992) . Therefore, since no acute effects were shown to be caused b y eCry3.1Ab, even at relativel y high dose levels, the eCry 3.1Ab protein is not considered toxic. Further, amino acid sequence comparisons showed no similarities between the eCry 3.1Ab protein and known toxic proteins in protein databases that would raise a safet y concern.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

23 **c ) Allergenicity Assessment**  Since eCry 3.1Ab is a protein, allergenic sensitivities were considered. Currently , no definitive tests exist for determining the allergenic p otential of novel proteins . Therefore, EPA uses a “ weight

- of

- the evidence ” approach where the following factors are considered: source of the trait; amino acid sequence similarity with known allergens; prevalence in food; and biochemical properties of the protein, including in vitro digestibility

in simulated gastric fluid (SGF), and gl yc osylation (as recommended b y CAC , 2003)
. Current scientific knowledge suggests that common food allergens tend to be resistant to degradation by acid and proteases; may be gl yc osyl ated; and present at high concentrations in the food. 1. **Source of the trait.** Bacillus thuringiensis is not considered to be a source of allergenic proteins. 2. **Amino acid sequence.** A comparison of the amino acid sequence of eCry3.1Ab with known allergens s howed no significant overall sequence similarity or identity

at the level of eight contiguous amino acid residues. This is the appropriate level of sensitivity to detect possible IgE epitopes without high false positive rates. 3. **Prevalence in food.** E xpressi on level analysis of eCry.1Ab protein is present at relatively low levels
. T he expression has been shown to be in the parts per million range. Thus, d ietary exposure is expected to be correspondingly low. 4. **Digestibility.** The eCry 3.1Ab protein was rapidly digested in less than 30 seconds in simulated mammalian gastric fluid containing pepsin (pH 1.2) after incubation at 37°C
. 5. **Glycosylation.** The eCry3.1Ab protein expressed in corn was shown not to be gl yc os yl ated. 6. **Conclusion.** Considering all of the avail able information, EPA has conclude d that the potential for eCry 3.1Ab to be a food allergen is minimal.

**3 . Aggregate Exposures**  In examining aggregate exposure, EPA consider s available information concerning exposures from the pesticide residue in food and all other non

- occupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses). The Agency

has considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of consumers) to the pesticide chemical residue and to other related substances. First, with respect to other related substances, the eCry 3.1Ab protein is a chimeric Bacillus thuringiensis protein, composed of portions of Cry1Ab and mCry 3A proteins, both of which are registered PIPs that were previously assessed as having a lack of mammalian toxicity at high levels of exposure. Exemptions from the requirement of a tolerance al read y have been established for Cry1Ab in food and mCry 3A in maize [see 40 CFR § 174.511, effective Apr. 25, 2007 and 40 CFR § 174.505, effective Apr. 25, 2007, respectivel y]
. Second, and specific to the eCry 3.1Ab protein, these considerations include diet ary exposure under the tolerance exemption and all other tolerances or exemptions in effect for the plant

- incorporated protectant chemical residue and exposure from non

-Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

24 occupational sources. Exposure via the skin or inhalation is not likely since the plant

- incorporated protectant is contained within plant cells, which essentially eliminates these exposure routes or reduces these exposure routes to negligible. The amino acid similarity assessment included similarity to known aeroallergens. It has been demon strated that there is no evidence of occupationally related respiratory sy mptoms, based on a health survey on migrant workers after exposure to Bt pesticides (Berstein et a l
. 1999). Exposure via residential or lawn use to infants and children is also not expected because the use sites for the eCry 3.1Ab protein are all agricultural for control of insects. Oral exposure, at very low levels may

occur from ingestion of processed corn products and, potentiall y, drinking water. However, oral toxicity

tes ting d one at a dose of 2 gm/kg showed no adverse effects. Furthermore, the expected dietary

exposure from corn is several orders of magnitude lower than the amounts of eCry 3.1Ab protein shown to have no toxicity . Therefore, even if negligible aggregate exposure should occur, the Agency

concludes that such exposure would present no harm due to the lack of mammalian toxicity and the rapid digestibility demonstrated for the eCry 3.1Ab protein. **4 .**  **Cumulative Effects from Substances with a Common Mechanism of Toxici**  **ty** Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify , or revoke a tolerance, the Agency consider “available information” concerning the cumulative effects of a particular pesticide’s residues and “other substanc es that have a common mechanism of toxicity .” Since eCry 3.1Ab is not considered toxic, EPA has not found eCry 3.1Ab to share a common mechanism of toxicity

with an y other substances, and eCry 3.1Ab does not appear to produce a toxic metabolite produced b y other substances. For the purposes of this tolerance action, therefore, EPA has assumed that eCry 3.1Ab does not have a common mechanism of toxicity with other substances. Following from this, therefore, EPA concludes that there are no cumulative effects asso ciated with eCry 3.1Ab that need be considered. For information regarding EPA’s efforts to determine which chemicals have a common mechanism of toxicity

and to evaluate the cumulative effects of such chemicals, see EPA’s website at http://www.epa.gov/pesticides/cumulative

. **5 .**  **Determination of Safety for U.S. Population, Infants and Children** The data submitted and cited regarding potential health effects for the eCry3.1Ab protein include the characte rization of the expressed eCry 3.1Ab protein in corn, as well as the acute oral toxicity , heat stability , and in vitro digestibility of the proteins. The results of these studies were used to evaluate human risk, and the validity , completeness, and reliabil ity of the available data from the studies were also considered. As discussed more full y in section B above, the acute oral toxicity data submitted supports the prediction that the eCry 3.1Ab protein would be nontoxic to humans. Moreover, eCry 3.1Ab showed no sequence similarit y to any known toxin. Because of this lack of demonstrated mammalian toxicity , no protein residue chemistry data for eCry 3.1Ab were required for a human health effects assessment. Even so, preliminary

expression level anal ys is showe d eCry 3.1Ab protein is present at relativel y low levels. Dietary

exposure is expected to be correspondingl y low.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

25 Since eCry 3.1Ab is a protein, its potential allergenicity is also considered as part of the toxicity assessment. Data considered as part of the allergenicity assessment include that the eCry 3.1Ab protein came from Bacillus thuringiensis which is not a known allergenic source, showed no sequence similarity to known allergens, was readil y degraded b y pepsin, and was not glycos yl ated when expresse d in the plant. Therefore, there is a reasonable certaint y that eCry 3.1Ab protein will not be an allergen. Considered together, the lack of mammalian toxicity at high levels of exposure to the eCry 3.1Ab protein and the minimal potential for that prote in to be a food allergen demonstrate the safet y of the product at levels well above possible maximum exposure levels anticipated in the crop. Finally , and specificall y in regards to infants and children, FFDCA section 408(b)(2)(C) provides that EPA shall assess the available information about consumption patterns among infants and children, special susceptibility

of infants and children to pesticide chemical residues, and the cumulative effects on infants and children of the residues and other substances w ith a common mechanism of toxicity . In addition, FFDCA section 408(b)(2)(C) provides that EPA shall apply an additional tenfold margin of safet y for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the data base unless EPA determines that a different margin of safety will be safe for infants and children. Based on its review and consideration of all the available information, as discussed in more detail above, the Agency

conclude s that there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to residues of the eCry 3.1Ab protein and the genetic material necessary for its production in corn. This include s all anticipated dietary exposures and all other exposures for which there is reliable information. The Agency has also concluded, again for the reasons discussed in more detail above, that there are no threshold effects of concern and, as a result, that an additional margin of safet y for infants and children is unnecessary in this instance. **6 .**  **Other Considerations** 
**a ) Analytical Method(s)**  EPA has determined that an anal yt ical method is not required for enforcement purposes when establishing an exempt ion from the requirement of a tolerance without an y numerical limitation. Nonetheless, Sy

ngenta has submitted validation method studies on two qualitative lateral flow strip kits for the analy

tical detection of eCry 3.1Ab protein in corn grain, leaf and see d corn matrices . Results showed the test kits are able to detect eCry 3.1Ab protein residues in corn with sufficient accuracy , precision, and sensitivity
. A validation study conducted by

an independent 3 rd party

laboratory confirmed the performance of Envi roL ogixTM QuickStixTM Kit for eCry 3.1Ab Corn Bulk Grain (Cat. No. AS TBD BG) and EnviroL ogixTM QuickStixTM Ki t for eCry 3.1Ab Corn Leaf and Seed (Cat. No. AS TBD LS ) . Lastl y, S yn genta has submitted documentation that the EnviroLogixTM QuickStixTM Kit for eCry 3.1Ab C orn Bulk Grain (Cat. No. AS TBD BG) received USDA GIPSA certification of the testBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

26 kit’s p erformance and manufacturer claims ( Certificate of Performance No. GIPS A 2009
- 011) . In lieu of an independent laboratory validation stud y, GIPSA certification is accep table to the Agency as confirmation of the test kit’s use as a suitable analy tical residue method for the qualitative detection of eCry 3.1Ab protein in bulk grain. **b )**  **Codex Maximum Residue Level** In making its tolerance decisions, EPA seeks to harmonize U.S. tolerances with international standards whenever possible, consistent with U.S. food safety standards and agricultural practices. In this context, EPA considers the international maximum residue limits (MRL s) established by the Codex Alimentarius Commission (Codex), as required b y FFDCA section 408(b)(4). The Codex Alimentarius is a joint U.N. Food and Agriculture Organization/World Health Organization food standards program, and it is recognized as an international food safet y standards

- setting organ ization in trade agreements to which the United States is a party . EPA may establish a tolerance that is different from a Codex MRL ; however, FFDCA section 408(b)(4) requires that EPA explain the reasons for departing from the Codex level. The Codex has n ot established a MRLfor Bacillus thuringiensis eCry 3.1Ab protein in corn.

**Ta ble 3**  **. Toxicological and Allergenicity Data**  **for Event 5307 Maize**  **STUDY TITLE STUDY TITLE**  **MRID NO.**  ECRY3.1AB
- 0208: Single

- dose Oral (Gavage) Toxicity Study in Mice with a 14
- Da y Observation Period **a** There was no evidence of toxicity resulting from a single oral dose of ECRY3.1AB

- 0208 test substance to mice via oral gavage after 14 day s. No adverse effects were seen and the LD 50 for males, females, and combined sexes in mice was greater than 2000 mg/kg (TOXICITY CATEGORY III, classified b y dose amount only). **CLASSIFICATION: ACCEPTABLE 477539**  **- 01** 

eCry 3.1Ab: Assessment of amino acid sequence homology

with known toxins **a** No significant sequence similarities with eCry 3.1Ab protein were identified with an y proteins known to be toxins. **CLASSIFICATION: ACCEPTABLE; Updated analysis presented in MRID No. 48442519.**  **477347 -**  **09**

eCry 3.1Ab: Assessment of amino acid sequence homology

with known or putative allergens **a** No significant sequenc e similarities of eCry 3.1Ab protein were identified with any

known proteins or putative allergens. **CLASSIFICATION: ACCEPTABLE; Updated analysis presented in MRID No. 48442518.**  **477347 -**  **10**

In vitro digestibility

of The eCry 3.1Ab protein from either microbial or plant sources **477347 -**  **11**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

27 **STUDY TITLE STUDY TITLE**  **MRID NO.**  eCry 3.1Ab protein as contained in test substance ECRY3.1AB
- 0208 and ev ent 5307 maize under simulated mammalian gastric conditions **b** was completel y digested within ten minutes in simulated human gastric fluid containing pepsin. No diges tion occurred in simulated gastric fluid that did not contain pepsin, showing it was responsible for the digestion. However, several data deficiencies and anomalous results were identified in this study and must be addressed by the registrant. **CLASSIFICA TION: SUPPLEMENTAL; Deficiencies**  **addressed in MRID No. 47734713.** Phosphomannose isomerase (Entrez Database Accession No AAA24109): Assessment of amino acid sequen ce homology

with known toxins **a** No significant sequence similarities of PMIwere i dentified with any proteins known to be toxins. **CLASSIFICATION: ACCEPTABLE; Updated analysis presented in MRID No. 48442521**  **477347 -**  **12**

Phosphomannose isomerase (Entrez Database Accession No AAA24109): Assessment of amino acid sequence homology

with known or putative allergens **a** No significant sequence similarities of PMIprotein were identified with any

known or putative proteins that are allergens.
**CLASSIFICATION: ACCEPTABLE; Updated analysis presented in MRID No. 48442521**  **477347 -**  **13**

Supplemental In formation for Stud y SSB
- 024
- 08: In Vitro Digestibility

of eCry 3.1Ab Protein as Contained in Test Substance ECRY3.1AB
- 0208 and in 5307 Maize Under Simulated Mammalian Gastric Conditions [EPA MRI D 47734711] **b** Using an enzy me kinetic approach, densitometric anal ys is of the SDS

- PAGE gels from the previously conducted in vitro digestibility

study in SGF for the microbial

- derived and the plant

- derived eCry 3.1Ab test substances showed rapid pepsin degradation rates (DT 50 = <1 minute and DT 50 = <2 minutes, respectiv el y). In addition, the data deficiencies that were identified in MRID No. 477347
- 11 were adequately addressed.
**CLASSIFICATION: ACCEPTABLE -**  **for the purposes of the EUP, but not Sec. 3 Registration; Deficiencies**  **addressed in MRID No. 48442523, 48442524 a nd 48442525.**  **480384 -**  **01**

eCry 3.1Ab (Entrez No significant sequence similarities in the overall amino acid **484425 -**  **18**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

28 **STUDY TITLE STUDY TITLE**  **MRID NO.**  database accession number ADC30135): Assessment of amino acid sequence similarity to known or putative allergens sequence of eCry 3.1Ab were identified when compared with sequences from known or putative protein allergens in the 2011 Food Allergy

Research and Resource Program (FARRP) AllergenOnline database using alignments of 80 or more amino acids. There were also no matches of eight of more contiguous amino acids in eCry 3.1Ab with allergen entries in the FARRP AllergenOnline database. **CLASSIFICATION: ACCEPTABLE** eCry 3.1Ab (Entrez database accession number ADC30135): Assessment of amino acid sequence similarity to known or putative toxins Bi oinformatic searches were conducted to compare the amino acid sequence of eCry 3.1Ab with that of known or putative protein toxins. The analy sis was conducted using the BLASTP search tool to compare the amino acid sequence of eCry 3.1Ab with 13,043,846 prot ein sequences in the NCBI Entrez® Database . The threshold value ( E

- value>0.079) for determining significance of matches was based on searches conducted with randoml y shuffled sequences of the amino acids comprising eCry 3.1Ab. Although 505 protein sequence s with significant similarity to that of eCry 3.1Ab were found, further evaluation from the records in the NCBI Entrez® Protein Database for identity and biological function, showed none were associated with known or putative human or mammalian toxins. **C LASSIFICATION: ACCEPTABLE**  **484425 -**  **19**

Phosphomannose isomerase (Entrez database accession number AAA24109): Assessment of amino acid sequence similarity to known or putative allergens Bioinformatic searches were conducted to determine if the amino acid seq

uence of PMIshared any

significant sequence similarities with known or putative allergen entries in the FARRP AllergenOnline database (2011). Overlapping stretches of 80 amino acids were used as query sequences, as were stretches of the PMI sequence cont aining 8 or more contiguous amino acids. The current search showed no additional amino acid similarities other than the previously reported 2004 s erum screening anal ys is study showing a lack of immune recognition to PMIprotein after one 8 amino acid ali gnment that was common to PMIand a food allergen α

parvalbumin from Rana species CH2001(reported in Rabe 2004; MRID No. 464252
- 01) was identified . This study reconfirms the conclusion that PMIshows no biologically relevant amino acid sequence similarit y to an y known or **484425 -**  **20**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

29 **STUDY TITLE STUDY TITLE**  **MRID NO.**  putative protein allergens. **CLASSIFICATION: ACCEPTABLE** Phosphomannose isomerase (Entrez database accession number AAA24109): Assessment of amino acid sequence similarity to known or putative toxins No significant sequence similariti es of PMIwere identified with any proteins known to be toxins.

**CLASSIFICATION: ACCEPTABLE 484425**  **- 21** 

In vitro digestibility

of eCry 3.1Ab protein as contained in test substance ECRY3.1AB
- 0208 under simulated mammalian intestinal conditions Sodium dodec yl sulfate poly acrylamide gel electrophoresis (SDS

- PAGE) and Western blot analy ses were used to evaluate the in vitro digestibility

of eCry 3.1Ab protein (produced from recombinant Escherichia coli ) in simulated mammalian intestinal fluid (SIF) containing p ancreatin. SDS

- PAGE results showed no bands representative of intact eCry 3.1Ab protein (Molecular weight: 74.8 kDa) following incubation for 1 minute at 35°C with SIF containing pancreatin (pH 7.5). The limit of detection for the SDS

- PAGE method was dete rmined to be 8 ng. Western blot results also showed no bands representative of intact eCry 3.1Ab protein in SIF (pH 7.5) after 1 minute incubation at 35°C. Immunoreactive fragments of eCry 3.lAb with molecular weights of approximately

56, 40, and 5 kDa were present at the conclusion of the 48 hour time course of the stud y. The protein bands were identified as degradation products of eCry 3.1Ab. The limit of detection for the method was determined to be 0.25 ng. The pancreatin stability and test material st ability

controls gave appropriate responses in both assay s. In conclusion, both assay s demonstrated the eCry 3.1Ab protein is susceptible to the proteol yt ic activit y of pancreatin and rapidl y degrades after one minute of incubation in simulated mammalian intestinal fluid. **CLASSIFICATION: ACCEPTABLE 484425**  **- 22** 

In vitro digestibility

of eCry 3.1Ab protein under simulated mammalian gastric Sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS

- PAGE), Western blot analy ses and densitometric anal ys es were used to evaluate the in vitro digestibility of eCry 3.1Ab protein (produced from recombinant Escherichia **484425 -**  **23 [This**  **report**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

30 **STUDY TITLE STUDY TITLE**  **MRID NO.**  conditions coli ) in simulated mammalian gastric fluid (SGF), containing pepsin. SDS

- PAGE results showed no bands representative of intact eCry 3.1Ab p rotein (Molecular weight: 74.8 kDa) following incubation for 30 seconds at 37°C with SGF containing pepsin (pH 1.2). Two faint bands with molecular weights of approximately 4 kDa and 5 kDa, respectively , were visible after incubation in SGF for 15 seconds; however these two bands diminished in intensity over time and were no longer detectable after incubation in SGF for 10 minutes. The limit of detection for the SDS

- PAGE method was determined to be 0.025  g. Densitometry anal ys is of the stained gels from S DS
- PAGE showed eCry3.l Ab is readily digested in SGF in less than 30 seconds. After incubation of eCry 3.1Ab in SGF for 15 and 30 seconds, 3% and 0% of the eCry 3.1Ab remained, respectivel y. Western blot analysis showed no immunoreactive intact or fragmented eCry 3.1Ab protein degradates after 30 seconds of incubation at 37°C with SGF containing pepsin (pH 1.2). The limit of detection for the SDS

- PAGE method was determined to be 0.4 ng. The pepsin stability and test material stability controls gave appropriate responses in both assay s. Collectively , the results demonstrated the eCry 3.1Ab protein is susceptible to the proteoly tic activity

of pepsin and rapidly degrades in less than 30 seconds after in vitro digestion in simulated mammalian gastric fluid. **CLASSI FICATION: ACCEPTABLE;**  T he data in this report were used to address the data deficiencies reported in MRID No: 477347
- 11 and 480384

- 01. **supersede s MRID**  **No. 477347**  **- 11]** 

Effect of buffer ionic strength on the in vitro digestibility

of eCry 3.1Ab protein under simulated mammalian gastric conditions Sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS

- PAGE), Western blot analy ses and densitometric anal ys es were used to evaluate the effect of buffer ionic strength on the in vitro digestibility of eCry 3.1Ab protein (produced from recombinant Escherichia coli ) in simulated mammalian gastric fluid (SGF) containing pepsin. The eCry 3.1Ab protein was prepared in 10 mM ammonium bicarbonate buffer (0.01 M, low ionic strength) or 95 mM gl ycine, 50mM sodium phosphate buffer (0.15 M, high ionic strength). SDS
- PAGE results showed no bands representative of intact **484425 -**  **24**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

31 **STUDY TITLE STUDY TITLE**  **MRID NO.**  eCry 3.1Ab protein (MW: 74.8 kDa) following incubation for 30 seconds at 37°C with SGF containing pepsin (pH 1.2) for the low ion ic strength buffer, whereas the high ionic strength buffer showed slower degradation. Densitometry anal ys is of the stained gels from SDS

- PAGE showed the eCry3.1Ab protein prepared in low ionic strength buffer was readil y digested b y pepsin in SGF with only 6% of the intact protein remaining after 15 seconds and none remaining after 30 seconds. The eCry 3.1Ab prepared in high ionic strength buffer was digested at a much slower rate with 15% intact protein remaining after incubation with pepsin for 1 minute. Western blot results showed a similar finding of no bands representative of intact eCry 3.1Ab protein (MW: 74.8 kDa) following incubation for 30 seconds at 37°C with SGF containing pepsin (pH 1.2) for the low ionic strength buffer, while the high ionic stre ngth buffer showed slower degradation. After incubation in the high ionic strength buffer for 1 minute, 15% of the eCry 3.1Ab remained undigested. The pepsin stability

and test material stability controls gave appropriate responses in both assay s. Collectiv el y, these results demonstrated the buffer strength affects the rate at which the eCry 3.1Ab protein is digested by pepsin. **CLASSIFICATION: ACCEPTABLE;** T he data in this report were used to address the data deficiencies reported in MRID No: 477347
- 11 and 4 80384
- 01. A comprehensive overview of the assessment of the in vitro digestibility

of eCry 3.1Ab protein under simulated mammalian gastric conditions. This report summarizes the studies conducted to assess the digestibility

of eCry 3.1Ab in simulated gastric fluid (SGF) containing pepsin. The proteoly tic degradation of eCry 3.1Ab was assessed b y sodium dodecyl sulfate pol ya cryl amide gel electrophoresis (SDS

- PAGE), Western blot, and densitometric anal ys is. The initial studies showed that the microb ially

- and plant

- expressed purified eCry 3.1Ab protein samples were moderatel y digested in SGF containing pepsin. These protein samples were diluted with a high ionic strength buffer, 95 mM gl ycine, 50 mM sodium phosphate, since it was not feasible to dilut e the plant

- derived eCry 3.1Ab in 10 mM ammonium phosphate buffer. Each assay showed the eCry 3.1Ab protein band was p resent at the 5 min. time point, but no longer visible at the 10 min. time point. **484425 -**  **25**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

32 **STUDY TITLE STUDY TITLE**  **MRID NO.**  Additional studies were conducted to compare the diges tibility of the microbiall y - produced eCry 3.1Ab in low and high ionic strength buffer. Although, eCry 3.1Ab is rapidl y digested in both buffers, digestion occurs more rapidly in the low ionic strength buffer. Densitometric anal ys es showed only 6% remained a fter 15 seconds and 0% intact eCry 3.1Ab remained after incubation in SGF for 30 seconds. After incubation in the high ionic strength buffer for 1 minute, 15% of the eCry 3.1Ab remained undigested. These studies support the conclusion the buffer strength affects the rate at which the eCry 3.1Ab protein is digested b y pepsin. Moreover, the additional studies using the lower strength ionic buffer demonstrate the eCry 3.1Ab derived from transformation Event 5307 maize is expected to be rapidl y digested under ty pical mammalian gastric conditions. **CLASSIFICATION: ACCEPTABLE;** The previously conducted studies compiled in this report were used to address the data deficiencies identified in MRID No. 477347
- 11 and 480384
- 01 reviewed in this memorandum. **a P**  **reviously Reviewed in US EPA 2010a b**  **Previously Reviewed in US EPA 2010b 7)**  **Bt11 x MIR162 x MIR604 x TC1507 x 5307 Corn** The molecular characterization of combination PIP Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn product and the genetic material neces sary for their production in the plant demonstrated the stability and integrit y of the intended transgenic DNA insertions of c ry1Ab , vip3Aa20 , mCry 3A, cry1F and eCry 3.1Ab from Bt11, MIR162, MIR604, TC1507 and 5307 corn events, respectivel y, when combined through traditional plant breeding. In addition, the combination PIP product protein confirmed comparable protein expression levels in various plant tissues matrices at select developmental growth stages for the combination PIP plant tissues com pared to its parental events. These results confirm the equivalence of the biochemical characteristics, thus demonstrating no increase in exposure to the presence and amounts of Cry 1 Ab, Vip3Aa20, mCry 3A, Cry 1F and eCry 3.1Ab proteins. Laboratory

assay s using sensi tive target pest species showed no enhanced toxicity to the target pest, demonstrating the lack of sy nergism among the Cry

proteins expressed in the combination PIP product (US EPA 2012 a ). In addition, previously investigated protein interaction studies us ing various subsets of protein combinations showed a lack of s ynergism, which includes: Cry 1Ab + mCry 3A for Bt11 xBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

33 MIR604 (MRID No. 467956

- 04; reviewed in US EPA 2007); Cry 1Ab + Vip3Aa20 + Cry 1F for Bt11 x MIR162 x TC1507 (MRID No. 482352

- 04, reviewed in U S EPA 2011a); and a protein mixture of Cry 1Ab + Vip3Aa20 + Cry 1F against a protein mixture of mCry 3A + Cry 34/35Ab1 for Bt11 × DAS
59122
- 7 × MIR604 × TC1507 corn (MRID No. 482023

- 17; reviewed in US EPA 2011b). Inaddition, Vip proteins are known to act in dividually to affect a ty

pical midgut pathology

in susceptible insects like previously

studied Bt delta

- endotoxins (L ee, et al. 2003). Thus, n o sy nergistic action or interaction of these proteins is known or expected to occur from exposure to the combinati on PIP product. These results, when combined with the results of previous studies that show no sy nergism among the components in each mixture, and the molecular characterization, and protein expression analy ses confirm the biochemical functional equivale nce of the Cry proteins expressed in the combination PIP and its constituent parental maize events. In conclusion, the submitted data provide justification for bridging the existing results and conclusions from the individual PIP events that are alread y contained in the Agency ’s database (US EPA 2001a, 2001b, 2006, 2007, 2011 a, 2011b and 2012b) Since the mode of action for these proteins does not suggest a s yn ergistic activity in combination for mammalian species, the individual parental event data for C ry 1Ab, Vip3Aa, Cry 1F , eCry 3.1Ab and mCry 3A proteins support the inclusion of the Cry 1Ab [40 CFR § 174.511] , Vip3Aa [40 CFR § 174.501], Cry 1F [40 CFR § 174.504], eCry3.1Ab [40 CFR § 174.532] and mCry 3A [40 CFR §174.505] PI P proteins expressed in the combin ation PIP product under the existing exemptions from the requirement of a tolerance for residues on food. Therefore, the toxicological and allergenicity data can be bridged to support the finding of reasonable certaint y of no harm to exposure of Cry 1Ab, Vi p3Aa20, Cry 1F , eCry 3.1Ab and mCry 3A proteins and the genetic material necessary for their production in Bt11 x MIR162 x MIR604 x TC1507 x 5307 to the U.S. population, including infants and children. This includes all anticipated dietary exposures and all other exposures for which there is reliable information.

**TABLE 4**  **. Summary of Submitted Data in support for Sec. 3 Registration of**  **Combination PI**  **P Bt11 x MIR162 x TC1507 Corn Product**  **Study Type/Title Summary**  **MRID #** Comparison of Transgenic Prote in Concentrations in Event Bt11, Event MIR162, Event MIR604, Event TC1507, Event The purpose of this stud y was to compare protein expression

by e nz ym e - linke d immunosorbent assay s (ELISA) in a combination PIP product (Bt11 x MIR162 x MIR604 x TC1507 x 5307 x GA21) with expression in corresponding, near - isogenic hy

brids derived from the individual transformation events: Bt11, MIR162, MIR604, TC1507, 5307, and G A21. **484429 -**  **01**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

34 **TABLE 4**  **. Summary of Submitted Data in support for Sec. 3 Registration of**  **Combination PI**  **P Bt11 x MIR162 x TC1507 Corn Product**  **Study Type/Title Summary**  **MRID #** 5307, Event GA21, and Bt11 × MIR162 × MIR604 x TC1507 × 5307 x GA21 Maize Hy brids Tissue samples were collected from each of five replicate

planted blocks of maize of each individual h ybrid line and the c ombination PIP product , at different developmental stages. Tissues anal yz ed included leaves, roots, kernels, whole plants, and pollen. Results showed eleven statistically significant differences (out of 33 statistical analy ses) were seen between the concentrations of the transgenic proteins expressed in the maize plant tissues of the individual

- event hy brids and the combination PIP product , however these differences were generally small and were not consistent across tissue t ypes or developmental stages. Overall, the majority of the transgenic protein concentration s were similar between the c ombination PIP product and the corresp onding individual event hy brid with the exception for the statistically

significant difference (~ 60

- 80% greater) in the concentrations of Cry1Ab protein in the combination stack corn leaf tissue compared to the corresponding leaf tissue of Event Bt11. Th e data provided b y the registrant on the statistically

significant difference (~50

- 70% greater) indicate no biological significance. The concentration of PMIand PAT were significantly higher in the combination PIP product. However these results were exp ected due to the insertions of multiple copies of the pmi and pat gene in the combination PIP due to the conventional crossing of the single parental events containing these genes. **Classification: ACCEPTABLE**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

35 **TABLE 4**  **. Summary of Submitted Data in support for Sec. 3 Registration of**  **Combination PI**  **P Bt11 x MIR162 x TC1507 Corn Product**  **Study Type/Title Summary**  **MRID #** Bt11 × MIR162 × MIR604 x TC1507 × 5307 x GA21 Maize: Comparitive Southern Blot Analy sis Molecular anal ys es (restriction enzy me digests and Southern blots) were performed to compare the integrity of the transgenic inserts in the individual maize lines (Event Bt11, Event MIR162, Event MIR60 4, Event TC1507, and Event 5307) with the transgenic inserts in the Combination PI P Product, Bt11 x MIR162 x MIR604 x TC1507 x 5307 x GA21 , which was created through conventional plant breeding techniques. Plants from Bt11, MIR162, MIR604, TC1507, 5307, the combination PIP product, and a non

- transgenic near

- isogenic control maize were grown under standard greenhouse conditions. All plants were individually anal yz ed by

real

time Poly merase Chain Reaction (PCR) to confirm the presence of the appropriate gene s for the transgenic lines and the absence of these genes from the non

- transgenic, near

isogenic, negative control plants. L eaves from each confirmed maize line were then pooled and genomic DNA was extracted for further anal ys es. Data from the Southern bl ot analy ses of the individual events Bt11, MIR162, MIR604, TC1507, 5307, and the combination PIP product confirmed the integrit y of the inserts from the individual events as they were incorporated into the combination PIP product during conventional breedi ng. **Classification: ACCEPTABLE 48**  **4429 -**  **02**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

36 **TABLE 4**  **. Summary of Submitted Data in support for Sec. 3 Registration of**  **Combination PI**  **P Bt11 x MIR162 x TC1507 Corn Product**  **Study Type/Title Summary**  **MRID #** Bt11 × MIR162 × MIR604 x TC1507 × 5307 x GA21 Maize: Test and Control Substance Characterization of Event Bt11 Maize, Event MIR162 Maize, Event MIR604 Maize, Event TC15 07 Maize, Event 5307 Maize, Event GA21 Maize, Bt11 × MIR162 × MIR604 x TC1507 × 5307 x GA21 Maize, and Nontransgenic Maize The purpose of this stud y wa s to determine the identity ,

purity , and stabilit y of the test materials and the identity and purity of the control material , where the test materials are seed s from Bt11 maize, MIR 1 6 2 maize, MI R604 maize, TC1507 maize, 5307 maize, GA21 maize, and the recombinant PIP product ( Bt11 × MI R 1 6 2 × MIR604 × TC1507 × 5307 × GA21 maize ).T he control material is seed f rom a non - transgenic maize hy brid (5XH751/NP2222 maize), near ly isogenic to the combination PIP product
. Individual plants grown from the test and control substances were anal yz ed for the presence or absence of transgenic inserts by real

- time Polymerase Ch ain Reaction (PCR) anal ys is. Results obtained from real

- time PCR analy sis were confirmed b y gel

- based, event

- specific, PCR analy sis. The resulting PCR products were anal yz ed b y agarose gel electrophoresis to confirm that amplicons of the expected band size were produced, thereb y confirming the identity, purity , and stabilit y of each batch of recombinant seeds. **Classification: ACCEPTABLE 484429**  **- 03** 

Bt11 x MIR162 x MIR604 x TC1507 x 5307 Corn: Supplemental In formation for MRID 48442901 The purpose of the su bmitted data was to provide supplemental information to address identified data deficiencies noted in MRID 48442901. The increase in Cry 1AB protein expressed in Bt11 x MIR162 x MIR604 x TC1507 x 5307 x GA21 leaf tissues was noted in Ray bould (2011). The maximum increase in exposure to Cry 1AB from cultivation of Bt11 x MIR162 x MIR604 x TC1507 x 5307 x GA21 maize is predicted to be about 1.8X the EECs from Event Bt11 maize, which is well within the margins of exposure for foliar and soil organisms.
**C lassification: Supplemental**  **48802801**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

37 **8) Bt11 x MIR604 x TC1507 x 5307**  The molecular characterization data of combination PIP Bt11 x MI R604 x TC1507 x 5307 corn product demonstrated the stability and integrity of the intended transgenic DNA insertions o f c ry1Ab , mcry3A , cry1F and ecry3.1Ab from the constituent parental events Bt11, MI R604, TC1507 and 5307 corn events, respectivel y , when combined through traditional plant breeding. In addition, ELI SA anal yses of the combination PIP plant and its parental events tissues showed comparable levels of CryAb, mCry 3A, Cry 1F and eCry 3.1Ab protein expression in various plant tissue matrices tested at selected developmental growth stages. These results confirm the genetic stability and similar expression levels of the Cry proteins in the combination PIP and its constituent parental maize events. Diet incorporation feeding assay s were conducted to determine whether various doses of lepidopteran

active protein mixtures (Cry 1AB, Vip3a20, and Cry 1F) and coleopteran

- active protein mixture (mCry 3a and eCry 3.1Ab) interact when used in combination against European corn borer (ECB) and Colorado potato beetle (CPB) (MRID NO. 48442908; reviewed in US EPA 2012a). Results showed the coleopteran

- active protein mixture does n ot affect the potency of the lepidopteran

- active mixture against ECB, and the lepidopteran

- active mixture does not affect the potency of the coleopteran

- active against CPB. In addition, previously

investigated protein interaction studies using various subsets of protein combinations showed a lack of s ynergism, which includes: Cry 1AB + mCry 3A for Bt11 x MIR604 (MRID No. 467956
- 04; reviewed in US EPA 2007); and a protein mixture of Cry 1Ab + Vip3A a20 + Cry 1F against protein mixture of mCry 3A + Cry 34/35Ab1 fo r Bt11 x DAS
- 59122
- 7 x MIR604 x TC1507 corn (MRID No. 482023

- 17; reviewed in US EPA 2011). In addition, Vip proteins are known to act individually

to affect a t ypical midgut pathology in susceptible insects like previously studied Bt delta

endotoxins (Lee et al. 2003 and Mostafa et al. 2003). Therefore, no s yn ergistic activity of these proteins is known or expected from exposure to the combination PIP product on susceptible pest species. These results, when combined with the results of previous studies th at show no synergism among the components in each mixture, and the molecular characterization, and protein expression analy ses confirm the biochemical functional equivalence of the Cry proteins expressed in the combination PI P and its constituent parental maize events. In conclusion, the submitted data provide justification for bridging the existing results and conclusions from the individual PIP events that are alread y contained in the Agency ’s database (US EPA 2001a, 2001b, 2006, 2007, 2011, and 2012b). Th e data also support the inclusion of the PIP Cry proteins expressed in the combination PIP product under the existing exemptions from the requirement of a tolerance for Cry 1Ab, mCry 3A, Cry 1F and eCry 3.1Ab protein residues in corn [40 CFR § 174.511 , 174 .505, 174.504, 174.532, respectivel y] . Thus, the toxicological and allergenicity data from the individual food safet y assessments of events B t11, MIR604 , TC1507 and 5307 corn can be bridged toBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

38 support the finding of reasonable certainty of no harm to the combined exposure of these Cry proteins and the genetic material necessary for their production in Bt11× MIR604 × TC1507 and 5307 maize to the U.S. population, incl uding infants and children. This includes all anticipated dietary exposures and all other e xposures for which there is reliable information. **TABLE 5**  **. Summary of Submitted Data in support for Sec.3 Registration of Combination PIP Bt11 x MIR604 x TC1507 x 5307**  **Study Type/ Title Summary**  **MRID#** Com parison of Transgenic Protein Concentrations in Event Bt11, Event MIR162, Event MIR604, Event TC1507, Event 5307, Event GA21, and Bt11 × MIR162 × MIR604 x TC1507 × 5307 x GA21 Maize Hy brids The purpose of this study was to compare protein expression by enzyme linked immunosorbent assay s (ELISA) in a com bination PIP product (Bt11 x MIR162 x MIR604 x TC1507 x 5307 x GA21) with expression in corresponding, near

- isogenic hybrids derived from the individual transformation events: Bt11, MIR162, MIR604, TC1507, 5307, and GA21. Tissue samples were collected from each of five replicate planted blocks of maize of each individual hybrid line and the c om bination PIP product , at different developmental stages. Tissues analyzed included leaves, roots, kernels, whole plants, and pollen. Results showe d 11 statistically significant differences (out of 33 statistical analyses) were seen betw een the concentrations of the transgenic proteins expressed in the maize plant tissues of the individual

- event hybrids and the com bination PIP product , however these differences were generally small and w ere not consistent across tissue types or developmental stages. Overall, the majority each transgenic protein concentration w as similar between the c om bination PIP product and the corresponding individual event hybrid. The exception w as the statistically significant difference (~ 50

- 70% greater) in the concentrations of eCry3.1Ab protein expressed in Bt11 x MIR604 x TC1507 x 5307 root issues compared to the corresponding root tissue results of Event 5307. The eCry3.1 Ab protein is reported in root samples (V5 stage) at a mean of 73.48 μg/g DW (52.03

– 111.06 μg/g DW) for the combination PIP Bt11 x MIR604 x TC1507 x 5307 product and at a mean of 40.29 μg/g DW (25.78

- 77.85 μg/g DW) for single parental event 5307. The data provided by the registrant on the statistically significant difference (~50

- 70% greater) indicate no biological significance . While no statistical comparison of PAT protein concentrations between the combination PIP product and the corresponding indi viduals

- event hybrids (Bt11 and TC1507) was conducted, the concentrations of PAT in the tissues of the combination PIP product w ere generally similar to the TC1507 or Bt11 hybrids. Similarly, no statistical comparison of PMI protein concentrations betwee n the combination PIP Product and the corresponding individual

- event hybrids (MIR604 and 5307) was conducted, but the concentrations of PMI in the combination PIP product were always higher than either the individual MIR604 or 5307hybrids, as expected. **C lassification: ACCEPTABLE**  **48443001**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

39 **TABLE 5**  **. Summary of Submitted Data in support for Sec.3 Registration of Combination PIP Bt11 x MIR604 x TC1507 x 5307**  Com parative Southern Blot Analysis of Bt11 x MIR604 x TC1507 x 5307 with the individual Events Bt 11 Maize, MIR604 Maize, TC1507 Maize, 5307 Maize and GA21 Maize Molecular analyses (restriction enzyme digest and Southern blots) w ere

performed to compare the integrity of the transgenic inserts in the individual maize lines (Event Bt11, Event MIR604, Event TC1507, and Event 5307) with the transgenic inserts in the combination PIP product, which was produced through conventi onal plant breeding techniques. Plants from Bt11, MIR604, TC1507, 5307, the combination PIP product, and non

- transgenic near

- isogenic control maize were grown under standard greenhouse conditions. All plants were individually analyzed by real

- time Polyme rase Chain Reaction (PCR) to confirm the presence of the appropriate genes for the transgenic lines and the absence of these genes from the non

- trangenic, near

- isogenic, negative control plants. Leaves from each confirmed maize line were then pooled and g enomic DNA was extracted for further analysis. Data from the Southern analyses of the individual events Bt11, MIR604, TC1507, 537, and the combination Pip product confirmed the intergrity of the inserts from the individual events as they were incorporated into the combination Pip product during conventional breeding. **Classification: ACCEPTABLE 48443002** 

Bt11 x MIR604 x Tc1507 x 5307 x GA21 Maize: Test and Control Substance Characterization of Event Bt11 Maize, Event MIR604 Maize, Event TC1507 Maize, Eve nt 5307 Maize, Event GA21 Maize, Bt11 x MIR604 x TC1507 x 5307 x GA21 Maize, and Nontransgenic Maize The purpose of this study was to determine the identity, purity, and stability of the test materials and the identity and purity of the control material, where the test materials are seeds from Bt11 maize, MIR604 maize, TC 1570 maize, 5307 maize, GA21 maize, and the recombinant Pip product (Bt11 x MIR604 x TC1507 x 5307 x GA21 maize). The control material is seed from a nontransgenic maize hybrid (5XH751/NP 2222 maize), nearly isogenic to the recombinant PIP Product. Individual plants grown from the test and control substances were analyzed for the presence or absence of transgenic inserts by real

- time Polymerase Chain Reaction (PCR) analysis. Results obtai ned from real

time PCR analysis were confirmed by gel

- based, event

- specific, PCR analysis. The resulting PCR products w ere analyzed by agarose gel electrophoresis to confirm that amplicons of the expected band size were produced, thereby confirming the id entity, purity and stability of each batch of recombinant seeds. **Classification: ACCEPTABLE 48443003** 

Bt11 x MIR604 x TC1507 x 5307 Corn: Supplemental Information for MRID 48443001 The purpose of the submitted data w as to provide supplemental information to address identified data deficiencies noted in MRID 48443001. Review

of the data indicate that significant increased exposure of nontarget organisms to the Cry1Ab, mCry3A, Cry1F, and 3Cry3.1Ab proteins is not expected as a result of cultivation of Bt11 x MIR604 x TC1507 x 5307 corn plants. **Classification: Supplemental 48802701** Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

40 

### R**EFERENCES** Bernstein , I. L., Bernstein , J
. A
. , Miller , M
. , Tierzieva , S
. , Bernstein , D
. I
. , Lummus , Z
. , Selgrade , M
. K
. , Doerfler DL, Seligy

VL. (1999) Immune responses in farm w orkers after exposure to Bacillus thuringiensis pesticides. Environ Health Perspect
. 107(7):575

- 82
. CAC ( 2003 ) Alinorm 03/ 34: Joint FAO/WHO Food Standard Programme. Codex Alimentarius Commission , Twenty
- Fifth Session, 30 July 2003. Rome, Ital y. Appendix I II: Guideline for Conduct of Food Safety

Assessments of Foods Derived from Recombinant

- DNA Plants; Appendix IV: Annex on Assessment of Possible Allergenicity. CAC , 47

– 60. Ge, A., D. Rivers, R. Milne, and D.H. Dean. (1991) Functional Domains of Bacillus th uringiensis Insecticidal Cry stal Proteins. Refinement of Heliothis virescens and Trichoplusia ni Specificity Domains on Cry 1A(c). J. Biol. Chem
. 266: 17954
- 17958. Geiser M., Schweizer S, Grimm C. (1986) The hy pervariable region in the gene s coding for e ntomopathogenic crys tal proteins of Bacillus thuringiensis : nucleotide sequence of the kurhd1 gene of subsp. kurstaki HD
- 1 Gene 48: 109
- 118
. Herman R. A., Storer N.A., Gao Y. (2006) Digestion assay s in allergenicity ass essment of transgenic proteins. Environ mental Health Perspectives. 114 :1154

– 1157
. Hofte, H., and Whitley H.R. ( 1989 ) In secticidal Crystal Proteins of Bacillus thuringiensis
. Microbiol
. Rev. 53: 242
- 255. Honee, G., D. Convents, J. Van Rie, S. Jansens, M. Peferoen, and B. Visser. ( 1991 ) The C

- Te rminal Domain of the Toxic Fragment of a Bacillus thuringiensis Crystal Protein Determines Receptor Binding. Mol. Microbiol . 5: 2799
- 2806. Li, J., J . Carroll, and D.J. Ellar. (1991) Cry stal Structure of Insecticidal δ

- Endotoxin from Bacillus thuringiensis at 2.5 A resolution. Nature 353: 815
- 821. Murray EE, Lotzer J, Eberle M. (1989) Codon usage in plant genes. Nucleic Acids Res 17:477

–498. Nakamura, K., K. Oshie, M. Shimizu, Y. Takada, K. Oeda, and H. Ohkawa. (1990) Construction of Chimeric Insectici dal Proteins Between the 130

- kDa and 135

- kDa Proteins of Bacillus thuringiensis subsp. aizawai for Analysis of Structure

- Function Relationship. Agric. Biol. Chem. 54: 715
- 724. NCBI (2011) Entrez Nucleotide database. Bethesda, MD: National Center for Biote chnology In formation, National Library of Medicine, National Institutes of Health, http://www.ncbi.nlm.nih.gov/entrez/query .fcgi?db=Nucleotide.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

41 Negrotto, et al . (2000) The use of phosphomannose

- isomerase as a selectable marker to recover transgenic maize plants ( Zea mays L. via Agrobacterium transformation). Plant Cell Reports , 19: 798
- 803. Sekar V, Thompson DV, Maroney MJ, Bookland RG, Adang MJ. (1987) Molecular cloning and characterization of the insecticidal cry stal protein gene of Bacillus thuringie nsis var. tenebrionis
. Proc Natl Acad Sci USA 84:7036

– 7040. Sjoblad, R. D., McClintock, J.T., and Engler, R. (1992) Toxicological Considerations for Protein Components of Biological Pesticide Products, Reg. Toxicol. Pharmacol . 15(1): 3

- 9. US EPA (2001) B iopesticides Registration Action Document for Bacillus thuringiensis ( Bt) Plant

In corporated Protec tants, dated October 21, 2001. US Environmental P rotection Agency , Washington, D
. C. US EPA (2006) Biopesticides Registration Action Document for Bacillus thuringiensis ( Bt) Modified Cry 3A Protein and the Genetic Material Necessary for its Production (Via Elements of pZM26) in Event MIR604 Corn (OECD Unique Identifier: SYN
- IR6 Ø 4
- 8 ), updated September 2010. US Environmental Pro tection Agency , Washington, D.C. US EPA (2010a) Review of Product Characterization and Human Health Data for Plant

- In corporated Protectant Bacillus thuringiensis ( Bt) eCry 3.1Ab insect control protein and the genetic material necessary for its production in Event 5307 maize (Zea mays ) [ EPA Reg. No. 67979
- EUP
- I] in support for an Experimental Use Permit and Temporary Exemption from the Requirement of a Tolerance ; submitted by Sy ngenta Seeds, Inc. Memorandum from A. Waggoner through J. Kough to M. Mendelsohn, dated May 25, 2010
. US Environmental Protection Agency , Washington, D.C. US EPA (2010b) Review of Human Health Data in support for an Experimental Use Permit for Field Testing of Event 5307 corn [EPA Reg. No. 67979

- EUP
- I] expressing Bacillus thuringiensis ( Bt) eCry 3.1Ab protein and a Petition for Temporary Tolerance Exemption from the Requirement of a Tolerance for eCry 3.1Ab protein; submitted by Syngenta Seeds, Inc. Memorandum from A. Waggoner through J. Kough to M. Mendelsohn, dated March 12, 2010. US Environmental Protection Agency , Washington, D.C. Walters FS, deFontes CM, H art H, Warren GW, Chen JS. (2010) Lepidopteran

- active variable

- region sequence imparts coleopteran activity in eCry 3.1Ab, an engineered Bacillus thuringiensis hy brid insecticidal protein. Appl Environ Microb 76: 3082
- 3088.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

42 **C. Environmental Risk Assessment for eCry3.1Ab as Expressed in Event 5307 Corn**  The paragraphs below describe the process and rationale developed b y BPPD for evaluating hazard of PIPs to nontarget organisms. This process is described in several of BPPD’s documents and is presented again here as background information. To minimize data requirements and avoid unnecessary tests, risk assessments are structured such that risk is determined first from estimates of hazard under “worst

- case” ex posure conditions. A lack of adverse effects under these conditions would provide enough confidence that there is no risk and no further data would be needed. Hence, such screening tests conducted earl y in an investigation tend to be broad in scope but r elatively simple in design, and can be used to demonstrate acceptable risk under most conceivable conditions. When screening studies suggest potentiall y unacceptable risk additional studies are designed to assess risk under more realistic field exposure c onditions. These later tests are more complex than earlier screening studies. Use of this “tiered” testing framework saves valuable time and resources b y organizing the studies in a cohesive and coherent manner and eliminating unnecessary lines of investi gation. Lower tier, high dose screening studies also allow tighter control over experimental variables and exposure conditions, resulting in a greater ability to produce statistically reliable results at relatively low cost c

. Tiered tests are designed to first represent unrealistic worst case scenarios and ONLY progress to real world field scenarios if the earlier tiered tests fail to indicate adequate certainty of acceptable risk. Screening (Tier I) non

- target organism hazard tests are conducted at ex posure concentrations several times higher than the highest concentrations expected to occur under realistic field exposure scenarios. This has allowed an endpoint of 50% mortality to be used as a trigger for additional higher

- tier testing. Less than 50% mortality

under these conditions of extreme exposure suggest that population effects are likely to be negligible given realistic field exposure scenarios. The EPA uses a tiered (Tiers I

- IV ) testing sy stem to assess the toxicity of a PIP to representativ e non target organisms that could be exposed to the toxin in the field environment. Tier I high dose studies reflect a screening approach to testing designed to maximize any toxic effects of the test substance on the test (non

- target) organism. The screen ing tests evaluate single species in a laboratory setting with mortality as the end point. Tiers II

– IV generall y encompass definitive hazard level determinations, longer term greenhouse or field testing, and are implemented when unacceptable effects are seen at the Tier I screening level.

c Non
- target invertebrate hazard tests often are conducted at exposure concentrations several times higher than the maximum concentrations expected to occur under realistic exposure scenarios. This has customarily allow ed an endpoint of 50% mo rtality to be used as a trigger for ad ditional higher

- tier testing. Lower levels of mortality under these conditions of extreme exposure suggest that population effects are likely to be negligible given realistic exposure scenarios. Thus, it follows that the observed proportion of responding individuals can be compared to a 50% effect to determine if the observed proportion is significantly lower than 50%. For example, using a binomial approach, a sample size of 3 0 individuals is sufficient to allow

a treatment effect of 30% to be differenti ated from a 50% effect with 95% confidence using a one

- sided Z test. A one
- sided test is appropriate because only effects of less than 50% indicate that further experiments are not needed to evaluate risk.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

43 Testing methods which utilize the tiered approach were last published b y the EPA as Harmonized OPPTS Testing Guidelines, Series 850 and 885 (EPA 712

- C
- 96
- 280, February 1996) d . These guidelines, as defined in 40 CFR 152 .20, apply to microbes and microbial toxins when used as pesticides, including those that are naturall y occurring, and those that are strain

- improved, either by natural selection or by deliberate genetic manipulation. Therefore, PIPs containing microbial toxins are also covered b y these testing guidelines. The Tier I screening maximum hazard dose (MHD) approach to environmental hazard assessment is based on some factor (whenever possible >10) times the maximum amount of active ingredient expected to be available to terrestrial and aquatic non

- target organisms in the environment (EEC) e . Tier I tests serve to identify

potential hazards and are conducted in the laboratory at high dose levels which increase the statistical power to test the hy potheses. Elevated test doses, therefore, add certaint y to the assessment, and such tests can be well standardized. The Guidelines call for initial screening testing of a single group or several groups of test animals at the maximum hazard dose level. The Guidelines call for testing of one treatment group of at least 30 animals or three groups of 10 test animals at the screening test concentration. The Guidelines further state that the duration of all Tier I tests should be approximately

30 day s. Some test species, notabl y non - target insects, may be difficult to culture and the suggested test duration has been adjusted accordingl y. Control and treated insects should be observed for at least 30 day s, or in cases where an insect species cannot be cultured for 30 day s, until negative control mortality

rises above 20 percent. Failing the Tier I (10 X EEC) screening at the MHD dose does not necessarily indicate the presence of an unacceptable risk in the field but it triggers the need for additional testing. f A less than 50% m ortality effect 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 estimation before deciding if the risk is acceptable or unacceptable. Where potential hazards are detected in Tier I testing (i.e. mortality is greater than 50%), additional information at lower test doses is required which can serve to confirm whether an y effect might still be detected at more realistic field [1X EEC] concentrations and routes of exposure g

.

d OPPTS Testing Guidelines, Series 850 and 885 we bsite: http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/ 885Microbial_Pesticide_Test_Guidelines/Series e The dose margin can be less than 10x where uncertainty in the syste m is low or where high concentrations of test material are not possible to achieve due to test organism feeding habits or other factors
. High dose testing also may not be necessary where many species are tested or tests are very sensitive, although the tes t concentration used must exceed 1X EEC
. f It is notable that that the 10 X EEC MHD testing approach is not equivalent to what is commonly known as “testing at a 10X SAFETY FACTOR” where any adverse effect is considered significant. Tier I screen testing i s not ‘safety factor testing’. In a “ 10X safety factor” test any adverse effect noted is a “level of concern”, whereas in the EPA environmental risk assessment scenario any adverse effect is viewed as a concern only at 1X the field exposure. g The 1X E EC test dose is based on plant tissue content and is considered a high worst case dose (sometimes referred to as HEEC) . This 1X EEC is still much greater than any amount which any given non

- target organism may be ingesting in the field because most non

- tar get organisms do not ingest plant tissue.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

44 When screening tests indicate a need for additional data, the OPPTS Harmonized Guidelines call for testing at incre mentall y 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 Eff ect Concentration (L OEC), No Observed Adverse Effect Level (NOAEL) , or reproductive and behavioral effects such as feeding inhibition, weight loss, etc. In the final anal ys is, 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. These tests offer greater environmental realism, but they may have lower statistical power. Appropriate statistical methods, and appropriate statistical power, must be emplo yed to evaluate the data from the definitive tests. Higher levels of replication, the number of test species, and/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. L C 50 , ED 5 0 , or LD 50 >10 X EEC) is sufficient to evaluate adverse effects, making lower field exposure dose definitive testing unnecessary . It is also notable that the recommended >10X EEC maximum hazard dose level is a highl y conservative factor. The published E PA Level of Concern [L OC] is 50% mortality at 5X EEC h (USEPA 1998). Validation: The tiered hazard assessment approach was developed for the EPA by the American In stitute of Biological Sciences (AIBS) and confirmed in 1996 as an acceptable method of environmental hazard assessment by

a FIFRA Scientific Advisory Panel (SAP) on microbial pesticides and microbial toxins. The December 9, 1999 SAP agreed that the Tiered approach was suitable for use with Plant

- Incorporated Protectants (PIPs); however, this p anel recommended that, for PIPs with insecticidal properties, additional testing of beneficial invertebrates closel y related to target species and/or likely to be present in GM crop fields should be conducted. Testing of Bt Cry proteins on species not clos ely related to the target insect pest was not recommended, although it is still performed to fulfill the published EPA non

- target species data requirements. In October 2000, another SAP also recommended that field testing should be used to evaluate population

- level effects on non

- target organisms. The August 2002 SAP, and some public comments, generally

agreed with this approach, with the additional recommendation that indicator organisms should be selected on the basis of potential for field exposure to the subject protein (USEPA 2000, 2001, 2002, and 2004). Chronic studies: D elay ed adverse effects and/or accumulation of toxins through the food chain are not expected to result from exposure to Bt PIP proteins, and these protein toxins are not routinel y t ested for chronic effects on non

- target organisms. However, the 30 day test duration requirement does amount to subchronic testing when performed at field exposure test doses. Proteins also do not bioaccumulate. The biological nature of proteins makes the m readil y s usceptible to metabolic, microbial, and abiotic degradation once they

are ingested or excreted into the environment. Although there are reports that

h Th e established peer and EPA Science Board review ed guidance on screening test levels of concern is 50% m ortality at 5X environmental concentration
. The appropriate endpoints in high dose limit/screening testing are based on mortality of the treated, as compared to the untreated (control) non

- target organisms. A single group of 30 test animals ma y be tested at the maximum hazard dose
.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

45 some proteins (Cry

proteins) bind to soil particles, it has also been shown that these proteins are degraded rapidl y b y soil microbial flora upon elution from soil particles. Conclusion: The tiered approach to test guidelines ensures, to the greatest extent possible, that the Agency requires the minimum amount of data needed to make scientificall y sound regulatory decisions. The EPA believes that maximum hazard dose Tier Iscreening 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 co nfidence. The Agency expects that Tier 1 testing for short

- term hazard assessment will be sufficient for most studies submitted in support of PI P registrations. However, if long range adverse effects must be ascertained, then higher

- tier longer

- term fi eld testing will be required. As noted above, the October 2000 SAP and the National Academ y of Sciences (NAS 2000) recommended testing non

target organisms directly

in the field. This approach, with an emphasis on testing invertebrates found in corn fields, wa s also recommended by the August 2002 SAP and was supported by several public comments. Based on these recommendations, the Agency has required field studies on long term invertebrate population/community and Cry protein accumulation in soils as a conditio n of registration due to the lack of baseline data on the potential for long

- term environmental effects from the cultivation of PIP
- producing plants. Since the commercialization of Bt crops, the number of field studies published in scientific literature i n combination with the post

- registration field studies submitted to the Agency has accumulated to a level where empirical conclusions can be made. As a result, the issue of long range effects of cultivation of these Cry proteins on the invertebrate commun ity structure in Bt crop fields has since been adequately addressed. Specificall y, a meta - anal ys is i of the data collected from 42 field studies indicated that non

target invertebrates are generall y more abundant in Bt cotton and Bt maize fields than in no n - transgenic fields managed with insecticides (Marvier et al ., 2007). In addition, a comprehensive review of short and long term field studies on the effects of invertebrate populations in Bt corn and cotton fields indicated that no unreasonable adverse e ffects are taking place as a result of wide scale Bt crop cultivation (Sanvido et al. 2007). Another review of field tests published to date concluded that the large

- scale studies in commercial Bt cotton have not revealed an y unexpected non

- target effects other than subtle shifts in the arthropod community caused by

the effective control of the target pests (Romeis et al ., 2006). Slight reductions in some invertebrate predator populations are an inevitable result of all pest management practices, which re sult in reductions in the abundance of the pests as prey
. Overall, the Agency

is in agreement with the conclusions of these studies and collectivel y, these results provide extensive data to support that Bt crops have not caused long term environmental e ffects on a population level to organisms not targeted b y Bt proteins. Based on these considerations, regulatory testing of the specialist predators and parasitoids of target pests may eventually be considered unnecessary . i This research was funded by Environmental Protection Agency grant CR

- 832147
- 01 . The Bt crop non - target effects database can be found on the National Center for Ecological Analysis and Synthesis (NCEAS)
. Website. ( http://delphi.nceas.ucsb.edu/btcrops/).Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

46 **1 . Environmental Exposure A**  **ssessment** Two separate SAP reports (October 2000 and August 2002) recommended that non

- target testing of Bt Cry proteins should focus on invertebrate species exposed to the crop in which the protein(s) will be expressed. Following SAP recommendations, th e EPA determined that non

- target organisms with the greatest exposure potential to Cry protein in transgenic cotton fields are beneficial insects that feed on cotton pollen and nectar, particularl y lepidopteran insects, and soil invertebrates. While EPA’s risk assessments of Bt cotton have focused primaril y on these taxa, BPPD recognizes that exposure to other nontarget organisms can occur and has required testing on representative species. The EPA risk assessment is centered on adverse effects at the fie ld exposure rates, which are ty pically based on protein expression levels within the plant for PIPs. Although it is recommended that non

target testing be conducted at a test dose 10X the EEC whenever possible, the test dose margin can be less than 10X wh ere uncertainty in the s ys tem is low or where high concentrations of test material are not possible to achieve due to test organism feeding habits. BPPD may also allow for testing at lower doses in cases where many species are tested or tests are very sen sitive, although the concentration used must exceed the EEC. For the purposes of the non

- target organism studies submitted in support of eCry 3.1Ab expressed in Event 5307 corn, the test material dose levels were based on an estimated concentration of eCry 3.1Ab protein expressed in the tissue(s) with the highest concentration based on p rotein expression studies (MRI D 44842509). **2 . Ecological Effects Data for**  **eCry3.1Ab Expressed in Event 5307 Corn** In the absence of PIP

- specific risk assessment guidance, EPA requires applicants for PIP registrations to meet the 40 CFR Part 158 data requirements for microbial toxins. These requirements include testing on birds, mammals, nontarget insects, honey bee, plants, and aquatic species, and information has been submitted to address these requirements. Li mit dose testing on representative organisms from several taxa was performed in support of the eCry 3.1Ab Section 3 FIFRA registrations. As stated above, BPPD’s risk assessments focus more greatl y on beneficial no ntarget invertebrates, since they are most closely

related to organisms susceptible to the insecticidal action of Bt toxins. The eCry 3.1Ab protein is meant to target species within the order Coleoptera (e.g., beetles). Bt toxins are known ty pically to have a limited host range, however, to address any unforeseen change in activity spectrum as a result of laboratory protein sy nthesis and to fulfill the published registration data requirements EPA requires that test species used for non - target insect evaluati ons should include several invertebrate species that are not related to the target pests. Earthworm studies are also recommended. The toxicity of the eCry 3.1Ab protein has been evaluated on several species of invertebrates including the lady beetle ( Coleom egilla maculata ), carabid beetle ( Poecilus cupreus ), rove beetle ( Aleochara bilineata ), predatory bug ( Orius laevigatus ), an aquatic invertebrate shredder species ( Gammarus fasicatus ), honey bee ( Apis mellifera ), and earthworm ( Eisenia fetida ). Reproductiv e and/or developmental observations were also examined in the lady beetle, rove beetle, carabid beetle, predatory bug, and honey

bee studies.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

47 Since exposure may

also occur to other nontarget organisms, EPA has received data to compl y with th e Agency ’s published data requirements on other nontarget organisms. A study to determine soil degradation of eCry 3. 1Ab has also been submitted. The individual results for nontarget organism and soil degradation testing for eCry 3.1Ab are summarized in Table 1
. The stud ies are described in more detail below, and full reviews of each study can be found in the individual Data Evaluation Records. The October 2000 SAP recommended that while actual plant material is the preferred test material, bacteria

- derived protein is a lso a valid test substance, particularl y in scenarios where test animals do not normally consume cotton plant tissue and where large amounts of Cry protein are needed for maximum hazard dose testing. In support of the Event 5307 registrations, test substances used in the submitted studies included bacteria

- produced purified eCry 3.1Ab protein as well as plant tissue (grain) from Event 5307 corn. Comparative anal ys es that were previously submitted for the EUP showed the equivalence of the bacterially

- produce d and purified eCry 3.1Ab and the eCry 3.1Ab proteins produced in Event 5307 corn . **Table 6**  **. Summary of environmental effects studies and**  **scientific rationales for eCry3.1Ab as expressed in**  **Event 5307 corn submitted to comply with data requirements publis**  **hed in 40 CFR § 158. 2150.**  **Data Requirement**  **OPPTS**

**Guideline Test Substance**  **Results Summary and Classification MRID**  **No.** Avian dietary testing, broiler chicken, Gallus domesticus 885.4050 Event 5307 corn grain A 49
- day dietary study showed no adverse e ffects to broiler chickens when fed starter, grow er, and finisher diets composed of 54%, 58%, and 63% Event 5307 corn containing eCry3.1Ab, respectively. The amount of protein contained in the diets was lower than expected. **Classification: Supple**  **mental** 48442527

Avian inhalation testing 885.4100 N/A Not required. Avian oral testing indicates that eCry3.1Ab is not toxic to birds. N/A
Avian oral testing 850.2100 Microbially

produced eCry3.1Ab No adverse effects were observed in Northern

bobwhite ( Colinu s virginianus ) exposed to an acute oral dose of 900 mg eCry3.1Ab/kg

- bw after 14 days of observation. The oral LD 50 is >900 mg eCry3.1Ab/kg

- bw
. **Classification: Acceptable** 48442529

Wild mammal testing 885.4150 N/A Not required. Adverse effects were not o bserved in an acute oral toxicity study in which mice were dosed with eCry 3.1Ab at 2000 mg/kg body weight. This laboratory study is sufficient for use in determining risk to wild mammals. 47753901

Freshwater fish testing, Channel catfish 885.4200 Event 5307 corn grain Consumption of Event 5307 grain at the

exposure level tested (41% of the diet) had no adverse effects on survival, body weight, and feed:weight gain ratio of channel catfish ( Ictalurus punctatus ) after 28 days of exposure. **Classification: Acceptable**  48442531

Freshwater 885.4240 Lyophilized No adverse effects on survival or reproduction 4 8442533Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

48 **Data Requirement**  **OPPTS**

**Guideline Test Substance**  **Results Summary and Classification MRID**  **No.** invertebrate testing, Gammarus fasicatus Event 5307 corn leaves, V6

- V8 stage of Gammarus fasicatus exposed to Event 5307

corn leaf material for 5 days
. **Classification: Acceptable** Estuarine and marine animal testing 885.4280 N/A Not required. Exposure in marine and/or estuarine environments is not expected to be significant. N/A
Non
- target plant testing 885.4300 N/A Not required. Bt and its proteins are not known plant pathogens or toxins, and adverse effects in plants are not anticipated. Exposure to nontarget plants is expected to be minimal. N/A

Non
- target insect testing, carabid beetle, Poecilus cupreus 885.4340 Microbially

produced eCry3.1Ab N o adverse effects on survival w ere observed in

carabid beetle larvae fed 400 μg eCry3.1Ab/g diet. Emerged adults fed eCry3.1Ab had significantly low er body

w eight than controls. The LC 50 wa s >400 μg eCry3.1Ab/g diet
. **Classification: Acceptable** 48442539

Non
- target insect testing, lady beetle, Coleomegilla maculata 885.4340 Microbially

produced eCry3.1Ab or Event 5307 maize pollen No adverse effects on survival, development

time, or adult weight in C. maculata fed 500 μg eCry3.1Ab/g diet (nomina l) for 21 days. Results of groups fed pollen are inconclusive. The LC 50 is >500 μg eCry3.1Ab/g diet. **Classification: Acceptable** 48442532

Non
- target insect testing, rove beetle, Aleochara bilineata 885.4340 Microbially

produced eCry3.1Ab No adverse effe cts on adult survival or number

of F1 progeny were observed in adult A. bilineata fed 400 μg eCry3.1Ab/g diet (nominal) for 35 days. The LC 50 is >400 μg eCry3.1Ab/g diet **Classification: Acceptable** 48442536

Non
- target insect testing, predatory bug, Orius laevigatus 885.4340 Microbially

produced eCry3.1Ab No adverse effects on survival of nymphs fed

400 μg eCry3.1Ab/g diet (nominal) until adulthood in a 12

- day

laboratory study . The LC 50 is >400 μg eCry3.1Ab/g diet **Classification: Acceptable** 48442537

Ho neybee testing, adults Apis mellifera 885.4380 Microbially

produced eCry3.1Ab No adverse effects on survival or brood

development were observed when honey bee adults fed on a sucrose solution of 50 μg eCry3.1Ab/g for 5 days in a 21
- day semi

- field bioassay
. **Classification: Acceptable** 48442534

Honeybee testing, adults Apis mellifera 885.4380 Microbially

produced eCry3.1Ab No adverse effects on brood development were

observed when honey bee adults fed on a sucrose solution of 50 μg eCry3.1Ab/g for 5 days i n a 24
- day semi

- field bioassay. Results for cumulative mortality in adults are uncertain due to an uncontrolled factor in the study. **Classification: Supple**  **mental** 48442538Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

49 **Data Requirement**  **OPPTS**

**Guideline Test Substance**  **Results Summary and Classification MRID**  **No.** Earthw orm toxicity, Eisenia fetida 850.6200 Microbially

produced eCry3.1Ab No adve rse effects on survival or body weight

were observed in earthworms fed 50 μg eCry3.1Ab/g of soil dry weight after 14 days. The LC50 w as determined to be > 50 μg eCry3.1Ab/g of soil and the NOAEC was 50 μg eCry3.1Ab/g of soil. **Classification: Acceptable** 484 42530

Soil fate and degradation 885.5200 Microbially

produced eCry3.1Ab Based on the measured bioactivity in Colorado

potato beetle (

Leptinotarsa decemlineata ), eCry3.1Ab was shown to lose approximately half of its insecticidal activity betw een 7 and 14 days. Little activity was apparent after 14 days; eCry3.1Ab is not likely to persist in soil. **Classification: Supple mental**  48442535

**a . Non**  **- target Organism Study Summaries for**  **eCry3.1Ab 1**  **. Avian Wildlife**  Avian Dietary Toxicity Heritage broiler chicks ( Gallus domesticus ) were fed diets prepared from either Event 5307 corn grain (expressing eCry 3.1Ab protein), a near

- iso line nontransgenic corn grain, or a commercially

- available North Carolina corn grain (NCSU 2007 ) . Feeding continued for 49 day s , through provision of starter, grower, and finisher diets, to evaluate if diet prepared with Event 5307 corn grain caused adverse effects on survival, body

weight, feed consumption, feed conversion, or carcass yield. The diets were standardized to a similar nutrient content, with the corn content of the diets ranging from 52% to 64% by weight. Based on a separate diet anal ys is (MRID 48442528, which was determined to be acceptable), the starter, grower, and finisher 5307 diet contained a mean of 2.13, 0.34, and 0.38 μg eCry 3.1Ab/g diet , respectivel y , which is less than the amount expected
. At test end, there were no treatment

- related statistically

significant differences in mortalit y, body weight, feed consumption, feed conversion, or carcass yi eld among bird s fed the 5307, nontransgenic, or NCSU 2007 corn grain diets. This study indicates that relativel y l ong - term dietary exposure to eCry 3.1Ab at these levels is not expected to result in adverse effects in birds; however, the exposure levels are very low. MRI D 48442527. Avian Oral Toxicity A laboratory

study was conducted to determine the acute toxicity of a single oral dose of eCry 3.1Ab protein to Northern bobwhite ( Colinus virginianus ). Birds received either a nominal dose of 900 mg eCry 3.1Ab/kg body

we ight via a gelatin capsule or an empt y gelatin capsule only (control group). The birds were observed for 14 day s, and body weight and feed consumption were determined. There were no mortalities, and all birds appeared normal in appearance and behavior duri ng the test. There were noBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

50 apparent treatment - related effects on body weight or feed consumption. The acute oral LD 50 was >900 mg a.i./kg
. MRID 48442529.

Avian Inhalation Toxicity Avian inhalation toxicity

testing is ty

pically not required for Bt corn event registrations, since exposure to Cry proteins v ia inhalation is not expected. Avian inhalation testing is not required for eCry 3.1Ab. **2 . Wild mammals**  An acute oral toxicity

study with mice (MRID 47753901) has been submitted to EPA in support o f the Event 5307 experimental use permit. In this study , mice were dosed with microbially produced eCry 3.1Ab at 2000 mg/kg bod y weight . Treatment related mortalities and clinical signs of treatment

related effects were not observed during the study or at n ecropsy . This study has bee n determined to be acceptable. There is no indication that laboratory species would not be representative of wild mammals in this case; therefore, this information is suitable for use in assessment of risk to wild mammals, and no additional testing with wild species is necessary
.

**3 . Aquatic species**  Freshwater animals A laboratory study was conducted to determine the effects of consumption of grain from Event 5307 maize on survival, body

weight and feed:weight gain ratio of channel catfish ( Ictalurus punctatus ). Grain was incorporated into diet at 41% and fed to 20 juvenile channel catfish for 28 day s; control fish were fed a diet containing near

- isogenic corn grain incorporated at the same percentage. Fish were fed an amount of diet daily

that was approximately

equal to 8% of their total initial biomass for 14 day s. The fish were then weighed and the amount fed was adjusted to 8% of the day 14 total biomass for the remainder of the study . No mortalities or phy sical or behavioral abnormalities were observed among the treatment or control fish during the 28

- day exposure. At test termination , no significant differences were observed in total biomass, weight gain, or feed:weight gain ratio. Based on the results, effects to freshwater fish are not expected at the level of exposure tested. MRID 44842531. Because of concerns raised in a study with the leaf shredding (caddis fl y) trichopteran, Lepidostoma liba (Rosi

- Marshall, et al. 2007), EPA is requiring registrants of Bt co rn and cotton to submit freshwater invertebrate toxicity

studies to reduce uncertainty regarding this issue. A study with a leaf shredding freshwater isopod, Gammarus fasciatus , was submitted to determine the effects of ingestion of Event 5307 leaf materia l collected from corn plants at the V6

- V8 growth stage (based on information in MRI D 48802603) on the survival of adult s . Percent survival of gammarids at test termination after five days was 88% for insects receiving Event 5307 leaf material and 80% for c ontrols receiving near isoline leaf material. Consumption of the leaf material also did not differ between these two groups. Based on the results, c onsumption of Event 5307 leaf material is not expected to have adverse effects on freshwater invertebrates. MRID 48442533.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

51 Estuarine and marine animals No significant direct exposures of estuarine and marine animals to eCry 3.1Ab are anticipated as a result of agricultural production of corn containing eCry 3.1Ab. Studies with estuarine and/or marine fish and invertebrates are not required for eCry 3.1Ab.

**4 .**  **Terrestrial and aquatic plant species** T he active ingredient , eCry 3.1Ab is part of a larger group of insect toxin s produced a naturall y - occuring Bacillus thuringiensis (

Bt 

- endotoxin s ) that have n ot shown toxicity to plants. Effects to nontarget plants in the terrestrial and aquatic environments are not expected and testing is not required.

**5 . Invertebrate species**  A laboratory toxicity

study was performed with larvae of a carabid gr ound beetle , Poecilus cupreus
. Beetle larvae (n=40 per treatment) were fed an artificial meat

- based diet containing either 400 μg eCry 3.1Ab protein/g diet , water (negative control), or teflubenzuron (a chitin sy nthesis inhibitor, positive control) for 59 d ay s. The exposure level tested is approximately 10 times the average expression rate of eCry 3.1Ab in leaves of Event 5307 corn plants. Overall mortalit y was 10% in the control, 8% in the eCry 3.1Ab treatment , and 100% in the positive control . Corrected mort ality did not differ significantl y between the groups receiving eCry 3.1Ab and the water control; however, the mean weights of emerged adult s in the control and eCry 3.1Ab treatments were 65.7 mg and 52.7 mg, respectivel y, and these differed significantly
. D evelopment time was slightly longer for beetles receiving eCry 3.1Ab , though this was not statistically significant. Because of the significant difference in adult body weight, this study indicates adverse sublethal effects at ~8X EEC
. MRID 48442539. Anoth er laboratory

study was conducted to determine the dietary toxicity of microbially

produced eCry 3.1Ab and Event 5307 corn pollen (which expresses eCry 3.1Ab) to the predatory lad ybird beetle ( Coleomegilla maculata ). Groups of C. maculata larvae were provide d diet containing 500 μg eCry 3.1Ab/g diet for 21 day s. The test also included a group fed Event 5307 corn pollen, an assay control group (diet onl y), a positive control group (potassium arsenate), and a non

- transgenic corn pollen control group. Compared to the assay control group, the weight of adults in the eCry 3.1Ab group was significantl y higher (~20%), and development time (day s to pupation, day s to adulthood) was significantl y shorter, but these differences were not considered to be adverse effects. R esults with the test groups fed pollen indicate a possible problem with this portion of the study ; however, the results of the experiment with the microbially

- produced eCry 3.1Ab are sufficient to show that eCry 3.1Ab does not have adverse effects on this in sect species at the exposure level tested . The L C 50 for this species is > 500 μg eCry 3.1Ab/g diet. MRID 48442532. A laboratory study

was conducted with a third Coleopteran species, the rove beetle ( Aleochara bilineata ). The purpose of the study was to ass ess the effects of eCry 3.1Ab protein on the survival and reproduction of adult beetle s . Adults of this beetle are predominantly entomophagous and their larvaeBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

52 are endoparasites of fl y pupae. In this study, eCry3.1Ab was incorporated into an artificial meat

- based diet at 400 μ g eCry 3.1Ab /g diet . A negative control group received diet treated with water, and a positive control group received diet treated with teflubenzuron (a chitin synthesis inhibitor). Adults were fed their respective diet for five weeks, d uring which survival was observed. Afterward, observations were made on the number of F 1 progen y that emerged from fl y pupae placed in the test arenas
. Mortality of adults after 35 day s was 17.5% in the controls and 20% in the eCry 3.1Ab groups, and did not differ significantl y. No difference in the number of F 1 progen y produced was observed between these groups. Mortality was onl y 18.8% in the positive control group; however, no offspring were produced in this group, which was expected. The stud y indicates that at the level tested, eCry 3.1Ab does not produce adverse effects. The LC 50 for this species is >4 00 μg eCry 3.1Ab/g diet . MRID 48442536. An additional study

was conducted with the predatory bug , Orius laevigatus . Ny mphs were provided an artificial me at - based diet containing a nominal concentration of 400 μg eCry 3.1Ab/g diet
. An untreated control diet and a toxic reference diet containing teflubenzuron were also included in the test. All ny mphs developed to adults or died by test day 12. Pre
- imaginal m ortality was 0% and 5% in the control and test groups, respectively , which was not significantly different. Mortality

was 100% in the toxic reference group. Based on the results, eCry 3.1Ab had no adverse effects on O. laevigatus when provided at a rate of 400 μg/g diet. The L C 50 for this species is >4 00 μg eCry 3.1Ab/g diet . MRID 48442537. A semi

- field study

with honey

bee ( Apis mellifera ) was also conducted to determine the effects of eCry 3.1Ab on adults and on brood development. Adults were fed a sucrose solution containing 50 μg eCry 3.1Ab/g for 5 day s. A sucrose solution

- onl y assay control and a sucrose solution containing fenox ycarb as a positive control were also included. Mortality was observed for eight day s prior to feeding. The test diets were prov ided to the bees daily within the hive for five day s and then observations of mortality and brood development were made up to 22 day s after initial feeding. Mortality was monitored with a dead bee trap placed on the hive, and flight intensity (the number o f worker bees entering and leaving the hive) was also observed. Brood development was monitored after the initial feeding day

and assessed using a brood development index based on expected and observed development stage at the time of observation (brood i ndex). An additional development index (compensation index) was calculated to account for eggs or larvae that were placed in cells as a result of a previous mortality . Mortality and development indices did not differ significantly between the negative control group and the group exposed to eCry 3.1Ab. Significant mortality and brood losses were observed in the positive control compared to the negative control. Based on the results adverse effects on honey bee adults and brood development are not expected whe n hives are exposed to eCry 3.1Ab at 50 μg eCry 3.1Ab under these conditions
. MRID 44842534. The effects of eCry 3.1Ab on honey bees was also observed in another semi

- field study in which bees were similarl y exposed to 50 μg eCry 3.1Ab/g in a sucrose solution for five day s. A negative control and a positive control containing diflubenzuron were also included. The endpoints evaluated were: a) the pre - imaginal mortalit y of pre

- marked brood s ; b) the number of dead adult bees, larvae and pupae expelled from the hi ve; and c) an assessment of whether the treatments affected the relative proportions of brood and food cells within the hives over the duration of the test (24 day s)
. For cells that contained eggs at the time of initial feeding, mortalit y was 25.5% in the (negative) control, compared with 38.2%Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

53 (17.0% corrected mortality) in the eCry 3.1Ab treatment group and 99.8% in the toxic reference treatment group. For cells that contained larvae, mortality was mortalit y was 16.8% in the control group, compared with 2. 3% (0.0% corrected mortality) in the eCry 3.1 Ab treatment group and 100% in the toxic reference treatment group. Mortality between the negative control and eCry 3.1Ab treated groups was not significant. The numbers of dead bees and brood deposited in the dea d bee traps during the treatment and post

- treatment periods were 22.4, 12.0 and 25 .3 per hive for the control, eCry 3.1Ab and toxic reference treatments, respectivel y. The stud y authors noted that wasps were taking cadavers out of the bee traps, which may have affected the results. However, the stud y reliabl y indicates that overall hive condition and brood development are not affected by eCry 3.1Ab at the level tested under the exposure conditions. MRID 48442538. Toxicity to a non - arthropod invertebrate was also examined in a study t o determine the toxicity of eCry 3.1Ab to the earthworm ( Eisenia fetida ). Artificial soil was treated at a rate of 50 μg eCry 3.1Ab/g of soil dry

weight, which was intended to represent approximately 10X the average expression rate of eCry 3.1Ab in roots of Event 5307 plants. Control artificial soil was treated with purified water onl y. Ten adult earthworms were placed on the soil surface in each jar, and the jars were maintained under controlled conditions for 14 day s. At test end, none of the earthworms in either group had died, and all showed normal condition and behavior. The change in mean earthworm weight was

- 9.1% in the control group, and

- 7.2% in the test material group, and the difference between these was not statistically significant. Worms likely lost weight because no food was included in the treatment. The no

- observed

effect concentration in this study was 50 μg eCry 3.1Ab/g soil dry weight. The presence and activit y of e Cry 3.1Ab in the artificial soil used in the test we re confirmed in a separate study
. MRID 48442530. **b . Soil Fate Studies for**  **e Cry**  **3. 1Ab**  Soil organisms may

be exposed to eCry 3.1Ab through contact with corn roots (by direct feeding), plant root exudates, incorporation of above

- ground plant tissues into so il following harvest, or b y soil

deposited pollen. Some evidence suggests that soils that are high in clays and humic acids are more likely to bind Cry

protein. However, neutral pH soils tend to have high microbial activit y and microbes contribute to Cry protein degradation. In addition, a study on the release of Cry

proteins in the root exudates of Bt cotton has shown that no Cry proteins were detected immunologicall y or b y larvicidal assay in an y soil or h ydroponic solution in which Bt cotton had been grown (Saxena and Stotzky , 2001). The weight of evidence indicates that Cry proteins do not accumulate in soil to arthropod

- toxic levels. Because Cry

proteins are toxins derived from soil

- inhabiting bacteria, Bacillus thuringiensis , the degradation of eCry 3.1Ab from Event 5307 corn is expected to be similar to that of these proteins that are produced naturall y from soil bacteria. Nonetheless, BPPD required the following soil fate evaluations to support this registration. A bioassay

s ys tem (first instar larvae of the Colorado potato beetle , CPB ) was used to investigate the loss of insecticidal activity of eCry 3.1Ab protein in soil over 120 day s under laboratory conditions. The test soil, characterized as a loam, was collected from the top six inches of s oil over an area of approximately

four acres in Newtown, IN. The site had been used for growing corn over the three previous y ears, and was currentl y being used for soybeans. Soil samples were dosed with aBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

54 concentration of approximately 3.125 mg eCry 3.1Ab/mL
. Controls were dosed with 1 mLof the buffer. Additional incubation bottles, containing dry weight equivalents of soil, were designated for biomass measurements
. All samples were mixed well, and maintained in an incubation chamber under aerobic condit ions at a temperature of 15

- 30°C. Each incubation bottle was connected to hy dration bottle and airflow s ys tem. The bottles were checked every one to two weeks, and moisture levels were adjusted with purified water to maintain proper h ydration levels. Treated soils were incubated for 0, 1, 3, 7, 14, 29, 60, 90, and 120 day s (NOTE: the 90

- day treated soils were dosed incorrectl y, and results f rom the sample were not used). Untreated controls were incubated for 0, 60 and 120 day s. Results from the 60 and 120 day

control incubations were also not available. Following incubation, the soil samples were incorporated into a commerciall y available CPB diet at a concentration of 10% (w/v)
. A total of 150 μL of soil/diet mixture was fed to each of 24 larvae. The conta iners with larvae were sealed and in th e dark at ambient temperature. Mortality readings were taken dail y starting at 72 hours and continuing through 120 hours. Corrected mortality was 72% , 63%, 48%, and 40% on Day s 0, 1, 3, and 7, and then were

- 4%, 3%, 1 % and 0% for Day s 14, 29, 60, and 120. The data are not sufficient to calculate a half

- life. However, based on the data provided, the half

- life of the eCry 3.1Ab in the soil tested appears to be between 7 and 14 day s. This is comparable to half lives obser ved with other Cry

proteins (e.g., see MRID 48442526). The study design could be improved to provide a more robust estimate of soil half life, and a range of soil ty pes should have been tested , including those with high clay and humic acid content, due to their known binding affinity for proteins. The data do show that eCry 3.1Ab is not likely to persist in loam soil s . However, additional studies would be useful in evaluat ing insecticidal protein degradation, accumulation, and persistence in a variety of soi l ty pes and also for refining the estimate of the DT 50 for eCry3.1Ab in soil. Based on FIFRA Scientific Advisory Panel recommendations and public comments, the Agency has required three ye ar soil fate studies for the currently registered Cry

protein produ cing crops grown in a variet y of soils and environmental conditions, a s a condition of registration. , A comprehensive review of all available scientific data on ecological effects of commercially grown GM crops over ten y ears was published in 2007 (Sanvid o, et al . 2007). The review concluded “none of the laboratory or field studies suggest accumulation of Bt

- toxins in soil over several ye ars of cultivation” and “experience from commercial cultivation indicates that Bt

- toxin will not persist for long perio ds under natural conditions.” The Agency

agrees with these conclusions. Collectively , the long

- term field studies for Bt crops also confirm the previous SAP conclusion that “bioaccumulation is not expected to occur with transgenic proteins because biodegr edation mechanisms for proteins are ubiquitous ” (US EPA, 2000). More importantly , the numerous laboratory studies that demonstrated rapid protein degradation in soil of Bt proteins produced in Bt crops (when performed under realistic environmental conditi ons) can be considered predictive that Bt protein in soil is not likely to persist or accumulate in soil after continuous cultivation. In light of these published findings and the relatively rapid degra d ation of e Cry 3. 1Ab protein in soil as demonstrated in the insect bioassay described above, there is no indication that the eCry 3.1Ab protein expressed in Event 5307 corn is likely to persist in soil after continuous cultivation. Therefore, no additional long

- term field studies are required for these PI P p roducts.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

55 **c . Supplemental Information**  Sy ngenta submitted a summary of nontarget organism testing with eCry 3.1Ab (MRID 44842526), which summarized the information above, and included an assessment of ecological risk and some discussion of environmental f

ate. These summaries were determined to be supplemental, and relevant information was utilized in this risk assessment. **3 . Environmental Risk Assessment for eCry3.1Ab** 
**a . Effects to Nontarget Wildlife, Invertebrates, and Plants**  **1 . Avian**  **Wildlife** Birds are known to forage in corn fields, and may be exposed to eCry 3.1Ab. An estimate of dietary exposure for birds is provided in MRID 44842526, which is based on estimates of dail y dietary dose based on consumption of Event 5307 grain and mean expression levels (fresh weight) in this plant tissue using a method developed by Crocker et al . (2002). The worst case exposure, based on 100% consumption of grain was estimated to be 1.94 mg eCry 3.1Ab/kg body weight for birds with body weights ranging fr om 22g (e.g., a sparrow) to 953g (e.g., a pheasant) . Exposure through other foods is not well characterized , however, and to account for uncertainties related to their possible consumption, a more conservative estimate of exposure may be determined using estimates of protein expression in tissues that express the highest amounts (e.g., leaves). Based on the method and mean fresh weight expression levels used in MRI D 44842526, assuming 100% consumption of a diet containing material with an eCry 3.1Ab concentration equivalent to the mean fresh weight expression level in leaves (51.74 μg/g) , a worst - case exposure would range from 5.69 to 18.11 mg eCry 3.1Ab/kg body weight for birds with the same body

weights. An avian oral toxicity

study was submitted, wherein the LD 50 for eCry 3.1Ab was determined to be >900 mg eCry 3.1Ab/kg body

weight. This exposure level is much higher than the maximum exposure estimates discussed above, and minimizes any uncertainties related to exposure in the field. Additional data provided in the study with broiler chickens shows that longer

- term dietary exposure at low levels is unlikely to affect the parameters examined in that study . Based on these data, cultivation of Event 5307 corn expressing eCry 3.1Ab is not anticipated to cause adv erse effects in birds.

**2 . Wild mammals**  Wild mammals also forage in corn fields and may be exposed to e Cry 3. 1Ab . Sy ngenta provided an estimated maximum dose of 1.82 mg eCry 3.1Ab/kg body weight for mammals, based on the same methods as described above for birds. Using these methods and the mean fresh weight expression in leaves as given above, a maximum exposure for mammals would be 17.07 mg eCry 3.1Ab/kg body weight.Bacillus thuringiensis eCry3.1Ab Corn

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56 Toxicity testing with laboratory

mice showed no adverse effects with exposure to e Cry 3. 1Ab at a dose of 2000 mg/kg bod yw eight . This amount is much higher than concentrations that wild mammals are expected to encounter in the field ; therefore, an y uncertainties related to exposure are minimized by testing at this level
. B ased on this infor mation, adverse effects to wild mammals are not expected as a result of the registration and use of eCry 3.1Ab as expressed in Event 5307 corn.

**3 . Freshwater animals**  BPPD previously

determined that exposure to Cry proteins in the freshwater environment were expected to be low, based on the assumption that exposure in aquatic environments primarily resulted from pollen deposition. In light of published studies showing reduced growth in caddis flies exposed to anti

lepidopteran Cry 1A protein (Rosi

- Marshal l et al. 2007), concerns were raised regarding the potential for exposure to shredder invertebrate species via consumption of corn plant litter in aquatic environments. EPA determined (see USEPA 2010a) that post - harvest crop residue i s the most likely rout e of exposure to aquatic organisms (Carstens et al. 2011) ; however, their exposure to biotech crops is likel y to be low, since the concentrations of Cry proteins in post

- harvest crop tissues are limited temporally and spatially (Swan et al. 2009, Jensen et al. 2010, Wolt and Peterson 2010, Carstens et al. 2011 )
. Nonetheless , because of concerns that have been raised over this issue, EPA has required aquatic invertebrate testing. Studies lasting 7

- 10 day s with Daphnia spp. have ty picall y been required. Studies with shredder species would test directly

the invertebrate species expected to be affected by Bt corn plant litter that may enter streams. A study

with a shredder species ( Gammarus fasicatus ) was submitted that showed that this species of isopod is n ot affected when exposed to Event 5307 corn leaves. The leaves were collected during a growth stage in which leaves express higher amounts of the eCry 3.1Ab protein compared to other growth stages. Event 5307 corn leaves also express greater amounts of eCry 3.1Ab compared to other tissues (based on MRI D 48442509). Therefore, the isopods in this study were exposed to eCry 3.1Ab at or near a maximum EEC. Additionally the study with channel catfish described above found no effects of eCry3.1Ab in this species whe n fed Event 5307 corn grain at 41% in the diet. Based on the results of this study , adverse effects to freshwater fish and invertebrates are not anticipated as a result of the registration and of eCry 3.1Ab as expressed in Event 5307 corn.

**4 . Estuarine**  **and marine animals** As discussed above, s ignificant amounts of e Cry 3. 1Ab use of Event 5307 corn are not expected to reach marine/estuarine areas. Therefore, BPPD concludes that adverse effects to fish and invertebrates in these environments as a result of the registration and cultivation of corn containing eCry 3.1Ab are not expected because exposure is expected to be minimal
.

**5 .**  **Terrestrial and aquatic plant species** T he active ingredient , eCry 3.1Ab is part of a larger group of insect toxin s produc ed a naturall y - occuring Bacillus thuringiensis (

Bt 

- endotoxin s ) that have n ot shown toxicity to plants. Effects to nontargetBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

57 plants in the terrestrial and aquatic environments are not expected, and BPPD concludes that adverse effects to terrestrial and a quatic plants as a result of the registration and use of eCry 3.1Ab in Event 5307 corn are not anticipated.

**6 . Invertebrate species**  Nontarget insects are expected to receive exposure primarily through consumption of Bt cotton pollen and/or nectar, though they may also consume pest insects that feed on cotton plant tissue or rarel y other corn plant tissues themselves. The principal exposure route of soil

- dwelling invertebrates, such as collembola, earthworms, and rove beetles, to eCry3.1Ab i s assumed to be from decomposing plant tissue and plant exudates in soil. Studies submitted to EPA that examined the effects of eCry 3.1Ab in lad y beetle, rove beetle, predatory bug, and earthworm showed no adverse effects at concentrations well above those that would be encountered by nontarget insects in the environment, even if directly consuming tissues with the highest levels of eCry3.1Ab protein expression. No mortality was observed in carabid beetles fed eCry 3.1Ab at 400 μg eCry 3.1Ab/g diet (~8X EEC); however, adult body weight was significantly

reduced at this exposure level. Effects in this study are not necessarily

unexpected, since eCry 3.1Ab is a coleopteran

- active protein. This sublethal effect could affect individual reproductive success and poss ibly population size for sensitive species, and additional testing to establish a sublethal endpoint (e.g., EC 50 or NOAEC) would reduce an y uncertaint y related to its observation in this study . Since other studies with nontarget coleopteran species showed no adverse effects with exposure to eCry 3.1Ab, and because exposure is expected to be much lower in the field, BPPD does not anticipate commercial cultivation of Event 5307 corn plants to cause significant effects to populations of nontarget insects and other invertebrates in the field, including coleopteran species. T wo studies examining the effects of eCry 3.1Ab on adult honey bees, honey

bee brood development, and overall hive health showed no adverse effects to honey bees at exposure levels to eCry 3.1Ab above those expected to be encountered in the environment. Therefore risk to honey bees as a result of the proposed registrations is also expected to be minimal. It should be noted that effects to invertebrates that are recognized as corn pests (i.e. , insects that feed detrimentally

on corn plants) that are not targeted by the eCry 3.1Ab protein (but are controlled by other means) are not included in BPPD’s assessment of risk to nontarget insects.
**b .**  **Effects on Soil Microorganisms** Numerous pu

blished studies indicate that exposure to Cry protein s produced in Bt PIP crop plants does not adversel y affect soil microorganisms (Sanvido et al ., 2007). Although a minimal transient increase and shift in microbial populations may result from the presenc e of transgenic plant tissue in soil, no adverse effects have been attributed to the Cry protein s . In addition, the soil degradation study discussed above shows that the eCry 3.1Ab degrades quickly in soil.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

58 With regard to the impact of geneticall y engin eered crops on soil, EPA has previously noted (USEPA 2010a) that agricultural practices themselves cause large changes in soil and soil microbial composition. Furthermore, factors such variations in seasons and weather, plant growth stage, and plant variet ies, independent of being genetically engineered, are also responsible for significant shifts in soil microbial communities. Most studies with geneticall y engineered crops to date have shown minor or no effects on soil microbes bey ond the variation caused by the factors listed above.
**c . Horizontal Transfer of Transgenes from**  **Bt Crops to Soil Organisms**  The EPA has evaluated the potential for horizontal gene transfer (HGT) from Bt crops to soil organisms and has considered possible risk implications if such a transfer were to occur. Genes that have been engineered into Bt crops are mostly found, or have their origin, in soil

- inhabiting bacteria. Soil is also the habitat of other toxin

- producing bacteria, and transfer of these genes and/or toxins to othe r microorganisms or plants has not been detected. Furthermore, several published experiments, that were conducted to assess the likelihood of HGT, have been unable to detect gene transfer under t ypical environmental conditions. Horizontal gene transfer to soil organisms has only been detected with very promiscuous microbes under laboratory conditions designed to favor transfer. As a result of these findings and the fact that the Bt toxin engineered into Event 5307 is derived from soil

- inhabiting bacteria , the EPA has concluded that the risk of HGT of transgenes found in Event 5307 corn expressing eCry 3.1Ab is low. **d . Gene Flow and Weediness Potential**  The movement of transgenes from the host plant into weeds has been a significant concern for EPA due to the possibility

of novel exposures to the pesticidal substance. This concern has been considered for each of the B t plant

- incorporated protectants currently registered , and was extensively reexamined in 2010 (see USEPA 2010 a ), and EPA believes that the se concerns have been satisfactoril y addressed. The Agency has determined that there is no significant risk of gene capture and expression of any Bt endotoxin by

wild or weedy

relatives of corn in the U.S., its possessions or territories. In addition, the USDA/APHI S has made this same determination under its statutory authority under the Plant Pest Act. The FIFRA EPA Scientific Advisory Panel meeting held on October 18

- 20, 2000, further discussed the matter of gene flow and offered some issues for consider ation in this matter. The panel agreed that the potential for gene transfer between corn (maize) and any receptive plants within the U.S., its possessions and territories was of limited probability and nearly

risk free. Based on these extensive anal ys es, the potential for gene flow and development of weediness is expected to be low for Event 5307 corn expressing eCry 3.1Ab protein.
**e . Conclusions**  BPPD concludes that significant adverse effects on birds, mammals, nontarget insects, honey bees, freshwate r and marine/estuarine fish and invertebrates, and terrestrial and aquatic plants are not expectedBacillus thuringiensis eCry3.1Ab Corn

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59 as a result of the use of Event 5307 corn expressing eCry 3.1Ab. EPA has also determined that there is no significant risk of gene capture and expression of e Cry 3.1Ab protein b y wild or weed y relatives of corn within the U.S. or its territories. Available data do not indicate that Cry proteins have an y measurable adverse effect on microbial populations in the soil, nor has horizontal transfer of genes from tran sgenic plants to soil bacteria been demonstrated. **f . Impacts on Endangered Species**  The primary

route of exposure to eCry 3.1Ab protein in corn is through ingestion of corn tissue. Since e Cry 3.1Ab protein is not expected to have adverse effects on mammals, birds, plants, freshwater and estuarine/marine fish and invertebrates , nontarget insect s (see discussion below) and other invertebrate species at the Estimated Environmental Concentration (EEC) , a “No Effect” determination is made for direct and ind irect effects to federally listed threatened and endangered (“listed”) species. In addition, EPA does not expect that any threatened or endangered plant species will be affected by outcrossing to wild relatives or b y competition with such entities. Hybrid corn does not exist in the wild, nor are there wild plants that can interbreed with corn in the United States. Because of the selectivity of eCry 3.1Ab protein for some coleopteran species, there exists some concern for listed species in the order Coleopte ra. The potential risk to listed threatened or endangered coleopteran species has been previousl y addressed for Bt Cry proteins in corn ( USEPA 2009, 2010b, 2010c) . At present, 18 coleopteran species are listed as threatened or endangered, and 16 of these o ccur in counties in which corn is grown. Eight of these species are subterranean, and exposure to Cry proteins expressed in plant tissues is not expected in these environments. The remaining species existing above ground, in terrestrial or aquatic habitats , are habitat specialists that are largely excluded from agricultural areas due to their specific habitat requirements. While habitats of some of these species may occur nearb y to agricultural areas where Event 5307 corn may be cultivated, exposure via con sumption of Event 5307 corn tissue is not anticipated due to their specific food requirements that would exclude consumption of corn plant material (i.e., they are predators/scavengers or consume specific food plants). BPPD also has previously determined that exposure to Cry proteins in pollen outside of areas where Bt corn is cultivated is limited (USEPA 2010a). Furthermore, the protein expression study for Event 5307 corn (MRID 48442509), which is pending review, shows that very little eCry 3.1Ab is expre ssed in Event 5307 pollen, so exposure to eCry 3.1Ab resulting from pollen that lands on food items within or outside of areas where Event 5307 corn is grown is expected to be very low. As discussed in previous assessments of coleopteran

- active Cry protei ns, the American burying beetle ( Nicrophorus americanus ) is the only species that may

potentiall y utilize areas where corn is grown. Exposure to this species has been discussed previously , and BPPD determined that this species is not expected to be exposed because it largel y feeds on carrion (USEPA 2010c). Based on the available information, exposure of listed coleopteran species to eCry3.1Ab protein produced b y Event 5307 corn is not expected. Therefore, a “No Effect” determination is made for direct eff ects to these species. Since effects on other insects or other animal or plant taxa are not anticipated, asBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

60 discussed above, a “No Effect” determination is also made for indirect effects to listed coleopterans and for effects to their critical habitat. **D. Environmental Risk Assessment for eCry3.1Ab, Cry1Ab, mCry3A, and Cry1F Expressed**  **Bt1 1**  **x MIR604**  **x TC1507**  **x 5307**  **Corn Hybrid 1**  **. Background** Sy ngenta has appl i ed for a FIFRA Section 3 Registration for a combined trait corn product Bt11 x MIR6 04 x TC1507 x 5307 (EPA File Sy mbol 67979

- EU) that confers lepidopteran and coleopteran insect resistance and herbicide tolerance (with the Event GA21 trait)
. The combined trait corn Bt11 x MIR604 x TC1507 x 5307 product was developed by crosses (via tradi tional breeding methods) with each of the following i ndividual PIP events : Bt11 corn (expressing Cry 1Ab), MI R604 (expressing mCry 3A), TC1507 (expressing Cry 1F) and 5307 (expressing eCry 3.1Ab). T his registration is supported by acceptable data approved prev iousl y for the individua l parental lines, and data for the eCry 3.1Ab protein expressed in Event 5307.,. A s ynergism study involving the Cry 1Ab , Vip3Aa20, and Cry 1F proteins w as previousl y s ubmitted and reviewed by EPA (MRID No. 48235204; reviewed in USEPA 2011) . To support the current applications for these stacks, a s ynergism study was submitted to study potential interactions in between mCry 3A and eCry 3.1Ab proteins. Another study was also submitted that tested the interaction of the combination of Cry 1 Ab , mCry 3A, Vip3Aa20, Cry 1F and eCry 3.1Ab proteins in susceptible lepidopteran and coleopteran species. These studies provide support for bridging to data for the individual PIP events that are already contained in the Agency ’s database.
**2 . Environment**  **al Assessment** The hazard of each of the individual events in Bt11 x MIR604 x TC1507 x 5307 has been addressed for previous registrations and for Event 5307 as discussed above. EPA has previously determined that the database for ecological risk assessment is complete for the Cry 1Ab, Cry 1F, and mCry 3A proteins expressed in this hy

brid, and also for eCry 3.1Ab based on the assessment above. Sy nergism is a unique concern with the environmental risk assessment of stacked PIPs is the potential for sy nergistic eff

ects that may result from interaction between the Cry proteins contained in the hy brid plants. Additionally , stacked h ybrids are assessed for protein expression comparability with their individual parent lines to ensure that exposure is not significantl y h igher in the hy brid. Therefore, this assessment contains an evaluation of the potential for sy nergism in the Bt11 x MIR604 x TC1507 x 5307 corn hy brid and a discussion of protein expression comparability
. **a. Event Bt11 (lepidopteran active) Environmenta**  **l Risk Assessment Summary** Potential adverse effects to non

- target organisms by the Cry 1Ab protein expressed by event Bt11 have been previousl y reviewed and was recentl y updated ( USEPA 2010a ). The following is a summary of the Bt11 e nvironmental risk asse ssment.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

61 The Cry 1Ab protein expressed in Bt11 corn is intended to control lepidopteran pests. The hazard of the Cry 1Ab protein was evaluated for the lady bird beetle , green lacewing, parasitic hy menoptera, collembolan, Daphnia spp., honey bee, earthworm, M onarch butterfly , and birds, mammals, fish, and non - target plants
. S oil degradation/persistence studies were also evaluated. Additionally , gene flow and weediness assessments via pollen and Cry protein DNA uptake b y plants and soil microorganisms previous ly

have been performed and are discussed extensively in the update to the Cry 1Ab BRAD (USEPA 2010a )
. This document also discusses field survey s of effects on nontarget insects and effects on nontarget lepidopterans that were conducted for several Bt Cry p rotein registrations in corn. Based on this information, E PA concluded that risk to non

- target wildlife, aquatic, and soil organisms is not expected from Cry 1Ab expressed in Event Bt11 corn. At present, the Agency

is aware of no identified significant adverse effects of Cry 1Ab on the abundance of non

- target organisms in any population in the aquatic or terrestrial field environment. In addition, no direct or indirect effects on Federall y listed endangered and threatened species or effects on their critica l habitat are expected . Further, the EPA has determined that there is no significant risk of gene capture and expression of Cry 1Ab protein by wild or weedy

relatives of corn in the U.S., its possessions, or territories , available data do not indicate that Cry proteins have an y m easurable adverse effect on micr obial populations in the soil, nor has h orizontal transfer of genes from transgenic plants to soil bact eria been demonstrated. In conclusion, the risk assessment found no risk to the environment f rom cultivation of Event Bt11 corn expressing Cry 1Ab.
**b. Event TC1507 (lepidopteran active) Environmental Risk Assessment Summary**  Potential adverse effects to non

- target organisms by Cry 1F protein were reviewed and updated in the Cry 1F BRAD, in 2010 (USE PA 2010a). The following is a summary of the Cry 1F environmental risk assessment. EPA performed risk assessments on plants, wild mammals, birds, fish, aquatic invertebrates, earthworms, terrestrial non

- target insects (including honey bee, parasitic wasps, green lacewings, lad ybird beetle, springtails [Collembola toxicity /reproduction], and monarch butterflies), as well as field evaluations of the effects of Cry 1F exposure on non
- target insects in corn fields, soil degradation/persistence studies, and an en dangered species impact assessment, particularl y for Lepidoptera . In addition, gene flow and weediness assessments via pollen and Cry protein DNA uptake b y plants were also performed. Cry 1F protein in soil has been shown to degrade rapidl y to very low l e vels. EPA concluded that there is sufficient information to believe that there is no risk from the uses of Cry 1F corn to non
- target wildlife, aquatic, and soil organisms. At present, the Agency

is aware of no identified significant adverse effects of Cry1 F protein on the abundance of non

- target organisms in any population in the aquatic o r terrestrial field environment. Field testing and field census data submitted to the Agency show minimal to undetectable changes in the beneficial insect abundance or d iversity . To date the available field test data show that compared to crops treated with conventional chemical pesticides, the transgenic crops have no detrimental effect onBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

62 the abundance of non

- target invertebrate populations. Inaddition, no direct or i ndirect effects on Federall y listed endangered and threatened species or effects on their critical habitat are expected
. The EPA has reviewed the potential for gene capture and expression of Cry 1F protein by wild or weed y relatives of corn in the United St ates, its possessions or territories and has found that there is no significant risk in the United States, its possessions or territories (USEPA 2010a). In conclusion, the risk assessment found no risk to the environment from cultivation of Event TC1507 corn expressing Cry 1F protein.
**c. Event MIR604 (coleopteran active) Environmental Risk Assessment Summary**  A risk assessment was performed for modified Cry 3A (mCry 3A) protein expressed in Event MIR604 corn when it was originally registered, and the BRAD for this PIP was also updated in 2010 (USEPA 2010b). EPA examined data on birds (Northern bobwhite, broiler chicken), mammals (from laboratory studies submitted for toxicology review), freshwater fish (rainbow trout), lady beetle, carabid beetle, rove beetle, insidious flower bug, honey bee, and earthworm. Data were not required for estuarine/marine fish and invertebrates due to expected lack of exposure. Similarly , data were not required for freshwater invertebrates because mCry 3A was not detectable in MIR60 4 corn pollen, and exposure was expected to be minimal in these environments. A soil degradation study was also submitted. Based on these data, EPA performed an assessment of the risk of mCry 3A to nontarget organisms. Inaddition, gene flow and weediness assessments were performed when MIR604 was initially registered, which was extensively updated in 2010. Based on the data submitted, EPA concluded that adverse effects to nontarget organisms, including listed species, are not expected as a result of the cu ltivation of Event MIR604 corn plants expressing mCry 3A protein. Based on recommendations from a Science Advisory Panel meeting that was convened to comment on EPA’s risk assessment and proposed findings for the mCry 3A registration, EPA required a full

- sca le field study to examine long

- range effects of cultivation of corn expressing mCry3A on invertebrate communities as a condition of registration (USEPA 2010b). This study has been submitted and a review is pending. At present, the Agency

is aware of no id entified significant adverse effects of mCry 3A protein on the abundance of non

- target organisms in any population in the aquatic o r terrestrial field environment. In addition, no direct or indirect effects on Federall y l isted endangered and threatened spec ies or effects on their critical habitat are expected
. The EPA has reviewed the potential for gene capture and expression of mCry 3A protein by wild or weed y relatives of corn in the United States, its possessions or territories and has found that there is no significant risk in the United States, its possessions or territories ( USEPA 2010b). In conclusion, the risk assessment found no risk to the environment from cultivation of Event MIR604 corn expressing m Cry 3A protein. **d. Event 5307 (coleopteran active)**  **Environmental Risk Assessment Summary**Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

63 The full risk assessment for eCry 3.1Ab is provided above; a summary of this assessment is presented here. The eCry 3.1Ab protein expressed in Event 5307 corn is a new active ingredient that has not been registered. Ap plication for its registration has been made concurrentl y with that for this Bt11 x MI R604 x TC1507 x 5307 corn hybrid. S yn genta has submitted studies with Northern bobwhite, broiler chicken, mammals (from laboratory studies), channel catfish, a Gammarus s pecies (an aquatic shredder invertebrate), lad ybird beetle, rove beetle, carabid beetle, predatory bug, honey

bee, and earthworm to assess the effects to nontarget organisms. A soil persistence stud y was also submitted. Based on the data provided, BPPD found that no adverse effects to nontarget birds, mammals, fish, aquatic invertebrates, nontarget insects and honey bees, and nontarget plants were assessed are expected as a result of the cultivation of Event 5307 corn plants expressing eCry 3.1Ab. Additiona lly , no direct or indirect effects on Federall y listed endangered and threatened species or effects on their critical habitat are expected
. The potential for gene capture and expression of Cry proteins has been extensively examined by

EPA ( USEPA 2010a ). Th is analy sis is relevant to the eCry 3.1Ab protein as well, and it was determined that the risk of gene flow and development of weediness is low. In conclusion, the risk assessment found no risk to the environment from cultivation of Event 5307 corn expre ssing eCry 3.1Ab protein.
**e. Synergism Studies**  The purp ose of s yn ergism studies i s to characterize the potential for interaction between the Bt Cry proteins contained in combined trait corn event Bt11 x MIR604 x TC1507 x 5307 . T o bridge the ecological effect s and environmental fate data of the individual parental events to the combined PI P products, the combined trait PIP product must be demonstrated as biochemically and functionall y equivalent to their respective parental PI P events. The functional equivale nce is established b y demonstrating that the effects of the pesticidal mixture of the combined PIP product on a susceptible pest species are comparable to the effects of each protein tested individually. Interactions between the test materials can be asses sed by comparing the larval mortality observed for the mixed proteins with the predicted responses based on the bioassay of each protein individually , via diet

- incorporation sensitive insect bioassay s. If there is no greater mortality than expected over th e range of concentrations in a sensitive pest species , it is likely that there will be no sy

nergism of the mixture against non

- target organisms and the effect of a mixture of proteins on non

- target organisms can be predicted from the effects of the individ

ual proteins alone. The hy pothesis of no s yn ergism of the insecticidal proteins when combined in Bt11 x MIR604 x TC1507 x 5307 was tested in three stages: 1) a test for s ynergism among the lepidopteran

- active proteins (Cry 1Ab and Cry 1F), 2) a test for s yn ergism among the coleopteran

- active proteins (mCry 3A and eCry 3.1Ab), and 3) a test for s ynergism between the mixture Cry 1Ab + Cry 1F and the mixture mCry 3A + eCry 3.1Ab. **1. Bioassays for Interaction Between Cry1Ab and Cry1F** Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

64 Tests for s ynergism among Cry1Ab and Cry 1F were previously conducted in two bioassay s (MRID 48235204): one using European corn borer ( Ostrinia nubilalis , ECB), which is sensitive to Cry 1Ab and Cry 1F, and one using fall army worm ( Spodoptera frugiperda , FAW), which is sensitive to Cry 1F only
. Both bioassay s also included an additional component, Vip3Aa20, which is present in Event MIR162 corn, and is active against FAW but not ECB. Vip3Aa20 was included because interactions among Cry 1Ab, Vip3Aa20, and Cry 1F were also being investigated as par t of the development of a different stacked h ybrid corn. This study was previously reviewed and found to be acceptable b y EPA (USEPA 2011)
. In each bioassay , freshl y - hatched larvae were exposed to the proteins incorporated into commercial insect diet. Th e treatments consisted of various individual concentrations of microbially

- produced Cry 1Ab, Vip3Aa20, and Cry 1F, and of mixtures of microbially

- produced Cry 1Ab + Vip3Aa20 + Cry 1F. The protein concentrations used were chosen to give a range of predicted res ponses to the mixtures, based on preliminary

tests and historical data. The presence of sy nergism or antagonism among the proteins was assessed b y comparing the observed mortality of larvae on diets containing the protein mixtures with the predicted mortal ity

based on bioassay s using each protein individually . The predicted responses were calculated based on an assumption of independent action, using an extension of the Colby method (Colby , 1967) as described in the study report. Based on the results for ECB and FAW, there was no tendency for an excess or deficit in observed mortality in an y test at any time, indicating that there is no s ynergism or antagonism among Cry 1Ab, Vip3Aa20, and Cry 1F. **2. Bioassays for Interaction Between mCry3A and eCry3.1Ab**  Tests for interactions between coleopteran

- active mCry 3A and eCry 3.1Ab insecticidal proteins were conducted with similar bioassay s using the Colorado potato beetle ( Leptinosara decemlineata , CPB) (MRID 48442909). Microbially

- produced mCry 3A and eCry 3.1Ab protei n test substances were used in a series of concentrations chosen to give a range of predicted responses to the combined proteins based on preliminary

tests and historical data. The presence of sy nergism or antagonism among the proteins was assessed b y comp aring the observed mortality of larvae on diets containing the protein mixtures with the predicted mortalit y based on bioassays using each protein individually. As in the bioassays previously performed to assess the interaction of Cry 1Ab, Vip3Aa20, and Cry 1F (MRID 48235204, discussed above), the predicted responses were calculated based on an assumption of independent action using an extension of the Colby method (Colby 1967). This study

was classified as acceptable. The observed mortality

results were low er than expected in the combined protein mixture bioassay s in comparison to the predicted mortality calculations, which presumed an additive effect due to the independent action of mCry 3A and eCry 3.1Ab proteins. However, the results of the combined protein mixtures were comparable to the observed mortality for the proteins when tested individually against CPB, which corroborates the hy pothesis s ynergism is not expected between mCry3A and eCry3.1Ab insecticidal proteins when expressed in combination. The res ults also support the functional equivalence of the mCry 3A and eCry3.1Ab insecticidal proteins expressed in combined PIP Bt11 x MI R604 xBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

65 MIR162 x TC1507 x 5307 corn product with its respective parental corn events MIR604 and 5307 corn. Therefore, this comb ination of proteins is not likely to cause unexpected increased effects in nontarget organisms. **3. Bioassays for Interaction of Cry1Ab, Cry1F, mCry3A, and eCry3.1Ab**  Diet incorporation feeding assay s were conducted to determine whether a mixture of lepidopte ran - active proteins (Cry 1Ab and Cry 1F, which also included Vip3Aa20) and a mixture of coleopteran

- active proteins (mCry 3A and eCry 3.1Ab) interact when used in combination (MRI D 48442908). A study had been previousl y conducted to determine the dose

- response relationships of the lepidopteran

- active and coleopteran

- active proteins using ECB and CPB, and establish appropriate dosage levels for the interaction study

(MRID 48202317). Using these dosage levels, an interaction study was performed in which ECB and C PB were exposed to a mixture containing all of the lepidopteran

- and coleopteran

active proteins. The effect of the coleopteran

- active mixture on the insecticidal activity of the lepidopteran

- active mixture was determined using ECB assay s. Two doses of the lepidopteran

- active mixture (ECB 1 [lower dose] and ECB 2 [higher dose]) were used alone and in combination with the higher dose of the coleopteran

- active mixture (CPB 2). Mortalit y results for the lepidopteran

- active mixture alone were compared to those for the combined lepidopteran

- active and coleopteran

- active mixtures. The effect of the lepidopteran

- active mixture on the insecticidal activity of the coleopteran

active mixture was determined using CPB assay s. Two doses of the coleopteran

- active mixture (CPB 1 [lower dose] and CPB 2 [higher dose]) were used alone and in combination with the higher dose of the lepidopteran

- active mixture (ECB 2). Mortalit y results for the coleopteran

- active mixture alone were compared to those for the combined coleopteran

- active and lepidopteran

- active mixtures. For each interaction bioassay , F
- tests were used to assess the statistical significance of the effects of dose, inactive ingredient, and dose x inactive ingredient interaction at the 5% probabilit y level. In the interaction studies with ECB, the ECB 2 dose produced higher mortality than the ECB 1 dose, as expected. There were no statistically significant differences in ECB mortality from addition of the coleopteran

- active (ECB

- inactive) ingredient (CPB 2) and no statistically significant dose x ECB

- inactive ingredient interactions. Similarly , the CPB 2 dose produced higher mortality than the CPB 1 dose in CPB, as expected. There were no statisticall y significant differences in CPB mortality from addition of the lepid opteran - active (CPB

- inactive) ingredient (ECB 2) and no statistically significant dose x CPB

inactive ingredient interactions. The study reviewer noted that there was some increase in the activity in the CPB assay s that was related to the high ECB dose, but it is not statistically significant. Based on the results of the study , the combination of these insecticidal proteins does not indicate potential for s ynergistic activity in the two sensitive insect species tested. The results also support the functio nal equivalence of the combined PIP Bt11 x MIR604 x TC1507 x 5307 corn product with its respective parental corn events Bt11, MIR604, TC 1507 and 5307 corn, respectivel y. Therefore, the combination of these proteins is unlikely to produce unexpected effect s in nontarget organisms as a result of the cultivation of Bt11 x MIR604 x TC1507 x 5307 corn plants.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

66 **f. Protein Expression in**  **Bt11 x**  **MIR604 x**  **TC1507 x**  **5307 Corn Plants** The expression of Cry 1Ab, mCry 3A, Cry 1F, and eCry 3.1Ab individuall y in single

- trait Bt11 , MIR604, TC1507, and 5307 plants, respectivel y, was compared with the expression of each of these proteins in the combined trai t Bt11 x MIR604 x TC1507 x 5307 corn plants (MRID 48443001). This study is also discussed in the environmental risk assessment submitted by Sy ngenta for Bt11 x MIR604 x TC1507 x 5307 corn (MRID 48443004). The data show that several proteins are expressed at significantl y higher amounts in some tissues of the stacked h yb rid corn plants compared to the individual trait plants. In th e hy brid, Cry 1Ab is expressed at levels that are significantly higher in R1 leaves and in R1 whole plants, mCry 3A expression is significantly higher in pollen (also see discussion below), Cry 1F expression is significantl y higher in V5 leaves, and eCry3.1Ab is significantl y higher in V5 roots. EPA communicated concerns for these increased levels of expression in Bt11 x MIR604 x TC1507 x 5307 corn plants in a letter to Sy ngenta dated March 15, 2012, and asked S yn genta to provide further information to justif

y equivalence of protein expression. Sy ngenta provided a response (MRID 48802701), which discussed the relevance of these increased levels of expression to margins of exposure for nontarget organisms and overall exposure during the life of the plant. In the letter, EPA also raised concern over the presence of mCry 3A protein in pollen of the Bt11 x MIR604 x TC1507 x 5307 stack. In the original risk assessment for this protein, mCry 3A was not detectable in MIR604 corn pollen, and tests for effects of mCry 3 A in aquatic invertebrates were not required due to the expected lack of exposure. MRID 48802701 also contains Sy ngenta’s response for these concerns, which is based on a discussion of new detection methods that enhanced the sensitivity of detection assay s below the previous level of quantitation. Sy ngenta additionally

discussed the exposure of aquatic invertebrates, and concluded that the previous findings are still relevant to the risk assessment for Bt11 x MIR604 x TC1507 x 5307 corn. The comparative pr otein expression study for Bt11 x MIR604 x TC1507 x 5307 corn and S yn genta’s response to the Agency ’s concerns is currently under review, and conclusions of its biochemical equivalence to its respective parental events are to be provided in the product cha racterization memo for Bt11 x MIR604 x TC1507 x 5307 corn (USEPA 2012a). Provided that the expression study and supplemental information are found to be acceptable, a conclusion can be made that significant increased exposure of nontarget organisms to the Cry 1Ab, mCry 3A, Cry 1F, and eCry 3.1Ab proteins is not expected as a result of cultivation of Bt11 x MIR604 x TC1507 x 5307 corn plants. **g. Risk Assessment Conclusions for**  **Cry1Ab, mCry3A, Cry1F, and eCry3.1Ab as Expressed in**  **Bt11 x**  **MIR604 x**  **TC1507 x**  **5307 Corn** EPA has concluded that the individual proteins expressed in Bt11 x MIR604 x TC1507 x 5307 corn will have no adverse effect on nontarget organisms, do not persist in the soil, and pose no risk of gene flow and development of weediness in wild relatives. S ynergism studies submitted to EPA also show that increased toxicity

is not expected from the combination of these proteins. While protein expression data are currentl y under review, a significant increase in nontarget exposure is not anticipated. Therefore , a conclusion is made that cultivation of Bt11 x MIR604 x TC1507 x 5307 corn will not result in adverseBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

67 effects to nontarget organisms or the environment, and additionally will have no effect, direct or indirect, on Federally

listed threatened and endange red species or their designated Critical Habitats. This conclusion is dependent on a finding that the expression study for Bt11 x MIR604 x TC1507 x 5307 corn and its associated supplemental information are found to be acceptable and to support the conclusi on of no significant increase in exposure. **E**
. **Environmental Risk Assessment for Cry1Ab, Vip3Aa20,**  **mC ry3A,**  **Cry1F, and eCry3.1Ab**  **E xpressed Bt11**  **x MIR162 x**  **MIR604 x**  **TC1507 x**  **5307 Corn Hybrid**  **1 . Background**  Sy ngenta has appl i ed for a FIFRA Sect ion 3 Registration for a combined trait corn product Bt11 x MIR162 x MIR604 x TC1507 x 5307 (EPA File Symbol 67979

- EG) that confers lepidopteran and coleopteran insect resistance and herbicide tolerance (with the Event GA21 trait)
. The combined trait corn Bt11 x MIR162 x MIR604 x TC1507 x 5307 product was developed by crosses (via traditional breeding methods) with each of the i ndividual events
. The data to support this registration are based on data submitted and approved previousl y for the individua l pare ntal lines, as well as the data for the eCry 3.1Ab protein expressed in Event 5307, as discussed above. S ynergism studies involving these proteins were also submitted. These studies provide support for bridging to data for the individual PI P events that are alread y contained in the Agency’s database. **2 . Environmental Assessment**  The hazard of each of the individual events in Bt11 x MIR162 x MIR604 x TC1507 x 5307 has been addressed for previous registrations. Event 5307 is a new active ingredient, and the risk assessment performed for this active ingredient also applies to this stack. EPA has previously determined that the database for ecological risk assessment is complete for the Cry 1Ab, Vip3Aa20, Cry 1F, and mCry 3A proteins expressed in this hy brid, and also for eCry 3.1Ab based on the assessment above. Summaries of the risk assessments for the individual Cry 1Ab, mCry 3A, Cry 1F, and eCry3.1Ab are provided above in the risk assessment for the Bt11 x MIR604 x TC1507 x 5307 combined trait corn product. A summary of the environmental assessment of Vip3Aa20 is provided below. This assessment also addresses the potential for sy

nergism in the Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn h ybrid, as well as protein expression comparability between the combined trait h ybrid and corn plants expressing the individual traits only
. **a. Event MIR162 (lepidopteran active) Environmental Risk Assessment Summary**  Potential adverse effects to non

- target organisms by the Vip3Aa20 protein expressed by event MI R162 have been prev iousl y reviewed and was recentl y updated (USEPA 2009). The following is a summary of the MIR162 environmental risk assessment. The Vip3Aa20 protein expressed in MI R162 corn is a Bt
- derived vegetative insecticidal protein intended to control lepidopteran pests; however, it is not active against all primary lepidopteran cornBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

68 pests. This protein is known to act individually to affect a t ypical midgut pathology in susceptible insects like previously

studied Bt delta

- endotoxin s (Lee, et al. 2003). Although th e general sy mptoms display ed b y s ensitive lepidopteran larvae following ingestion of Vip protein s resembles that caused by Cry 1Ab proteins ( i.e., cessation of feeding, loss of gut peristalsis, overall paral ys is of the insect, and death ) (Yu , et al
. 1997 ; and L ee et al. 2008 ) , the Vip3Aa protein variants share no homology with Cry 1Ab and other known Cry proteins and the modes of action differ between the Vip and Cry proteins. Therefore, while event MIR162 is active against a wide spectrum of lepidopteran pes ts, it is intended for use in creating combinations with other PIPs instead of commercial distribution in single

trait seed . The potential hazard of Vip3Aa20 protein was evaluated for Northern bobwhite, broiler chicken, mammals (based on acute oral toxicol ogical laboratory studies on mice ), channel catfish, Daphnia magna , insidious flower bug, lad y beetle, green lacewing, rove beetle, collembolan, honey bee, and earthworm
. In support of the Event MIR162 registr ation, test substances used in the submitted studies included bacteri al

- produced purified Vip3Aa19 and Vip3Aa1 p rotein, plant

- expressed Vip3Aa20 protein as expressed in Event Pacha maize leaves, in addition to plant

- expressed Vip3Aa20 protein as expressed in Event MIR162 maize grain, pollen, and leaves
. Since biochemical and functional equivalence was shown among Vip3Aa1, V ip3Aa19 and Vip3Aa20 proteins, some of the studies conducted with Vip3Aa1 and Vip3Aa19 were bridged to the assessment for Vip3Aa20
. A soil degradation stud y was also submitted that a llowed EPA to conclude that Vip3Aa20 is not likely to persist in soil. Additionally , gene flow and weediness assessments were performed, and the potential for these events has also been discussed extensively for Cry proteins in corn in updated Bt corn BRAD s (e.g., see USEPA 2010). Based on this information, EPA concluded that risk to non

- target wildlife, aquatic, and soil organisms is not expected from Vip3Aa20 expressed in Event MI R162 corn. At present, the Agency

is aware of no identified significant a dverse effects of Vip3Aa20 on the abundance of non

- target organisms in any population in the aquatic or terrestrial field environment. In addition, no direct or indirect effects on Federall y l isted endangered and threatened species or effects on their crit ical habitat are expected. Further, the EPA has determined that there is no significant risk of gene capture and expression of Vip3Aa20 protein by wild or weed y relatives of corn in the U.S., its possessions, or territories, available data do not indicate that Cry proteins have an y measurable adverse effect on microbial populations in the soil, nor has horizontal transfer of genes from transgenic plants to soil bacteria been demonstrated. In conclusion, the risk assessment found no risk to the environm ent from cultivation of Event MIR162 corn expressing Vip3Aa20. **b. Synergism Studies**  The sy nergism studies discussed in the risk assessment of the Cry proteins expressed in the Bt11 x MIR604 x TC1507 x 5307 hybrid above (MRIDs 48442908 and 48442909 ) are al so applicable to the environmental risk assessment of Bt11 x MIR162 x MIR604 x TC1507 x 5307 hy brid corn because the Vip3Aa20 protein was also included in these studies. These studies showed no increase in toxicity to sensitive insect species as a result o f the combination of the Cry 1Ab, Vip3Aa20, mCry 3A, Cry 1F, and eCry 3.1Ab proteins. The results also support the functional equivalence of the combined PIP Bt11 xBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

69 MIR604 x MIR162 x TC1507 x 5307 corn product with its respective parental corn events Bt11, MIR604, MIR162, TC 1507 and 5307 corn, respectively
. Therefore, increased toxicity is not expected in nontarget organisms as a result of the combination of these proteins in Bt11 x MIR162 x MIR604 x TC1507 x 5307 hy

brid corn.
**c. Protein Expression in**  **Bt11 x**  **MI R162 x**  **MIR604 x**  **TC1507 x**  **5307 Corn Plants** The expression of Cry 1Ab, Vip3Aa20, mCry 3A, Cry 1F, and eCry 3.1Ab individually in single

- trait Bt11, MIR162, M IR 604, TC1507, and 5307 corn plants, respectivel y, was compared with the expression of each of these pro teins in the combined trai t Bt11 x MIR604 x TC1507 x 5307 corn plants (MRID 48442901). This study

is also discussed in the environmental risk assessment submitted by S yn genta for Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn (MRID 48442904). The data show that Cry 1Ab is expressed at levels that are significantl y higher in R1 leaves and in R1 whole plants. EPA communicated concerns for these increased levels of expression in Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn plants in a letter to Sy

ngenta dated Marc h 15, 2012, and asked S yn genta to provide further information to justify equivalence of protein expression between the single and combined trait plants. Sy ngenta provided a response (MRID 48802801), which discussed the relevance of these increased levels o f expression to margins of exposure for nontarget organisms and overall exposure during the life of the plant. In the letter, EPA also raised concern over the presence of mCry3A protein in pollen of the Bt11 x MIR162 x MIR604 x TC1507 x 5307 stack. As di scussed above, in the original risk assessment for this protein, mCry 3A was not detectable in MIR604 corn pollen, and tests for effects of mCry 3A in aquatic invertebrates were not required due to the expected lack of exposure. MRID 48802801 also contains S yn genta’s response for these concerns, which are identical to their justification and conclusion discussed above for Bt11 x MIR604 x TC1507 x 5307 corn. The comparative protein expression study for Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn and Sy ngenta’ s response to the Agency ’s concerns is currentl y under review, and conclusions of its biochemical equivalence to its respective parental events are to be provided in the product characterization memo for Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn (USEPA 2 012b). Provided that the expression study

and supplemental information are found to be acceptable, a conclusion can be made that significant increased exposure of nontarget organisms to the Cry1Ab, Vip3Aa20, mCry 3A, Cry 1F, and eCry 3.1Ab proteins is not exp ected as a result of cultivation of Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn plants. **d. Risk Assessment Conclusions for**  **Cry1Ab, Vip3Aa20, mCry3A, Cry1F, and eCry3.1Ab as Expressed in**  **Bt11 x**  **MIR162 x MIR604**  **x TC1507**  **x 5307 Corn**  EPA has concluded that the individual proteins expressed in Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn will have no adverse effect on nontarget organisms, do not persist in the soil, and pose no risk of gene flow and development of weediness in wild relatives. Sy nergism stu dies submitted to EPA also show that increased toxicity is not expected from the combination of these proteins. While data areBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

70 currentl y under review to show that these proteins are not expressed in Bt11 x MIR162 x MIR604 x TC1507 x 5307 hy

brid corn at lev els that significantly increase nontarget exposure above levels of concern, a significant increase in nontarget exposure is not anticipated. Therefore, a conclusion is made that cultivation of Bt11 x MIR604 x TC1507 x 5307 corn will not result in adverse effects to nontarget organisms or the environment, and additionally will have No Effect, direct or indirect, on Federally listed threatened and endangered species or their designated Critical Habitats. This conclusion is dependent on a finding that the exp ression study for Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn and its associated supplemental information are found to be acceptable and to support the conclusion of no significant increase in exposure. **IV. References**  Carstens, K.L., Anderson, J.A., B achman, P., DeShrijver, A., Divel y, G. Federici, B. Hamer, M., Gielkens, M., Jensen, P., Lamp, W., Raushen, S., Ridley , G., Romeis, J., Waggoner, A. 201 1
. Geneticall y m odified c rops and a quatic e cos ys tems: c onsiderations for e nvironmental risk a ssessment an d n on - target o rganism t esting. Transgenic Res., published online 26 November 2011. Colby , S.R. 1967. Calculating s ynergistic and antagonistic responses of herbicide combinations. Weeds 15:20
- 22.10. Crocker, D., Hart, A., Gurney , J., McCoy , C. 2002. Proj ect PN0908: Methods for estimating daily food intake of wild birds and mammals. Central Science Laboratory . Unpublished report to the (UK) Department for Environment, Food and Rural Affairs. Included as an appendix to US EPA MRID 46155615. Jensen, P.D., D ively , G.P ., Swan, C.M., L amp, W.O. 2010. Exposure and non

- target effects of transgenic Bt corn debris in streams. Environ. Entomol. 39(2):707

- 714. Lee, M.K., Walters, F.S. , H art, H., Palekar, N., Chen, J

- S. 2003 . The mode of action of the Bacillus thu ringiensis vegetative insecticidal protein Vip3A differs from that of Cry 1Ab δ

endotoxin. App. Environ. Micro. 69(8): 4648

- 4657. Marvier, M., McCreed y, C., Regetz, J. & Kareiva, P. 2007. A meta

- anal ys is of effects of Bt cotton and maize on nontarget invertebrates. Science 316: 1475

– 1477. National Academ y of Science (NAS). 2000. Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation is available from the National Academ y Press, 2101 Constitution Avenue, N.W., Lockbox 285, W ashington, DC 20055; (800) 624

- 6242 or (202) 334

- 3313 (in the Washington metropolitan area); http://www.nap.edu
. Romeis, J., Meissle, M., B igler , F . 2006. Transgenic Crops expressing BacillusBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

71 thuringiensis toxins and bi ological control. Nature Biotechnology 24: 63
- 71. Rosi

- Marshall E. J., Tank, J. L
. , Roy er, T. V. , Whiles, M. R., Evans
- White, M., Chambers, C., Griffiths, N. A. , Pokelsek, J. , Stephen , M.L
. 2007. Toxins in transgenic crop by products may

affect headwater stream ecos ys tems. PNAS : 104(41): 16204

– 16208. Sanvido, O., Romeis, J., Bigler, F. (2007). Ecological Impacts of Genetically Modified Crops: Ten Years of Field Research and Commercial Cultivation. Adv Biochem Engin/Biotechnol 107: 235

– 278. Saxena, D. and Stotzky , G. (2001) Bacillus thuringiensis ( Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil. Soil Biol. Biochem . 33: 1225

– 1230. Swan, C.M., Jensen, P.D. , Dively , G.P. , Lamp , W.O. . 2009. Processing of transgenic crop residues in stream ecos ys tems. J. App. Ecol. 46:1304
- 1313. United States Environmental Protection Agency (USEPA) (1998). “Guidelines for Ecological Risk Assessment.” EPA 630/R

- 95
- 002F. Wash ington, DC, USA. [Federal Register, May 14, 1998. 63(93): 26846

- 26924.] USEPA. 2000. SAP report No 99
- 06. Sets of scientific issues being considered b y the Environmental Protection Agency

regarding: Section I

- Characterization and Nontarget Organism Dat a Requirements for Protein Plant Pesticides. Dated February 4, 2000. Available at the EPA website: http://www.epa.gov/scipol y/ sap/1999/index.htm#december USEPA. 2001. SAP Report No. 2000
- 07. Sets of scientific issues being considered by the Environmental Protection Agency regarding: Bt plant

- pesticides risk and benefit assessments. Dated March 12, 2001. Web site: http://www.epa.gov/scipol y/ sap/2000/october/octoberfinal.pdf USEPA. 2002. SAP Report No. 2002
- 05. A set of scientific issues being considered b y the Environmental Protection Agency regarding: Corn rootworm plant

- incorporated protectant nontarget insect and insect resistance management issues. Dated November 6, 2002. http://www.epa.gov/scipoly /sap/2002/august/august2002final.pdf USEPA. 2004. SAP Report No.2004
- 05. Product characterization, human health risk, ecological risk, and insect resistan ce management for Bt cotton products. Dated August 19, 2004. http://www.epa.gov/scipoly /sap/meetings/2004/june/final1a.pdf USEPA, 2009. Biopesticides Registration Action Document

– Bacillus thuringiensis Vip3Aa20 Insecticidal Protein and the Genetic Material Necessary for Its Production ( via Elements ofBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

72 Vector pNOV1300) in Event MI R162 Maize (OECD Unique Identifier: SYN

- IR162
- 4), http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006599.pdf
. USEPA, 2010a. Biopesticides Registration Action Document

– Cry 1Ab and Cry 1F Bacillus thuringiensis (

Bt) Corn Plant

- Incorporated P rotectants, http://www.epa.gov/oppbppd1/biopesticides/pips/cry 1f

- cry 1ab

- brad.pdf

. USEPA, 2010 b . Biopesticides Registration Action Document

– Modified Cry 3A Protein and the Genetic Material Necessary

for its Production (Via Elements of PZM26) in Event MI R604 Corn SYN
IR604
- 8 , http://www.epa.gov/oppbppd1/biopesticides/pips/mcry 3a
- brad.pdf
. USEPA, 20 10 c . Biopesticides Registration Action Document

– Bacillus thuringiensis Cry 34Ab1 and Cry 35Ab1 Proteins and the Genetic Material Necessary for Their Production (PHP17662 T

DNA) in Event DAS

- 59122
- 7 Corn (OECD Unique Identifier: DAS

- 59122
- 7) , http://www.epa.gov/oppbppd1/biopesticides/pips/cry 3435ab1

- brad.pdf
. USEPA, 2011. Review of Molecular C haracterization data, Test S ubstance C haracterizations , Protein Expression Analy ses and S ynergis m Study for Combination PIP Bt11 x MIR162 x TC1507 corn, Agrisure TM 3100 [EPA Reg. No. 67979

- RL , OECD Unique ID. SYN

- BTØ11
- 1 x SYN
IR162
- 4 x DAS
- Ø15Ø7
- 1] in support for Sec. 3 Registration, submitted by Sy ngenta Seeds, Inc.

– Field Crops

- NAFTA
. Memorandum from A. Waggoner, through J. Kough, to J. Kausch. Dated March 25, 2011. US Environmental Protection Agency , Washington, D.C. USEPA, 2012a. Review of Molecular Characterization Data, T est Substance Characterizations and Protein Expression Ana ly ses for Combination PIP Bt 11 x MIR604, TC1507 x 5307 corn [EPA Reg. No. 67979
- EU; OECD Unique ID. SYN

- BTØ11
- 1 x SYN
- IR
- 604
- 5 x DAS
- Ø15Ø7
- 1 x SYN
- 05037
- 1] in support for FIFRA Section 3 Registration, submitted by Sy ngenta S eeds, Inc.

– Field Crops

- NAFTA . Memorandum from J. Facey , through J. Kough, to M. Mendelsohn. US Environmental Protection Agency , Washington, D.C. USEPA, 2012b. Review of Molecular Characterization D ata, Test S ubstance C haracterizations , Protein Expression Analy ses and S ynergis m Study for Combination PIP Bt11 x MIR162 x MIR604 x TC1507 x 5307 corn [EPA Reg. No. 67979
- EG , OECD Unique ID. SYN

- BTØ11
- 1 x SYN
IR162
- 4 x SYN
- IR
- 604
- 5 x DAS
- Ø15Ø7
- 1 x SYN
- 05307
- 1 ] in support for Sec. 3 Registration, submitted by

S yn genta Seeds, Inc.

– Field Cr ops - NAFTA. Memorandum from A. Balen, through J. Kough, to M. Mendelsohn. US Environmental Protection Agency, Washington, D.C. Wolt, J.D. and Peterson, R.K.D. 2010. Prospective formulation of environmental risk assessments: Probabilistic screening for Cry1 A(b) maize risk to aquatic insects. Ecotoxicol. Environ. Sa fety 73: 1182
- 1188.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

73 Yu, C. Mullins, M.A., Warren, G.W., Koziel, M.G. and Estruch, J.J. (1997). The Bacillus thuringiensis Vegetative Insecticidal Protein Vip3A Lyses Midgut Epithelium Cells of Susceptible Insects. Applied and Environmental Microbiology 63 (2): 532

- 53.
**F .**  **Insect Resistance Management (IRM) Background**  BPPD j has review ed the Insect Resistance Management (IRM) plan and supporting data submitted by Sy ngent a for two proposed commercial Bt corn products containing the new active ingredient eCry 3.1Ab (event 5307). The two products ( EPA Reg No. 67979
- EU and 67979

- EG) are “stacked py ramids” consisting of eCry 3.1Ab and previously

- registered Bt corn toxins for co ntrol of lepidopteran stalk borers and corn rootworm. Separate IRM volumes were submitted for each proposed product: Bt11 x MIR604 x TC1507 x 5307 (MRID# 484430
- 08) and Bt11 x MIR162 x MIR604 x TC1507 x 5307 (MRID# 484429
- 10). **Conclusions and Recommen dations**  1) Sy ngenta has submitted adequate data and modeling to demonstrate that the 5307 py ramided products ( Bt11 x MIR604 x TC1507 x 5307 and Bt11 x MIR162 x MIR604 x TC1507 x 5307) will have high durability with a 5% refuge in the Corn Belt (relative t o single toxin products deploy ed with 20% refuge). There should be strong durability for both of the target pest complexes: lepidopteran stalk/ear borers (European corn borer, Southwestern corn borer, and corn earworm) and corn rootworm. 2) For southern regions in which cotton is also grown, Sy ngenta proposed a 20% refuge for Bt11 x MIR162 x MIR604 x TC1507 x 5307 (67979

- EG) but a 50% refuge for Bt11 x MI R604 x TC1507 x 5307 (67979

- EU), presumably because the latter product has one fewer lepidopteran

- act ive toxin. Larger refuge sizes in this region are driven b y concerns with corn earworm (a corn and cotton pest). BPPD notes that a 20% refuge is compatible with the requirements for other similar Bt corn p yr amids (including two S yn genta products). Sy ng enta may be able to support a 20% refuge for the Bt11 x MIR604 x TC1507 x 5307 py ramid with a relatively

simple model (as was done to support the other registered p yr amids). 3) Sy ngenta did not propose a seed blend with initial product registration for ei ther of the two 5307 py ramids. BPPD notes that additional information and modeling may be necessary

to support such a refuge strategy
. 4) Sy ngenta provided data to demonstrate that the risk of cross resistance between eCry 3.1Ab and mCry 3A (S yn genta’s oth er registered rootworm toxin) should be low. The compan y did not, however, provide an assessment of the cross resistance potential between eCry 3.1Ab and other rootworm toxins
j The use of BPPD in this c hapter refers to the BPPD IRM Team consisting of Alan Reynolds and Jeannette MartinezBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

74 not registered b y S yn genta (i.e., Cry 3Bb1 and Cry34/35Ab1). BPPD recognizes that eCry 3.1Ab (an engineered toxin) should have significant structural differences with Cry 3Bb1 and Cry 34/35Ab1, and therefore, a low likelihood of cross resistance. But no information was provided in the submission to verify this assumption. Accordingl y, BPPD recommends that Sy ngenta provide additional data or anal ys is to address the cross resistance potential of eCry 3.1Ab with Cry 3Bb1 and Cry 35/35Ab1. As part of this effort, BPPD also recommends that S yngenta work to develop a eCry3.1Ab

- resistant colony (similar to the work done for mCry 3A
-- see Meihls et al. 2011). 5) Existing programs for resistance monitoring (lepidopteran pests), remedial action, and compliance assurance should be compatible with the 5307 py ramids. Additional data and information, however, are recommended for corn rootworm resistance monitoring (see #6 below). BPPD recommends that Sy ngenta update and submit grower education materials (e.g., technology use guides) to include the 5307 product line and the appropriate refuge requirem ents. In addition, Syngenta should submit annual IRM reports for resistance monitoring, compliance, and sales. 6) BPPD recommends that Sy ngenta submit the following additional data for corn rootworm resistance monitoring with the eCry3.1Ab toxin:  Baselin e susceptibility data;  A proposal for a plant

- based diagnostic assay including an investigation into the feasibility of using the “Sublethal Seedling Assay” (Nowatzki et al. 2008);  A monitoring plan for northern corn rootworm ( Diabrotica barberi );  Corn roo tworm damage guidelines (for unexpected pest damage investigations of eCry 3.1Ab corn). **Background** Sy ngenta Seed, Inc. has proposed to register three Bt corn products containing the new active ingredient event 5307, which expresses the eCry 3A.1Ab toxin. Applications were submitted for registration of Event 5307 alone as a breeding intermediate and in two stacked/py ramided end products (referred to as the “5307” p yr amids in this review). The proposed products are as follows:

 5307 (breeding intermediate) ; EPA Reg. No. 67979
- EE  Bt11 x MIR604 x TC1507 x 5307; EPA Reg. No. 67979
- EU  Bt11 x MIR162 x MIR604 x TC1507 x 5307; EPA Reg. No. 67979
- EG The eCry 3A.1Ab toxin in event 5307 was engineered from “variable regions” of mCry 3A and Cry 1Ab (Walters et al. 2010) . It is active against the corn rootworm (CRW) complex, including western corn rootworm (WCRW, Diabrotica virgifera virgifera ) and northern corn rootworm (NCRW, Diabrotica barberi ). MIR604 (mCry 3A) is also targeted against CRW and has been previously reg istered in a number of stacked and pyramided products (see BRAD

-- BPPD 2006).Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

75 Bt11 (Cry 1Ab) and TC1507 (Cry 1F) x TC1507 are effective against European corn borer (ECB, Ostrinia nubilalis ), the major lepidopteran target pest in the Corn Belt, as well as c orn earworm (CEW, Helicoverpa zea ), southwestern corn borer (SWCB, Diatraea grandiosella ), and fall arm ywo rm (FAW, Spodoptera frugiperda ). MIR162 (Vip3Aa20) is also targeted against the lepidopteran pests CEW, SWCB, and FAW but does not provide efficacy a gainst ECB. All three of the lepidopteran toxins have been previousl y registered and deplo yed in stacked/py ramided products (BRADs: BPPD 2001 and 2009a). Sy

ngenta has indicated that a number of other secondary lepidopteran pests will be targeted includ ing black cutworm ( Agrotis ipsilon ), dingy cutworm ( Feltia jaculifera ), lesser corn stalk borer ( Elasmopalpus lignosellus ), southern corn stalk borer ( Diatrea crambidoides ), common stalk borer ( Papaipema nebris ), sugarcane borer ( Diatrea saccharalis ), and western bean cutworm (WBCW, Richia albicosta ). Because 5307 (EPA Reg. No. 67979

- EE) is intended for breeding purposes only (and not for commercial corn production), a specific IRM plan is not required for the product (and it is not addressed further in this review). Although no refuge is ty pically planted with breeding corn, such registrations have been limited in terms of overall acreage (at county and national levels) to mitigate potential resistance. **Summary of Syngenta’s IRM Submission s**  **for 5307 Py**  **ramided Products** Two separate IRM plans were submitted for the stacked/p yr amided registrations containing the new active ingredient 5307, though much of the same data was shared between the two applications. Each of these listed studies listed is revie wed in this memorandum and the assessment of the shared data is applicable to both products. MRID# 484429
- 10: Insect Resistance Management Plan for Bt11 x MIR162 x MIR604 x

TC1507 x 5307 Maize MRID# 484430
- 08: Insect Resistance Management Plan for Bt11 x MIR604 x 5307 x TC1507

Maize The major components of Sy ngenta’s IRM plan included dose determinations, investigation of cross resistance, and simulation modeling. These sections and BPPD’s review of the overall IRM plan are described below. **1 . Dose Con**  **siderations** Lepidopteran Toxins Because all of the lepidopteran

- active toxins in the two proposed products have been previousl y registered, S yn genta did not conduct new dose studies for ECB, SWCB, or CEW. Rather, data (MRID# 480506
- 06) developed for a s eparate p yr amided product containing Cry 1Ab, Cry 1F, and Vip3Aa20 were cited. In conducting these studies, S yn genta employ ed the methodology prescribed by

the 1998Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

76 Science Advisory

Panel (SAP 1998), using techniques “1” and “4” for each of the insects. Sp ecifically , the two SAP procedures are as follows: 1) Serial dilution bioassay with artificial diet containing l yophilized tissues of Bt plants using tissues from non

- Bt plants as controls; 4 ) Survey

large numbers of commercial plants (using a controll ed infestation with a susceptible laboratory

strain ) in the field to make sure that the cultivar is at the LD 99.9 or higher to assure that 95% of heteroz ygotes wou ld be killed. Sy ngenta reported the results of these tests in their IRM submission for the r egistration of Bt11 x MIR162 x TC1507 corn (EPA Reg. No. 67979

- 15). Based on those data, Sy ngenta concluded that Bt11 x MIR162 x TC1507 corn has high dose expression for ECB and SWCB. This conclusion was supported by

the data obtained from the Bt11 x MIR 162 x TC1507 py ramid and also from the single toxin components (Bt11 and TC1507) which showed similar high dose activity . M IR 162 has no activity against ECB, but showed high activity for SWCB in one test (technique “4”) but not the other (technique “1”). Against CEW, Bt11 x MIR162 x TC1507 expresses an “effective” high dose as a py ramid, though the three single toxin events were shown to have less than high dose expression by themselves. Effective high dose indicates that the combined activity of the tox in in the pyramid produces complete mortality (though the single toxin constituents may allow some survival). In the current submission, Sy ngenta concluded that these Cry 1Ab and Cry 1F dose results should also be valid for Bt11 x MI R162 x MIR604 x TC1507 x 5307 and Bt11 x MIR604 x 5307 x TC1507 since field expression data show comparable toxin levels between the p yr amided and constituent single toxin products. The lepidopteran dose data were fully described in MRID# 480506
- 06 (reviewed in BPPD 2011). BPP D Review

- Lepidopteran Dose Considerations Data for lepidopteran dose expression were previously reviewed for the registration of Bt11 x MIR162 x TC1507 corn (see review in BPPD 2011). An excerpt of the main conclusions in BPPD’s review is as follows: BPPD generally

agrees with Sy

ngenta’s characterization of the dose profile for Bt11 x MIR162 x TC1507 corn
-- i.e., high dose activity against the major target pests (with CEW considered an “effective” high dose). Dose testing was not reported for FAW in this submission for Cry 1Ab or Cry 1F. The results obtained for the other toxin combinations in these studies, including the single trait (Bt11, MIR162, TC1507) and dual toxin (Bt11 x MI R162, Bt11 x TC1507) constituent products, support previously

- determine d dose conclusions for MI R162 (summarized in BPPD 2009a). BPPD questions, however, whether the sample sizes tested in SAP method #1 were adequate for a definitive “high dose” conclusion. Sy ngenta tested only

20
- 24 insects per treatment (withBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

77 100% mort ality

on Bt). It is unclear if these small scale tests are sufficient to conclude that each toxin will produce 99.99% mortality to the larger ECB and SWCB populations. BPPD notes that no new dose data were developed specificall y for the either of the “ 5307” p yr amided products. Sy

ngenta’s dose conclusions for these products are largel y supported by a reference to field expression data showing comparable levels of protein in the py ramided products to the single toxin events. These data were submitted as part of the “5307” applications, but were not summarized in the IRM volume. Field efficacy trials (another means to verify dose performance in p yramided/stacked products) against the major target pests were also not submitted or referenced in the report. However, provided that the protein expression levels are comparable (as described by S yn genta), BPPD agrees with the dose characterization of the “5307” p yr amided products as having a high dose for ECB and SWCB and an “effective” high dose for CEW. Roo tworm Toxins (

MRID# 484429

- 10, Appendix A) Dose studies for the CRW

- active toxins eCry3.1Ab and mCry 3A were conducted by Dr. Bruce Hibbard (USDA

- ARS, Columbia, MO). CRW larval mortality to single toxin and py

ramided events was assessed from artificiall y - infested plots in Central Missouri over a three y ear period. Selection intensity (survival relative to the control) was calculated to provide an indication of the effectiveness (dose) of each toxin and the py

ramid. Within each test field (1

- 3 locations w ith 3

- 5 replicates were tested per ye ar) plots were established of eCry 3A.1Ab (designated event SYN101 in the report), mCry 3A (MIR604), SYN101 x MI R604, and a non - Bt isoline. During the seasons prior to testing, soy beans were planted to reduce the possibi lity of wild CRW infesting the plots. Insecticides were not used in the test fields (including seed treatments) to avoid confounding factors, though the fields were otherwise managed with corn agronomic practices ty pical of the region. In the Bt test plo ts, each plant was verified for Bt expression using gene strip tests. Buffers (vegetation

- free areas) were established outside each test plot to prevent CRW larval movement between plots. Prior to testing, plots were thinned to 16 plants per row (3.05 m row length). WCRW eggs (obtained from wild populations) were artificiall y infested in each plot when the corn plants reached the V2

- V3 growth stage. The infestation rate was 1,380 eggs per 30.5 cm (row foot) for 2007
- 2008 tests, but was reduced to 875 e ggs/foot in 2009 testing to reduce potential density dependent mortality in the control plots (based on work b y Hibbard et al. 2010). Test plots were fitted with screen tents to collect emerging adult CRW approximately 5

- 6 weeks after infestation. After initial beetle emergence, all but four plants in each plot were cut off at 1 m and stripped of leaves (presumabl y for easier beetle collection). Beetles were collected 2

- 3 times per week for the duration of the study
. Survival of WCRW was assessed in ter ms of “selection intensity ,” or the percentage of beetles emerging from the Bt treatments relative to the non

- Bt control plots. In addition to WCRW, any northern corn rootworm (NCRW) and southern corn rootworm (SCRW) beetles (presumably from wild populat ions) were also collected from the field tents.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

78 The results from the trials showed that both the single toxin (SYN101 and MIR604) and py ramided (MIR604 x SYN101) treatments significantl y reduced WCRW and SCRW populations relative to control plots. NCRW b eetle emergence was also reduced b y the Bt treatments, though in most of the treatments sites no beetles were collected in the non

- Bt tents. The mCry 3A and eCry 3.1Ab toxins appeared to have a greater impact on WCRW emergence than SCRW. In terms of select ion intensity , single trait SYN101 reduced beetle survival b y 99.79%, while p yramided MIR604 x SYN101 lowered emergence b y 99.91% over the five test locations/years. MIR604 alone resulted in an average selection intensity of 97.84%. There appeared to be little difference in beetle counts or selection intensity between males and females. The study authors did not report selection intensity for SCRW (though the average beetle counts were provided), but noted selection intensities for NCRW of 97.13% (MIR604 ), 98.85% (SYN101) and 100% (MIR604 x SYN101). Based on the results, Sy ngenta concluded that the mCry 3A x eCry 3.1Ab pyramid expresses a “highly effective dose” for WCRW and NCRW. Data from the dose investigations are summarized in Table 1 below. To dete rmine whether each toxin was acting independently , the researchers conducted a statistical anal ys is of beetle emergence. Had the two toxins acted independentl y, 0.0982 beetles would have been expected to emerge from the MIR604 x SYN101 plots (based on the survival results from the single toxin plots). In the experiments, 0.8 beetles emerged (averaged across all five locations), a statistically higher amount than the 0.0982 expected.
**Table 7**  **.** Mortality

(Selection Intensit y) of CRW to eCry3.1Ab and mCry 3A in Single Toxin and Py ramided Toxin Deployments (compiled from data provided in Tables 3 and 4 of MRI D# 484429
- 10, Appendix A)

**Treatment SCRW**  **NCRW 1**  **WCRW**

Beetle Ct. 2 (avg.) Sel. Int.

(%) Beetle Ct. 2

(avg.) Sel. Int.

(%) Beetle Ct. 2

(avg.) Sel. Int.

(%) MIR604 13.1 b NR 0.2 b 97.13 19.0 b 97.84 SYN101 13.6 b NR 0.1 b 98.85 1.9 c 99.79 MIR604 x SYN101 10.6 b NR 0.0 b 100 1.9 c 99.91 Isoline 36.8 a NR 7.9 a 0 879.0 a 0 1 NCRW beetles were collected at only tw o of the five test sites. 2 Total beetle counts for both sexes are reported. For each treatment , m ean values with separate letters are statistically different by

Fisher’s Least Square Difference test. BPPD Review

- Corn Rootworm Dose Considerations Sy ngenta’s dose studies for CRW follow the me thodology employ ed for other rootworm

- active PIP toxins including Cry 34/35, mCry 3A, and Cry 3Bb1 (see methodology and data published in Storer et al.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

79 2006; Hibbard et al. 2010a, b; and data summarized in BPPD 2009b). BPPD agrees with S yn genta that the events containing eCry 3.1Ab have high activity against WCRW, although it is unlikely that the toxin is expressed at a “high dose” level (as defined b y the 1998 SAP). None of the other registered CRW toxins (Cry34/35, mCry 3A, and Cry 3Bb1) is known to express a true high dose for CRW, even in py ramided toxin deploy

ments. Still, eCry 3.1Ab greatl y reduced WCRW survival (>99%) and may be among the most efficacious toxins for rootworm registered to date. The study authors compared the eCry 3.1Ab results with data f rom other toxins and noted that selection intensity for Cry 34/35 was 96.47% (Storer et al. 2006) and 94.88% for mCry 3A (Hibbard et al. 2010b). Data analyzed for a separate product (SmartStax) showed selection intensit y ranges of 96.17
- 99.96 % for Cry 3Bb1 , 94.20
- 99.18 % for Cry 34/35 , and 98.22

- 99.97 % for Cry 3Bb1 x Cry34/35 py ramid (reviewed in BPPD 2009b )
. Fewer conclusions can be drawn for either NCRW or SCRW. Although the researchers reported high reductions in NCRW emergence (see Table 1), the data were based on only two locations (no beetles were collected in the other locations). No artificial infestations were performed, so it is assumed that the NCRW observed in the two locations were from wild populations. Even in the plots where NCRW were fo und, overall beetle numbers were low (7.9 avg. beetles/non

- Bt field). Similarly , SCRW collections presumabl y arose from feral populations, though beetles were fairl y numerous (36.8 avg. beetles/non

- Bt field). Although not reported, the selection intensit y appeared to be lower with SCRW than with either WCRW or NCRW (e.g., 71.2% for the p yr amid). However, because infestation was not controlled, any conclusions regarding the efficacy of eCry 3.1Ab against either NCRW or SCRW are limited. **2 . Cross Resista**  **nce** Lepidopteran Toxins Sy ngenta’s conclusions regarding potential cross resistance between Cry 1F, Cry 1Ab, and Vip3A were based upon data and rationales that were previously submitted for Bt11 x MIR162 x TC1507 (EPA Reg. No. 67979
- 15), a lepidopteran

- act ive p yr amid containing the same toxins as the proposed products. These cross resistance data were assessed in BPPD’s review of the Bt11 x MIR162 x TC1507 submission ( MRID# 480506
- 07 and

- 08; reviewed in BPPD 2011). Previously , the cross resistance potent ial for Vip3A was assessed for the initial registration of MIR162 (see discussion in BPPD 2009). For ECB, S yn genta cited a number of published studies to support the lack of cross resistance between Cry 1Ab and Cry 1F. Vip3A (MI R162) is not active against ECB and was therefore not considered in the cross resistance assessment. Sy ngenta noted the following points for ECB:  Resistance is most likely to result from a decrease in Bt (Cry) toxin binding with epithelial cells in the insect midgut. Other mechanis ms (e.g. metabolic adaptations) would likely be too weak to overcome the high levels of mortality causes by

PIP toxins in the field.  Midgut binding assay s have shown that Cry 1Ab and Cry 1F do not strongl y compete for the same binding sites in ECB (Hua et al . 2001).Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

80  Studies with Cry 1Ab and Cry 1F
- resistant ECB colonies did not show high levels of cross resistance to the reciprocal toxin (Pereira et al. 2008; Siqueira et al. 2004) Fewer data are available for CEW and the three toxins. Sy ngenta cited work with Cry 1F and Cry 1Ac (developed for Dow AgroScience’s WideStrike cotton product, see Storer 2002), based on the rationale that Cry 1Ab and Cry 1Ac share binding sites and likely would have similar patterns of cross resistance. Work with resistant colonies has been limited since stable field

- relevant resistance has not been established. However, Sy ngenta noted that a Cry1Ab

- resistant laboratory CEW strain was still susceptible to Vip3A (Konasale et al. 2008). [BPPD note: After review, BPPD believes Sy ngenta i ntended to reference Anilkumar et al. 2008, a study in which Cry 1Ac
- resistant CEW were tested.] Sy ngenta referenced previously

- submitted resistant colony studies for FAW (MRID# 480506
- 07 ; reviewed in BPPD 2011 ) . The compan y concluded that these data show ed incomplete cross resistance between Cry 1F and Cry 1Ab, but no cross resistance with Cry 1F and Vip3A. Overall, S yn genta concluded that: 1) Based on midgut binding characterizations and studies with resistant colonies, Cry 1F likely has “little or no pote ntial cross resistance” with Vip3A in CEW or FAW; 2) Similarly , there is low potential for “field relevant” cross resistance between Cry 1F and Cry 1Ab in ECB or FAW; 3) More limited information on CEW (midgut binding studies with Cry 1F and Cry 1Ac) suggests there is “potential for moderate levels of cross resistance between Cry 1F and Cry 1Ab.....however, this risk is highly mitigated by

the presence of Vip3A.”
BPPD Review

- Lepidopteran Cross Resistanc e Potential BPPD generally

agrees with the overall concl usion that there is low potential for cross resistance between the toxins among the lepidopteran pests, despite the cursory information provided by S yn genta in its previous submission ( MRID# 480506
- 07 and

- 08; see review in BPPD 2011). No new data were pr ovided with the two 5307 py ramid applications. Cry 1F and Cry 1Ab may

have some risk for cross resistance. Both are part of the Cry 1 famil y of Bt toxins that share some structural similarities and several studies have found cross resistance in various in sects between different toxins in the group (e.g., Denholf et al. 1993; Granero et al. 1993; Gould et al. 1995, Bolin et al. 1999; others). Nevertheless, the weight of evidence from midgut receptor binding characterizations and resistant colony work sugge sts that among the major corn pests (ECB and FAW), the potential for cross resistance between Cry 1F and Cry Ab1 should be low. There are other factors that may reduce the probability

for cross resistance among the targeted pests. As discussed in BPPD (201 1), recent research (Pereira et al. 2010) suggests that resistance to Cry 1F may be the result of a unique process different from identified resistance mechanisms in other Bt cry toxins. Also, an anal ys is of separate data b y BPPD (2010a) concluded that the re is low cross resistance potential between Cry 1Ab and Cry 1F for the major target pests of Bt corn.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

81 Fewer data and/or information regarding CEW were available or cited b y S yn genta. Previous binding work (Storer 2002) was conducted with Cry 1F and Cry 1Ac (data developed by Dow AgroSciences); Sy ngenta believes that Cry 1Ac and Cry 1Ab can be expected to have similar binding patterns. BPPD cannot confirm this h ypothesis with the information cited by S yn genta but notes that a separate analysis (BPPD 2010b) con cluded that it is likely that Cry1Ab and Cry 1F will have a low cross resistance potential in CEW. The presence of Vip3A in one of the proposed 5307 py ramids (Bt11 x MIR162 x MI R604 x TC1507 x 5307) will likely

help mitigate the potential risk of Cry 1F
- C ry 1Ab cross resistance for CEW and FAW. For example, a Cry 1Ac
- resistant CEW colony was tested and found to be cross resistant to Cry 1Ab, but not Vip3A (Anilkumar et al. 2008). Similarly , Cry 1F
- resistant FAW were even more susceptible to Vip3A than an uns elected colony (MRID# 480506
- 07 ; reviewed in BPPD 2011 )
. On the other hand, ECB may have a greater concern for cross resistance in a Bt11 x MI R162 x TC1507 lepidopteran py ramid because Vip3A is not active against the insect. Cry 1Ab and Cry 1F, therefore, must maintain separate pathway s of toxicity to realize the benefits of the py ramid. Sy ngenta has not addressed cross resistance potential with SWCB in this or previous submissions. It is commonly

assumed that SWCB behaves similar to ECB and that conclusi ons made for ECB can be applied to this insect as well. However, the 2010 SAP noted key differences between the insects, particularl y in response to Bt toxins (SAP 2011). To illustrate, Vip3A has at least some activity against SWCB while having no activi ty against ECB (BPPD 2009a). Therefore, it is unclear to BPPD whether the assumption that SWCB will also be at low risk for cross resistance with Cry 1Ab, Cry 1F, and/or Vip3A will have validity

for a lepidopteran py

ramid containing the toxins
. While the l ikelihood of cross resistance may be low in terms of modified midgut receptors, it is possible that other mechanisms of cross resistance (e.g., behavioral modifications or expression of cry

- proteases) could arise with p yramided Bt corn. The 2010 SAP concl uded that the prediction of low cross resistance potential between Cry 1F and the Cry 1A toxins for ECB and SWCB remains uncertain and outlined a number of epistatic mechanisms that could lead to increased survival on p yr amided Bt corn (SAP 2011). For thes e reasons, cross resistance should be considered in predictive modeling to evaluate the durability of a Cry 1F/Cry 1Ab/Vip3A py ramid (particularl y for worst case scenarios). Corn Rootworm Toxins Sy ngenta tested the cross resistance potential between eCry 3 .1Ab and mCry 3A by investigating midgut binding patterns in WCRW. Potential interactions of the toxins with WCRW binding sites were investigated in qualitative and quantitative competitive binding assay s. The qualitative binding studies were published in Walters et al. (2010). Brush border membrane vesicles preparations were made from first instar WCRW larvae and exposed to activated (ch ym otry

psin

- processed) mCry 3A or eCry 3.1Ab. Heterologous and homologous competition experiments were performed for each of the toxins. The resulting western blots showed that both unlabeled mCry 3A and eCry 3.1Ab competed with their respective labeled homologues. On the otherBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

82 hand, in the heterologous competitions unlabeled eCry 3.1Ab did not inhibit labeled mCry 3A binding , while unlabeled mCry 3A failed to diminish the binding of labeled eCry 3.1Ab. Based on these competition studies, the study authors concluded that mCry 3A and eCry 3.1Ab recognize different binding sites in WCRW. This novel activity is likely due to the ex change of Cry 1Ab and Cry 3A variable regions resulted in the engineered toxin (eCry 3.1Ab). A second set of experiments was conducted to quantify the level of competition among mCry 3A and eCry 3.1Ab for binding sites in WCRW. The studies were performed by D rs. Juan Jurat
- Fuentes and Siva Rama Krishna Jakka of the University of Tennessee (Appendix C of MRID# 484429
- 10). Both toxins were chy

motry psin

- activated and radiolabeled with I 125 for detection by photographic plates. In initial testing, both toxins we re shown to specifically bind to the BBMV preparations, which suggested limited binding sites in WCRW. The level of specific binding was higher for eCry 3.1Ab (70

- 80%) than for mCry 3A (50%). Three separate binding competition assay s were conducted: 1) la beled mCry 3A with unlabeled eCry 3.1Ab and mCry 3A; 2) labeled eCry 3.1Ab with unlabeled mCry 3A and eCry 3.1Ab, and 3) labeled mCry 3A with unlabeled mCry 3A (non

- activated) and eCry3.1Ab (activated). Results from the assay s showed little or no competition between mCry 3A and eCry 3.1Ab for BBMV binding sites. In the first competition, labeled mCry 3A binding was reduced slightly (to ~ 80%) when exposed to the higher concentrations of unlabeled eCry 3.1Ab. Homologous competition with unlabeled mCry 3A resulted in a significantly greater reduction in labeled mCry 3A binding (to ~ 40

- 60% at the two highest concentrations). The authors suggested that the low level of competition observed could be explained if there are multiple binding sites that recognize mCry 3A with one that is shared with eCry 3.1Ab. However, the shared binding site would have low affinity for eCry 3A.1Ab since competition was observed at only at the peak concentrations tested. No competition was observed in the second assay : eCry 3.1Ab binding was not reduced by even the highest concentrations of unlabeled mCry 3A. These results suggest that the specific binding site(s) for eCry 3.1Ab are not recognized by mCry 3A. Competition experiments with non

- activated protoxins showed similar patterns as were o bserved in the first competition, indicating that both chymotry psin

- activated and non

- activated mCry 3A and eCry 3.1Ab toxins recognize the same binding sites. Considering the results of both studies, Sy ngenta concluded that eCry 3.1Ab and mCry 3A maintain i ndependent binding sites in WCRW and that separate genetic mutations would be needed to develop resistance to both toxins. As such, the toxins are viable in a p yramided product for resistance management of CRW. BPPD Review

- Corn Rootworm Cross Resistanc e Potential Sy ngenta’s cross resistance analy sis was based on the assumption that Bt resistance will develop through an alteration of the toxin binding receptors in the target, a mechanism that has been documented for a number of pests (Ferre et al. 1991; Tabashnik 1994; L ee et al. 1995; Ferre & Van Rie 2002). Sy ngenta showed through an anal ysis of WCRW midgut binding activity and competitive binding assays that eCry 3.1Ab and mCry 3A largel y recognize unique binding sites. As such, cross resistance betwee n the two toxins is not expected according to the traditional Bt resistance model of binding site modification.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

83 This is perhaps not surprising, considering that eCry 3.1Ab is an engineered toxin with different structural elements (transferred from Cry 1Ab) than mCry 3A (Walters et al. 2010). Sy ngenta did not consider alternate resistance mechanisms or cross resistance scenarios in addition to toxin

- receptor binding. It has been suggested that resistance in CRW to Bt may evolve differentl y than lepidopteran pests on which the toxin

- receptor mechanism of resistance is based. Resistance to Bt in CRW may

involve multiple loci exerting small changes in susceptibility as opposed to a single locus creating high level resistance (as is assumed for lepidoptera) (S AP 2011). Several selection experiments with CRW have suggested that resistance may be incomplete (Meihls et al. 2008, 2011) or could occur at low levels (Lefko et al. 2008). The SAP also noted the potential for epistatic processes in which a single gene tic change (e.g., protease expression or regulation of cadherin levels) could confer broader resistance to Bt toxins. BPPD notes, however, that specific cases of epistasis (or broader mechanisms of cross resistance) have not y et been documented in CRW or other Bt corn pests. In addition, one study of a Cry 3Bb1

- resistant colony showed no evidence of cross resistance to Cry 34/35Ab1 (Gassmann et al. 2011). Sy ngenta’s assessment of cross resistance was limited to the novel eCry 3.1Ab and existing mCry 3A prote ins, both of which are proprietary to the company . No anal ys is of the other registered CRW toxins, Cry 3Bb1 and Cry 34/35Ab1, was included in the submission. Customarily , the registrant of a new PIP active ingredient will analy ze cross resistance for all e xisting toxins in the landscape and not just the toxin(s) in the proposed product or p yr amid (e.g., refer to the cross resistance discussion for mCry3A in BPPD 2006). BPPD recognizes that because eCry3.1Ab is an engineered toxin, it is likely to have sign ificant structural differences with Cry 3Bb1 and Cry 34/35Ab1 and perhaps different midgut binding patterns in CRW. But no information was provided in the submission to verify this assumption; therefore, BPPD recommends that Sy ngenta provide additional data or analy sis to address the cross resistance potential of eCry 3.1Ab with Cry 3Bb1 and Cry 35/35Ab1. In addition to midgut binding analy sis (as submitted by S yn genta), tests with resistant colonies can be useful to assess cross resistance, provided such colo nies are available. Resistant colonies have been selected with Cry 3Bb1 (Meihls et al. 2008), Cry 34/35Ab1 (Lefko et al. 2008), and mCry 3A (Meihls et al. 2011), though it is unknown whether such colonies could be accessed for future testing. Should they be available, BPPD recommends testing with the resistant colonies to improve the cross resistance anal ys is for mCry 3A. BPPD is unaware of any selection experiments that have been conducted for the relativel y new eCry3A.1Ab protein, though it is recommended that selection experiments be initiated with this protein as well. **3 . Simulation Modeling**  Lepidoptera Since the lepidopteran

- active toxins (Cry 1Ab, Cry1F, and Vip3Aa20) in Syngenta’s proposed 5307 products have been previously registered, the applicant cited existing modeling for CEW, ECB, and SWCB. For CEW, Sy

ngenta cited simulations conducted for the initial registration of Vip3Aa20Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

84 (MIR162). These simulations, described in BPPD (2009a), were used to support deploy ment of a 20% refuge in cotton

- growi ng regions, where the risk of resistance in CEW may be high. S yn genta did not conduct additional simulations for CEW because the compan y is proposing to maintain the existing 20% refuge standard for the new Cry 1Ab/Cry 1F/Vip3Aa20 py ramid. Modeling for ECB and SWCB was previousl y developed for the registration of Syngenta’s Bt11 x MIR162 x 1507 py

ramid (EPA Reg. No. 67979

- 15). The model (detailed in MRI D# 480506
- 06) was deterministic for two loci (Cry 1Ab and Cry 1F) and was similar in design to the single lo cus simulation created b y Caprio (1998). Vip3A was not included in the simulations since it is not active against ECB. Results from the modeling showed that resistance did not develop (within 100 y ears) to the Cry 1Ab/Cry 1F py

ramid with either a 5% or 20% refuge. S yn genta asserted that the model included a high degree of conservatism including assumptions that resistance carries no fitness costs, resistance is complete, no natural refuge in the s ys tem, and that single trait versions of Cry 1Ab and Cry 1F ar e present. The compan y concluded that the simulations support the use of Cry1Ab, Cry1F, and Vip3Aa20 in a py ramid with a 5% refuge. BPPD Review

- Lepidopteran Modeling Sy ngenta’s ECB/SWCB models were assessed for the registration of Bt11 x MI R162 x 1507 (see full review in BPPD 2011). BPPD generally agreed with Sy

ngenta that, under the modeling conditions investigated, Bt11 x TC1507 x MIR162 was highly durable relative to the corresponding single trait PIPs (i.e., resistance did not evolve within the ti me frame of the model). Furthermore, BPPD agreed that some of the parameter choices were conservative for the model, including assumptions of complete resistance, no fitness costs for resistance, and no natural refuge. Also, the exclusion of MIR162 (Vip3 Aa20) from the modeling adds conservatism for SWCB, which has susceptibility to the toxin. Despite these conclusions, BPPD’s anal ys is was limited by the cursory

description provided for the model (Appendix F, MRID# 480506

- 06), which lacked a number of cri tical details in the methodology description. BPPD had additional questions regarding some of the parameter assumptions and was unable to find descriptions of others. The scope of the modeling appeared to be limited, as no alternate scenarios (i.e., wors t cases hy potheses) or sensitivity

analy sis were conducted (or reported in the submission). For these reasons, it was difficult for BPPD to assess the relative durability of refuge options for Bt11 x TC1507 x MI R162 based solely on the limited modeling submitted by S yn genta. BPPD recommended that Sy ngenta provide an improved modeling anal ys is to address the concerns so that the expected durability of Bt11 x TC1507 x MIR162 could be confirmed (BPPD 2011). A revised modeling report was required as a term o f registration, which was subsequently submitted to EPA (MRID# 485509
- 02). As of the date of this memorandum, this submission has not y et been formally reviewed b y BPPD. Sy ngenta’s CEW model was submitted and reviewed for the registration of MIR162 (Vip3 Aa20). The model was designed b y Dr. Michael Caprio (Mississippi State University) and included several scenarios in which Vip3Aa20 x Cry 1Ab corn (with either 20% or 50% refuge) was planted in a landscape containing Bt cotton (Vip3Aa19 x Cry1Ab). Simulat ions with 20% or 50% corn refuge didBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

85 not evolve resistance during the 25

- ye ar model timeframe. BPPD’s assessment of the model concluded that it, coupled with other evidence, provided sufficient support to warrant the use of a 20% refuge for the MIR162 py

r amid in southern cotton

- growing regions (BPPD 2009a). Sy ngenta is proposing to employ the same 20% refuge strategy in cotton regions with the 5307 py ramids (containing Cry 1Ab, Cry 1F, and/or Vip3Aa20) and did not conduct any new CEW modeling for these new products. BPPD agrees that the modeling originall y submitted for MIR162 is adequate to support Sy ngenta’s proposed lepidopteran refuge strategy for the 5307 applications. Corn Rootworm Sy ngenta partnered with Dr. Michael Caprio (Mississippi State Unive rsity) to conduct simulations for CRW. Dr. Caprio (in collaboration with U.S EPA’s Office of Research development) had previously designed a stochastic model that was used for an analy sis of a “seed blend” IRM strategy (Caprio and Glaser 2010). For S yn ge nta’s simulations, Caprio modeled the time to resistance for a MIR604 (mCry 3A) x 5307 (eCry 3.1Ab) py ramid with a 5% block or seed blend refuge. Single trait events (MIR 604 and 5307) were also modeled as a mosaic with 20% block refuges. Caprio’s model employ ed a landscape structure with four 36 ha fields, with each of the fields including a 20 x 20 grid of patches (each 30m x 30m). The grid allowed for structured refuges to be placed within the Bt fields so that adult dispersal could be modeled. Field s were populated at the start of each y ear in the model run with the PIP crop and refuges. For the single toxin mosaic simulations, each of two toxins was randomly

assigned to two of the fields. Refuges were located at one side of the Bt field (randoml y selected for each field). Bt fields had a 30% probability of not having a refuge each ye ar of the simulation (corresponding with a non

- compliance rate of 30%). No partial non

- compliance was incorporated and Caprio noted that there was a small probability (0.81%) in any given y ear of there being no refuge in an y of the four fields (an unlikely scenario in the real world). If resistance to one of the two toxins developed, in subsequent y ears all fields were planted exclusively with the second toxin. Dual toxin simulations for the py ramid (with 5% structured refuge or seed blend) were modeled using the same landscape features, though no single toxin PIPs were included (all four fields were planted with the same dual gene PIP). Adult dispersal was modeled in a routine that included both population and individual parameters. For each dispersal routine, a certain proportion of the total population was determined to move at least one patch (randomly

drawn from binomial distribution). Movement on an individua l basis included vector (direction) and distance, both of which were randomly generated (dispersal distance was based on data developed b y Nowatski et al. 2003). A second routine covered “aging” in which individuals aged sequentially

by one day in the simulations. This feature allowed for the modeling of a developmental delay for insects exposed to PIP toxins, which was accomplished by holding back 12% of such individuals from progressing through life stages each day (resulting in an average overall delay of 7 day s for the population). Larval movement included a base movement rate (the same for both Bt and non

- Bt plants) and asy mmetrical movement away from Bt plants. Base larval movement was sampled for each simulationBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

86 from a distribution that ranged fro m 1 to 10% with 5% being the most likely value. As ymmetrical movement incorporated the genot ype of the insect and mortality on Bt, such that susceptible genot ypes (more likel y to be killed on Bt) had greater movement rates on Bt plants. The frequency (ratio) of asy mmetrical movement was determined for each simulation from a probability distribution (0.1 to 1.0, 0.5 most likely). Many of the other parameters were populated using uncertaint y distributions, as was done for larval movement. Values for toxi n mortality (used to derive genoty

pe fitness) were provided by S yn genta, while other biological parameters were carried over from previous modeling (Caprio and Glaser 2010). In formation on the parameter assumptions and model features is detailed in Table 2 below. **Table 8**  **.** Parameter Values and Assumptions for Syngenta’s (Caprio) Stochastic Model to Evaluate the Durability

of 5307 x MIR604 (taken from data provided in Table 1 and other information in Appendix D of MRID# 484429
- 10)
**Parameter Value or Assum**  **ption Notes** 

**Min Most Likely**  **Max** Initial R

- allele freq. 0.001 0.005 0.02 Same values for both toxins but drawn independently

for each locus Mortality due to toxin 1 0.95 0.9808 0.9997 Toxin not specified; assumed to be eCry 3.1Ab Mortality due to toxin 2 0.833 0.95 0.9906 Toxin not specified; assumed to be mCry 3A Overall mortalit y 0.99165 0.99904 0.9997 Base larval movement 0.01 0.05 0.1 Total movement for the lifetime of the larvae Asy mmetrical movement ratio 0.1 0.5 1.0 Insect aging One day per simulated day ; 12% on Bt plants have one day

developmental delay Cross resistance Not reported BPPD assumes to be zero Non compliance (structured refuges) 30% (no partial non

- compliance) Model duration 4440 day s (31.7 y ears) Definition of resistance R allele > 0.5 Model continued to run if resistance developed to one toxin

- all subsequent fields were assumed to be planted to 2 nd toxinBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

87 Several hundred simulations were conducted for each toxin deploy ment scenario with each simulation run for the e quivalent of 4440 day s (31.7 y ears). Resistance did not evolve to either toxin in the py ramid seed blend scenario (510 total simulations). Similarly , resistance did not evolve to the py ramid in the structured refuge scenario (535 simulations) though in three simulations resistance evolved to the second toxin. Resistance to both toxins evolved only in simulations with the single toxin mosaic scenario. The average time to resistance was 26.7 y ears for toxin 1, 28.7 y ears for toxin 2, and 29.9 ye ars for bo th toxins; however, resistance did not evolve to both toxins in 62.7% of the simulations (311 total). Caprio also presented a risk profile for the single toxin modeling with levels of risk ranging from 0% to 50%. For a 10% level of risk, the time to resi stance would be ≥ 25.3 y ears for both toxins. This means that 90% of the simulations evolved resistance after 25.3 ye ars, while in the other 10% of simulations resistance developed more quickl y (the researcher essentially assumes a 10% chance of being “wr ong” about the estimated durability). As the level of risk drops, the resistance values also drop (more outlying simulations are included); at the 0% level of risk the time to resistance was 17.4 y ears for both toxins (i.e., none of the simulations had re sistance develop earlier than 17.4 y ears). Results from the modeling are summarized in Table 3. Caprio (and S yn genta) concluded that the p yramid of mCry3A and eCry3.1Ab with 5% refuge (seed blend or block refuge) is more durable (“less risky”) than a mos aic of single toxin products with a 20% refuge. Given that resistance did evolve to toxin 2 in three simulations of the py ramid/structured refuge scenario, Caprio also concluded that the seed blend (in which resistance never evolved) was somewhat more dur able than the structured refuge. This was likely due to the non

- compliance rate (30%) that was assumed for structured refuges. **Table 9**  **.** Results from Syngenta’s modeling (taken from data provided in Table 2 and other information in Appendix D of MRI D# 48 4429
- 10)

**Scenario Simulations**  **Mean Time to Resistance (years) Toxin 1**  **Toxin 2 Both Toxins**  Single toxin mosaic: 50% toxin 1, 50% toxin 2 311 28.67 26.73 29.92 Py ramid with 5% structured refuge 535 No resistance * No resistance Py ramid with 5% seed b lend refuge 510 No resistance evolved to either toxin * Resistance evolved in three simulations to toxin 2. The time to resistance was not reported for these simulations.
BPPD Review

- Corn Rootworm Modeling Sy ngenta’s modeling largel y produced expect ed results, in that py ramided deploy

ment of toxins were relativel y more durable than single toxin PIP despite having lower percent refuges. This output isBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

88 consistent with other models that have analyzed py ramids as a resistance management approach for Bt crops (Roush 1998, Caprio 1998, Zhao et al. 2003, Gould et al. 2006, Onstad and Meinke 2010). Caprio’s stochastic model differs from other models by using a probabilistic approach for assigning parameter in which values are drawn from a distribution (with a most likely value) for each simulation. This allows the model to incorporate variability and uncertaint y associated with biological parameters (e.g., resistance allele frequencies, dose mortalit y, and movement). Man y s imulations are run for each scen ario from which a “risk profile” of estimated durability can be drawn for different levels of uncertaint y (selected b y the risk assessor). As the criterion for certainty in the durability

estimate is raised, the time to resistance decreases. BPPD has pre viously reviewed and utilized Caprio’s model in evaluations of other products (see BPPD 2010a) and has found it to be a rigorous risk assessment tool. While BPPD agrees with Sy ngenta regarding the overall conclusions, the modeling was limited in scope and the handling of some parameters. Three general scenarios were run that included landscapes of either single toxin or py

ramided toxin PIPs. The single toxin simulations included a mosaic of both traits (eCry 3.1Ab and mCry 3a) but both py ramid scenarios (s eed blend and block refuge) were run with no single trait PIPs in the landscape. This was a realistic choice in the case of eCry 3.1Ab since no products (single trait or pyramid) have been previously approved with the toxin. In the case of mCry 3A, however , single toxin PIPs expressing the trait have been registered since 2006 and would likely be grown along with 5307 py ramided products should they be registered. Single trait products can act as a “stepping stone” to resistance for p yramids, particularly i f non

- compliance with single trait refuge requirements is high. It is uncertain how the durability of the toxins would have been affected in a simulation including both single traits and p yr amids. BPPD expects that resistance would have evolved to the si ngle trait mCry 3A (as occurred in the single toxin mosaic simulations), though it is unknown whether resistance would have evolved to the p yr amid within the modeled time frame. One possible way to address this issue would be to increase the resistance all ele frequency for mCry 3A to account for previous selection to the single toxin. It is unclear, however, whether such an approach was considered in Sy ngenta’s simulations. The model duration (31.7 y ears) may have also limited the anal ys is between the 5% py ramid block and 5% py ramid seed blend refuges. For the vast majority of simulations (including the single toxin mosaics), resistance did not evolve during the model time period. In onl y three simulations (out of over 1,000 total) was resistance observe d in the py ramid toxin scenarios. Because of this, it is difficult to make an y relative comparisons between the p yr amid refuge options (i.e., blocks vs. blends); in essence, the only conclusion that can be drawn is that py ramided deploy

ments are superior to a mosaic of single toxin PIPs. The study

author (Caprio) suggested that blended refuge may be superior to block refuge for py ramids, though this was based on the three cases of resistance to one of the toxins that arose during the block refuge simulati ons. BPPD suspects, however, that this result is not statistically relevant given that only 0.56% of the simulations (3 of 535) showed resistance. Expanding the time horizon of the model could have helped to tease out differences between block refuge and seed blends and perhaps further flesh out the role non

- compliance play s on block refuge durability
.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

89 Though not explicitly

stated in the report, cross resistance was presumabl y assumed to be negligible and not included in the modeling. Data submitted by Sy ngenta (and reviewed earlier in this memorandum) support the assumption of little cross resistance potential through the traditional toxin

- midgut binding site model. BPPD cannot, however, entirel y eliminate the possibility of cross resistance through m eans other than toxin

- receptor interactions. The 2011 SAP identified epistasis, in which a single genetic change (e.g., protease expression or regulation of cadherin levels) exerts a broad effect, as a potential mechanism that could result in resistance to multiple toxins. Because of this, Sy ngenta could have assumed a low level of cross resistance as a worst case scenario in the modeling (it is unclear if the model assumed epistasis as part of genoty pe survival). This would likely have reduced the durabi lity of both traits in all scenarios, though the relative impact of cross resistance/epistasis on durability between seed blend and block refuges is unknown. Other potential parameters and considerations, such as mating (random/non

- random), pre

- ovipositio nal dispersal, oviposition, and density

- dependent effects, were not discussed in the report. These parameters can be important for relative evaluations of block and seed blend refuges, though S yn genta’s application proposes only

block refuge for the 5307 py ramids. BPPD assumes that these parameters were addressed in the same manner as Caprio’s previous modeling (Caprio and Glaser 2010), though this cannot be confirmed. Despite the limitations in the model and lack of clarity in the report, BPPD concludes that Sy ngenta’s modeling demonstrates high durabilit y of the mCry3A x eCry 3.1Ab p yr amid with a 5% block refuge. A number of other published and unpublished models have also shown that multi

- toxin PIPs with high efficacy and low cross resistance have supe rior durability to single toxin PIPs. On the other hand, additional information may be need to fully

assess the durability

of a 5% seed blend, including more complete descriptions of how parameters such as genot ypic fitness/movement, assumptions regarding heterozygote survival, and oviposition were addressed in the model. BPPD notes, however, that Sy ngenta has not proposed a seed blend strategy for 5307 corn at the time of their application. **4 . Proposed IRM Plan**  For Bt11 x MIR604 x TC1507 x 5307 (EPA Reg. No. 67979
- EU) and Bt11 x MIR162 x MIR604 x TC1507 x 5307 (67979

- EG ), Sy ngenta has proposed similar IRM strategies. The plans are based on reduced (5%) refuges for py ramided Bt corn and established paradigms for stewardship (i.e., resistance monitorin g, remedial action, and compliance). Refuge Separate refuge requirements were proposed for the Corn Belt (i.e., non

- cotton growing regions) and cotton - growing regions. Larger refuges have been required in cotton regions because of CEW, which can have up to two generations on corn before moving onto cotton during the same growing season. In the Corn Belt, resistance management is primaril y geared towards stalk borers (ECB and SWCB) and CRW.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

90 Corn Belt Refuge (non

- cotton growing regions) For both Bt11 x MIR604 x TC1507 x 5307 and Bt11 x MIR162 x MIR604 x TC1507 x 5307 , Sy ngenta proposes a 5% structured refuge. The refuge can be planted as either a “common refuge” for both ECB/SWCB and CRW or as two separate (5%) refuges for each pest complex. The commo n refuge must be planted adjacent to or within the Bt field. In

- field refuges must be at least four strips wide and if crop rotation is used, it must be employ ed for both the refuge and Bt fields. Insecticide treatments can be made in the refuge for corn rootworm larvae with soil insecticides. Non

- rootworm late season pests can be treated with foliar (non

- Bt) insecticides in the refuge provided adult CRW are not present and economic thresholds are reached. If adult CRW are present, both the refuge and B t field must be treated in the same manner. Economic thresholds are to be determined by local or regional professionals. S yn genta also proposed that “pests on the [Bt] maize acres can be treated as needed without having to treat the common refuge.” With the separate refuge option, discrete refuges (each 5%) must be planted for lepidoptera and rootworm. The lepidopteran refuge must be planted within 1⁄2 mile of the Bt field and in

- field strips must be at least four rows wide. Insecticides can be used in the refuge for 1) corn rootworm larvae (soil insecticides), or 2) corn borers (non

- Bt foliar applications) provided economic thresholds are reached. The corn rootworm refuge has essentially the same restrictions as the common refuge in terms of deplo ym ent (adjacent to or within the Bt field), in

- field strip width (at least four rows), and insecticide use for rootworm and other pests. Cotton

- Growing Regions Sy ngenta proposed different refuge requirements for each product in cotton regions, though both strategies maintain the common and separate refuge options. For Bt11 x MIR162 x MI R604 x TC1507 x 5307, Sy

ngenta proposed a 20% lepidopteran refuge while for Bt11 x MI R604 x TC1507 x 5307, a 50% lepidopteran refuge would be required. The common refuge (2 0% for Bt11 x MI R162 x MIR604 x TC1507 x 5307 and 50% for Bt11 x MIR604 x TC1507 x 5307) must be planted with the same deploy ment and insecticide use restrictions as the common refuge for the Corn Belt. For the separate refuges, both products must have a 20% corn rootworm refuge. Each product must also have a lepidopteran refuge: 20% for Bt11 x MI R162 x MIR604 x TC1507 x 5307 and 50% for Bt11 x MIR604 x TC1507 x 5307. The separate refuges must also be planted with the same restrictions as the separate r efuges for the Corn Belt region. Sy ngenta concluded that their proposal for the 5% refuge is supported by a number of factors, including submitted data reviewed in this memorandum. Specificall y, S yn genta cited the following justification:Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

91  EPA has set a precedent of lower refuge for p yr amided products for both lepidoptera and corn rootworm (e.g., MON 89034, SmartStax);  Two of the toxins in the py ramids, Bt11 (Cry 1Ab) and TC1507 (Cry 1F), are expressed at high dose levels for ECB and SWCB;  For CEW, the Bt11 x TC1507 x MI R162 py ramid offers three modes of action and a functional high dose;  For CRW, MIR604 (mCry 3A) x 5307 (eCry 3.1Ab) offers two effective modes of action;  Cross resistance potential for both lepidoptera and corn rootworm among the toxin groups i n the py ramids is low;  Simulation modeling demonstrates the durability of the Bt11 x MIR162 x MIR604 x TC1507 x 5307 py ramid relative to single toxin deploy ments. BPPD Review

- Refuge As discussed in the modeling section, BPPD concludes that there is ade quate scientific support to demonstrate that the 5307 py ramids will have high durability with a 5% structured refuge (relative to single trait PIPs with 20% refuge). S yn genta has not proposed a seed blend for an y of the 5307 products; BPPD notes that addi tional information and modeling may be necessary

to support such a refuge strategy
. Sy ngenta’s proposed language for the refuge requirement includes the following statement: “Pests on the Bt11 x MIR162 x MIR604 x TC1507 x 5307 [or Bt11 x MI R604 x TC1507 x 5307] maize acres can be treated as needed without having to treat the common refuge.” This language has not been specificall y included in the registration terms and conditions of similar Bt corn products, but the registrations do not prohibit the prac tice. Treatment of Bt acres should not increase the risk of resistance since most pest insects present would presumabl y be resistant to the toxin(s) (or otherwise have avoided lethal exposure to the toxin). Therefore, differential insecticide usage in wh ich the PIP field is treated but the refuge is not would not be expected to create a selection gradient disfavoring susceptible insects (as would be the case if only the refuge was treated). For cotton
- growing regions, Sy ngenta proposed a 50% lepidopteran refuge for Bt11 x MIR604 x TC1507 x 5307, while requesting a 20% refuge for Bt11 x MI R162 x MIR604 x TC1507 x 5307. BPPD assumes the difference is due to the fact that the latter product has three active toxins against lepidoptera (CEW in particular) whi le the former has two. CEW has been the primary target pest of concern in cotton areas (given its annual life cy cle on corn and cotton) and larger refuges (50%) were required for single toxin corn PI Ps in the region. However, EPA has approved a number of two toxin py ramids with a 20% refuge in cotton regions, including two S yn genta products (EPA Reg. Nos. 67979

- 12 and 67979

- 15) and another registrant’s product containing Cry1Ab and Cry 1F (29964

- 7). Sy ngenta may

be able to support a 20% refuge for the Bt1 1 x MIR604 x TC1507 x 5307 py ramid with a relativel y simple model (as was done to support the other registered p yr amids).Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

92 Sy ngenta’s refuge proposal did not specificall y define the cotton

- growing region that would require 20% (or 50%) refuges. Based on the paradigm established for previously

- registered Bt corn PIPs, this region should be defined as follows: Alabama, Arkansas, Georgia, Florida, Louisiana, Mississippi, North Carolina, South Carolina, Oklahoma (onl y the counties of Beckham, Caddo, Comanche, Custer, Greer, Harmon, Jackson, Kay , Kiowa, Tillman, Washita), Tennessee (onl y the counties of Carroll, Chester, Crockett, Dy er, Fay ette, Franklin, Gibson, Hardeman, Hardin, Hay wood, Lake, Lauderdale, L incoln, Madison, Obion, Rutherford, Shelby , and Tipto n), Texas (except the counties of Carson, Dallam, Hansford, Hartley , Hutchinson, L ipscomb, Moore, Ochiltree, Roberts, and Sherman), Virginia (onl y the counties of Dinwiddie, Franklin City , Greensville, I sle of Wight, Northampton, Southampton, Suffolk City , Surrey , Sussex) , and Missouri (only the counties of Dunkin, New Madrid, Pemiscot, Scott, Stoddard). Stewardship (Resistance Monitoring, Remedial Action, Compliance Assurance) Sy ngenta did not address resistance monitoring, remedial action, and refuge co mpliance for the 5307 py ramids other than to indicate that existing programs would be utilized for the new products. The existing stewardship framework developed for the lepidopteran toxins (Cry 1Ab, Cry 1F, and Vip3Aa20) has been coordinated b y the Agric ultural Biotechnology Stewardship Technical Committee (ABSTC) for over a decade. BPPD agrees that no additional information is needed for the inclusion of the 5307 products into the ABSTC programs for resistance monitoring, remedial action, and compliance assurance. Corn rootworm resistance monitoring has not been implemented by ABSTC and each registrant has developed a toxin

- specific plan (though there are common structural elements between plans). Sy ngenta’s CRW monitoring and remedial action plans wer e developed for the MIR604 product registrations (67979

- 5,
- 8; amended September, 2010). The general structure should be applicable to the 5307 registrations, however, additional information is recommended for the eCry 3.1Ab toxin. Baseline susceptibility data for eCry 3.1Ab were not addressed or submitted with the I RM volume. Also, a proposal for a diagnostic assay (to distinguish potentially resistant rootworm from susceptibles) was not included in the submission. BPPD recommends that Sy ngenta develop b oth baseline susceptibility data and a (plant

- based) diagnostic assay for eCry 3.1Ab as a term or condition of registration. Resistance monitoring for CRW has been challenged by the “non

- high dose” nature of the Bt toxins. CRW do not adapt well to artif icial diets and monitoring assay s based on toxin

- diet incorporation have been variable and may

be unreliable for detecting shifts in susceptibility . As part of the 2010 extensions of Bt corn registrations, BPPD notes that Sy ngenta was required to pursu e the use of the “Sublethal Seedling Assay ” (Nowatzki et al. 2008) as a potential diagnostic assay for mCry 3A. This technique involves assessing the rate of larval development on corn seedlings and was shown to be more sensitive to changes in susceptibili ty to Cry 34/35 (a separatel y registered CRW toxin). It is recommended that Sy

ngenta also investigate the feasibility of this approach for eCry 3.1Ab. In addition to the Sublethal Seedling Assay , the 2010 extension of mCry 3A required Sy ngenta to: 1) devel op a resistance monitoring plan for northern corn rootworm ( Diabrotica barberi ) and 2) establish cornBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

93 rootworm damage guidelines (for unexpected pest damage investigations). BPPD recommends that these requirements also be applied to the eCry 3.1Ab registrations. Sy ngenta’s submission indicated that they plan to submit the usual annual reports required for Bt corn (i.e., resistance monitoring, compliance, and sales) for the 5307 products. BPPD notes that Sy ngenta’s grower education materials (e.g., technol ogy use guides) will need to be updated to include the 5307 product line and refuge requirements. BPPD recommends that these materials be submitted to the Agency prior to or concurrent with the first commercial growing season of the products.
**References** Anilkumar, K.J., A
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- Simón, J
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- Carey , S
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- Resistant Cotton Bollworm Helicoverpa zea (Boddie)
. Appl. Environ. Microbiol. 74: 462
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. BPPD, 2001. Biopesticide Registration Action Document (BRAD): Cry 1Ab and Cry 1F Bacillus thuringiensis (Bt) Corn Plant Incorporated Protectants (updated September, 2010). Available at http://www.epa.gov/oppbppd1/biopesticides/pips/cry 1f

- cry 1a b

- brad.pdf BPPD, 2005. Bacillus thuringiensis Cry 3 4Ab1 and Cry 35Ab1 Protein s and the Genetic Material Necessary

for their Production in Event DAS
- 59122
- 7 Corn Biopesticide Registration Action Document (BRAD) . Available at http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006490.pdf
. BPPD, 2006. Biopesticide Registration Action Document (BRAD) : Modified Cry 3A Protein and the Genetic Mate rial Necessary for its Production (via elements of pZM26) in Event MIR604 Corn SYN
IR604
- 8 (updated September, 2010). Available at http://www.epa.gov/oppbppd1/biopesticides/pips/mcry3a

- brad.pdf
. BPPD, 2008. Biopesticide Registration Action Document (BR AD): Bacillus thuringiensis Cry 1A.105 and Cry2Ab2 Insecticidal Protein s and the Genetic Materia l Necessary for Its Production in Corn (updated September, 2010). Available at http://www.epa.gov/oppbppd1/biopesticides/pips/mon

- 89034
- brad.pdf BPPD, 2009a. Biopesticide Registration Action Document (BRAD): Bacillus thuringiensis Vip3Aa20 Insecticidal Protein and the Genetic Material Necessary for Its Production (via Elements of Vector pNOV1300) in Event MIR162 Maize (OECD Unique Identifier: SYN

- IR162
- 4) (updated March, 2009). Available at http://www.epa.gov/oppbppd1/biopesticides/i ngredients/tech_docs/brad_006599.pdf BPPD, 2009b. Review of Additional Insect Resistance Management (IRM) information for SmartStax (MON 89034 x TC1507 x MON 88017 x DAS 59122

- 7) Bt corn. A. Rey nolds memorandum to M. Mendelsohn, dated July

1, 2009.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

94 BP PD, 2010a. Science Assessment of Pioneer’s modeling addenda (MRIDs 478710

- 01, 478879
- 01, and 479108
- 01) in support of their application of registration for Optimum AcreMax1 Insect Protection . J. Martinez memorandum to M. Mendelsohn, dated February 4, 2010
. BPPD, 2010b. Review of Insect Resistance Management (IRM) considerations with a proposed reduced refuge for Bt corn event 1507 x MON 810 in the Corn Belt. A. Rey nolds memorandum to A. Sibold, dated October 20, 2010. BPPD, 2010c. EPA PRELIM INARY RISK ASSESSMENT of Monsanto’s and Dow’s 5% Seed Mixture Request for a Section (3) Full Commercial Registration of SmartStax Corn . J. Martinez memorandum to M. Mendelsohn, dated October 29, 2010. BPPD, 2011. Review of the Insect Resistance Management (IRM) p lan and supporting data for Bt corn event Bt11 x MIR162 x TC1507. A. Rey nolds memorandum to J. Kausch, dated March 2, 2011. Caprio, M. , 1998. Evaluating resistance management strategies for multiple toxins in the presence of external refuges. J. Econ. E ntomol. 91: 1021
- 1031. Caprio, MA and JA Glaser. 2010. “Simulation model evaluation of pest resistance development to refuge in the bag concepts related to Pioneer Submission”. Report generated for OPP/BPPD on January 22, 2010. Denholf, P., S. Jansens, M. Peferoen, D. Degheele, and J. Van Rie, 1993. Two different Bacillus thuringiensis delta

- endotoxin receptors in the midgut brush border membrane of the European Corn Borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae). Appl. Environ. Microbiol. 59: 1828
- 1837. Ferré, J., M. D. Read, J. Van Rie, S. Jansens, and M. Peferoen, 1991. Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor. Proc. Natl. Aca d. Sci. USA
. 88: 5119
- 5123. Ferré, J. and J. Van Rie, 2002. Biochemistry and genetics of insect resistance to Bacillus thuringiensis
. Annu. Rev. Entomol. 47: 501
- 533. Gassmann, A.J., J.L . Petzold

- Maxwell, R.S. Keweshan, and M.W. Dunbar, 2011. Field

- evolved resistance to Bt maize by western corn rootworm. PLoS ONE. 6(7): 1

- 7. Gould, F., A. Anderson, A. Rey nolds, L. Bumgarner, and W. Moar, 1995. Selection and genetic anal ys is of a Heliothis virescens (Lepidoptera: Noctuidae) strain with high level s of resistance to Bacillus thuringiensis toxins. J. Econ. Entomol. 88: 1545
- 59.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

95 Gould, F., M. B. Cohen, J. S. Bentur, G. C. Kenn edy , and J. Van Du yn, 2006. Impa ct of small fitness costs on pest adaptation to crop varieties with multiple toxins: a heuris tic model. J. Econ. Entomol. 99: 2091
- 2099. Granero, F., V. Ballester, and J. Ferre, 1996. Bacillus thuringiensis cry stal proteins CRY1Ab and CRY1Fa share a high affinity binding site in Plutella xylostella (L .). Biochem. Biophys. Res. Comm. 224: 779
- 783. Hibbard B.E., L.N. Meihls, M.R. Ellersieck, and D.W. Onstad, 2010a. Density

- dependent and density

independent mortality

of the Western corn rootworm: impact on dose calculations of rootworm

- resistant Bt corn. J. Econ. Entomol. 103: 77
- 84. Hibbard, B.E., T.L. Clark, M.R. Ellersieck, L.N. Meihls, A.A. El Khishen, V. Kaster, H. York

- Steiner, and R. Kurtz , 2010 b . Mortality of western corn rootworm larvae on M IR 604 transgenic maize roots: Field survivorship has no significant impact on survivorship of F1 progen y on MIR604. J. Econ. Entomol. 103: 2187
- 2196. Hua, G., L. Masson, J.L
. Jurat
- Fuentes, G. Schwab, and M.J. Adang, 2001. Binding Anal ys es of Bacillus thuringiensis Cry {delta}

- Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis
. Appl. Environ. Microbiol. 67: 872
- 879
. Lee, M.K., F. Rajamohan, F. Gould, and D.H. Dean, 1995. Resistance to Bacillus thuringiensis Cry 1A δ

- endotoxins in a laboratory

- selected Heliothis virescens strain is related to receptor alteration. Appl. Environ. Microbiol. 61 : 3836
- 3842. Lefko , S
. A
. , T
. M
. Nowatzki, S
. D
. Thomposon, R
. R
. Binning, M
. A
. Pascual, M
. L
. Peters, E
. J
. Simbro, and B
. F
. Stanley , 2008. Characterizing laboratory colonies of western corn rootworm (Coleoptera: Chry somelidae) selected for sur vival on maize containing event DAS

- 59122. J. Appl. Entomol. 132: 189
- 204 Meihls L
. N
. , M
. L
. Higdon, B
. D
. Siegfried, T
. A
. Spencer, N
. J
. Miller, T
. W
. Sappington, M
. R
. Ellersieck, and B
. E
. Hibbard , 2008. Increased survival of western corn rootworm on tran sg enic corn within three generations of on

- plant greenhouse selection. Proceed. Nat. Acad. Sci. 105: 19177
- 19182. Meihls L
. N
. , M
. L
. Higdon, M
. R
. Ellersieck, and B
. E
. Hibbard , 20 11
. Selection for resistance to mCry 3A
- expressing transgenic corn in western corn rootworm
. J. Econ. Entomol. 10 4 : 1045
- 1054
. Nowatzki, T.M., S.A. L efko, R.R. Binning, S.D. Thompson, T.A. Spencer, and B.D. Siegfried, 2008. Validation of a novel resistance monitoring technique for corn rootworm (Coleoptera: Chry somelida) and ev ent DAS
- 59122
- 7 maize. J. Appl. Entomol. 132: 177
- 188. Onstad, D. and L. Meinke, 2010. Modeling Evolution of Diabrotica virgifera virgifera (Coleoptera: Chry somelidae) to Transgenic Corn with Two Insecticidal Traits
. J. Econ Entomol. 103: 849
- 860
.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

96 Pe reira E.J., H.A. Siqueira, M. Zhuang, N.P. Storer, B.D. Siegfried, 2010. Measurements of Cry 1F binding and activity

of luminal gut proteases in susceptible and Cry 1F resistant Ostrinia nubilalis larvae (Lepidoptera: Crambidae). J. Invertebr. Pathol. 103: 1
- 7. Roush, R.T., 1998. Two toxin strategies for management of insecticidal transgenic crops: will py ramiding succeed where pesticide mixtures have not? Phil. Trans. R. Soc. Lond. 353 : 1 777
- 1 786
. Scientific Advisory

Panel (SAP), 1998. Transmittal o f the final report of the FIFRA Scientific Advisory Panel Subpanel on Bacillus thuringiensis (Bt) Plant

- Pesticides and Resistance Management, Meeting held on February

9
- 10, 1998. Report dated, April 28, 1998. (Docket Number: OPPTS

- 00231). Scientific Advisory

Panel (SAP), 2011. Transmittal of the meeting minutes of the FIFRA Scientific Advisory m eeting held on December 8

- 9 , 2011 to address scientific issues associated with insect resistance management for SmartStax refuge

- in

- a - bag, a plant

- incorporate d protectant (PIP) corn seed blend. Report dated, March 3 , 2011 . (Docket Number: EPA
- HQ
- OPP
- 2010
- 0772 ). Siqueira, H.A.A., D. Moellenbeck, T. Spencer, and B.D. Siegfried, 2004. Cross

- resistance of CrylAb

selected Ostrinia nubilalis (Lepidoptera: Crambid ae) to Bacillus thuringiensis d

- endotoxins. J. Econ. Entomol. 97: 1049
- 1057. Storer, N., 2002. Product durability plan for cotton expressing Cry 1F and Cry 1Ac insecticidal cry stal proteins from Bacillus thuringiensis . Unpublished report submitted to EPA by Dow Agrosciences. MRID# 458084
- 15. Storer, N., 2003. A spatially explicit model simulation western corn rootworm (Coleoptera: Chry somelidae) adaptation to insect

- resistance maize. J. Econ. Entomol. 96: 1530
- 1547. Storer, N., J.M. Babcock, and J.M. Edwards, 2006. Field measures of western corn rootworm (Coleoptera: Chry somelidae) mortalit y caused b y Cry 34/35Ab1 proteins expressed in maize event 59122 and implications for trait durability. J. Econ. Entomol. 99: 1381
- 1387. Tabashnik, B. E., 1994. Evolution of resistance to Bacillus thuringiensis . Annu. Rev. Entomol. 39: 47

- 79. Walters, F., C. deFontes, H. Hart, G. Warren, and J. Chen, 2010. Lepidopteran

- active variable

- region sequence imparts coleopteran activity in eCry 3.1Ab, an enginee red Bacillus thuringiensis hy brid insecticidal protein. Appl. Environ. Microbiol. 76 : 3082
- 3088. Zhao, J., J. Cao, Y. L i, H. Collins, R. Roush, E. Earle, and A. Shelton, 2003. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nature Biotechnology. 21: 1493
- 1497.Bacillus thuringiensis eCry3.1Ab Corn

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97 **G .**  **Benefits and Public Interest Finding**  **for Initial Registrations of Event 5307**  **Corn** The criteria for determining whether registration of a pesticide chemical is in the public interest are set forth in a Federal Register Notice dated March 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 criteria is met: i) the use is for a minor crop; (ii) the use is a replacement for another 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 l ack 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 b y currently registered pesticides; ii) the new pesticide is comparatively less risky to health or the environment than currently registered pesticides; or iii) the benefits (including economic benefits) from the use of the new active ingredient exceed those of alternative registered pesticides and other available nonchemical techniques. Sy ngenta has provided data to support its claim that 5307 CRW
- protected corn is in the public i nterest (MRID# 484425
- 41) . EPA’s anal ys is su pports the following conclusions: 1. Results of efficacy

trials conducted from 2006 to 2009 indicate that 5307 corn provides effective control of key rootworm pests of field corn , including western corn rootworm ( Diabrotica virgifera virgifera ), northern corn rootoworm ( Diabrotica barberi ), and Mexican corn rootworm (

Diabrotica virgifera zeae )
. Efficacy was measured in terms of root damage (i.e., crop protection) and adult emergence (i.e., population reduction). 2. The eCry 3.1Ab protein expressed in 530 7 is a novel mode of action for corn rootworm control. The protein is a chimera engineered from the mCry 3A and Cry 1Ab Bt proteins. It has unique biochemical properties and low cross resistance potential with other registered Bt PIPs targeting CRW (refer to the Insect Resistance Management chapter in this BRAD document). As such, 5307 is expected to provide insect resistance management (IRM) benefits for this and other CRW

- protected corn products (detailed in #3 below)
. 3. CRW is a highly

adaptive insect and has been able to overcome a number of cultural and chemical control regimes (Meinke et al. 1998, Spencer et al. 2005). Recent reports have suggested that CRW may be developing resistance to Cry 3Bb1, a separatel y - registered Bt toxin (Gassmann et al. 2 011, Gassmann 2012). Bt toxins for CRW control may have a higher risk of resistance than other PIPs because they are not expressed at “high dose” levels for the insect (Tabashnik and Gould 2012). On the other hand, the availability

of multiple toxins for CRW with unique modes of action (i.e., a toxin mosaic) may enhance IRM b y reducing the selection pressure on an y one toxin. The 5307 (eCry3.1Ab) protein would represent the fourth Bt toxin for CRW and would further expand the existing toxin mosaic. Furthermore, it is expected that 5307 will be included with other Bt proteins to provide multiple control mechanisms in a single product (known as a toxin “pyramid”). Simulation models predict that toxinBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

98 py ramids with unique modes of action are more durable ( less susceptible to resistance) than single toxin PIPs (Roush 1998, Zhao et al. 2003, Onstad et al. 2010). Sy ngenta has proposed two such py ramids with 5307 and a second CRW toxin, MIR604 (mCry 3A): a) Bt11 x MIR604 x TC1507 x 5307 and b) Bt11 x MIR162 x MIR604 x TC1507 x 5307. 4 . If 5307 corn is registered, it will be the fourth CRW
- protected Bt corn product on the market. The availability

of multiple CRW

- protected corn products will increase grower choice and price competition, likely resulting in lowe r seed prices for consumers and higher adoption rates. 5. Data provided b y S yn genta from a three

- year study showed that 5307 corn provided significantly greater crop yi eld (bushels/acre) than non

- Bt corn with or without insecticide use. Sy ngenta reporte d a yi eld benefit of 63 bushels/acre compared to non

- Bt corn without insecticide and 25 bushels/acre relative to non

- Bt corn treated with Force 3G insecticide. Yield from 5307 was comparable (statistically equivalent) to MIR604 corn (a separate CRW

- protec ted PIP registered by Sy ngenta). 6 . Registration of 5307 corn is expected to result in further reduction of chemical insecticide use b y growers. This is of special importance since many pesticides registered for CRW

- control are highly toxic to humans and the environment, while eCry3.1Ab

- expressing corn poses no foreseeable human health or environmental risks (refer to the Human Health Assessment and Environmental Assessment chapters in this BRAD document)
. Sy ngenta’s submission provided a summary of the c onventional insecticides used for CRW control; many of these products are “restricted use” due to high mammalian toxicity and bear environmental hazards statements warning of dangers to birds, mammals, fish, aquatic invertebrates, and bees. EPA previously conducted an assessment of these insecticides and their associated mammalian and environmental toxicities for Cry 3Bb1, a previously

- registered CRW PIP (see U.S. EPA 2010). This analy sis also demonstrated the high toxicity and environmental concerns of man y CRW insecticides used in field corn. REFERENCES Gassmann A
. J
. , J
. L
. Petzold

- Maxwell, R
. S
. Keweshan, and M
. W
. Dunbar , 2011. Field

- evolved resistance to Bt maize by western corn rootworm. PLOS one , Vol. 6 (7): 1

- 7
. Gassmann, A.J., 2012. Field

- evolved resistance to Bt maize by western corn rootworm : Predictions from the laboratory

and effects in the field. J. Invertebr. Pathol. Inpress. Meinke, L.J., B.D. Siegfried, R.J. Wright, and L.D. Chandler, 1998. Adult susceptibility of Nebraska western corn rootworm (Coleoptera: Chry somelidae) populations to selected insecticides. J. Econ Entomol. 91 : 594
- 600
. Onstad, D. and L. Meinke, 2010. Modeling Evolution of Diabrotica virgifera virgifera (Coleoptera: Chry somelidae) to Transgenic Corn with Two Ins ecticidal Traits. J. Econ Entomol. 103: 849
- 860.Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

99 Roush, R.T., 1998. Two toxin strategies for management of insecticidal transgenic crops: will py ramiding succeed where pesticide mixtures have not? Phil. Trans. R. Soc. Lond. 353: 1777
- 1786. Spencer, J.L ., E. Levine, S.A. I sard, and T.R. Mabry , 2005. Movement, dispersal and behaviour of western corn rootworm adults in rotated maize and soy bean fields. In: Vidal, S. et al. (Eds.), Western Corn Rootworm: Ecology and Management, CABI Publishing, Cambr idge, pp. 121

- 144. Tabashnik, B.E. and F. Gould, 2012. Delaying corn rootworm resistance to Bt corn. J. Econ Entomol. 10 5 : 767
- 776
. U
. S
. EPA. 2010. Biopesticide Registration Action Document for Bacillus thuringiensis Cry 3Bb1 Protein and the Genetic M aterial Necessary for Its Production (Vector PV

- ZM IR 13L) in MON 863 Corn (OECD Unique Identifier: MON

- ØØ863
- 5). http://www.epa.gov/oppbppd1/biopesticides/pips/cry 3bb1

- brad.pdf Z hao, J., J. Cao, Y. L i, H. Collins, R. Roush, E. Earle, and A. Shelton, 2003. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nature Biotechnology. 21 **II I**  **. REGULATORY POSITION FOR EVENT**  **5307 CORN**  Pur suant to section 3(c)(7)(C) of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) , the Environmental Protection Agency ( EPA ) may

conditionally

register a new pesticide active ingredient for a period of time reasonably sufficient for the generation and submission of required data that are lacking because insufficient time has elapsed since the imposition of the data requirement for those data to be developed. EPA may

grant such conditional registration only if EPA determines that (1) the use of the pesticide product during the period of the conditional registration will not cause an y unreasonable adverse effect on the environment, and (2) the registration and use of the pesticide during the conditional registration is in the public interest. EPA determines that all of these criteria have been fulfilled. The first criterion under FIFRA section 3(c)(7)(C) , as mentioned above , has been met because insufficient time has elapsed since the imposition of the requirements for the following data : 1) A dd itional data or analy sis to address the cross resistance potential of eCry 3.1Ab with Cry 3Bb1 and Cry 35/35Ab1. As part of this effort, BPPD also recommends that Sy ngenta work to develop a eCry 3.1Ab

- resistant colony (similar to the work done for mCry 3A
-- s ee Meihls et al. 2011). 2) Additional data for corn rootworm resistance monitoring with the eCry 3.1Ab toxin:  Baseline susceptibility data;  A proposal for a plant

- based diagnostic assay including an investigation into the feasibility of using the “Subleth al Seedling Assay ” (Nowatzki et al. 2008);  A monitoring plan for northern corn rootworm ( Diabrotica barberi );Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

100  Corn rootworm damage guidelines (for unexpected pest damage investigations of eCry 3.1Ab corn). The applicants submitted and/ or cited data suffic ient for EPA to determine that conditional registration of the Bacillus thuringiensis eCry 3.1Ab insecticidal protein and the genetic material necessary for its production (via elements of vector PSYN12274) in 5307 Corn (SYN

-  53  7
- 1) under FIFRA section 3(c )(7)(C) will not result in unreasonable adverse effects to the environment, as discussed above. The applicants submitted and/or cited satisfactory data pertaining to the proposed use. The human health effects and non - target organism effects data are consid ered sufficient for the period of the conditional registration. These data demonstrate that no foreseeable human health hazards or environmental effects are likel y to arise from the use of the product s and , furthermore, that the risk of resistance developi ng to the eCry 3.1Ab proteins during the conditional registrations is not expected to be significant. Registration of the Bacillus thuringiensis eCry 3.1Ab insecticidal protein and the genetic material necessary for its production (via elements of vector PSYN12274) in 5307 Corn (SYN

-  53  7
- 1) is in the public interest for the following reasons : 1. Results of efficacy

trials conducted from 2006 to 2009 indicate that 5307 corn provides effective control of key rootworm pests of field corn , including wester n corn rootworm ( Diabrotica virgifera virgifera ), northern corn rootoworm ( Diabrotica barberi ), and Mexican corn rootworm (

Diabrotica virgifera zeae )
. Efficacy was measured in terms of root damage (i.e., crop protection) and adult emergence (i.e., populat ion reduction). 2. The eCry 3.1Ab protein expressed in 5307 is a novel mode of action for corn rootworm control. The protein is a chimera engineered from the mCry 3A and Cry 1Ab Bt proteins. It has unique biochemical properties and low cross resistance po tential with other registered Bt PIPs targeting CRW (refer to the Insect Resistance Management chapter in this BRAD document). As such, 5307 is expected to provide insect resistance management (IRM) benefits for this and other CRW

- protected corn products (detailed in #3 below)
. 3. CRW is a highly

adaptive insect and has been able to overcome a number of cultural and chemical control regimes (Meinke et al. 1998, Spencer et al. 2005). Recent reports have suggested that CRW may be developing resistance to C ry 3Bb1, a separatel y - registered Bt toxin (Gassmann et al. 2011, Gassmann 2012). Bt toxins for CRW control may have a higher risk of resistance than other PIPs because they are not expressed at “high dose” levels for the insect (Tabashnik and Gould 2012). On the other hand, the availability

of multiple toxins for CRW with unique modes of action (i.e., a toxin mosaic) may enhance IRM b y reducing the selection pressure on an y one toxin. The 5307 (eCry3.1Ab) protein would represent the fourth Bt toxin for CR W and would further expand the existing toxin mosaic. Furthermore, it is expected that 5307 will be included with other Bt proteins to provide multiple control mechanisms in a single product (known as a toxin “pyramid”). Simulation models predict that to xin py ramids with unique modes of action are more durable (less susceptible to resistance) than single toxin PIPs (Roush 1998, Zhao et al. 2003, Onstad et al. 2010). Sy ngenta has proposed two such py ramidsBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

101 with 5307 and a second CRW toxin, MIR604 (mCry 3A ): a) Bt11 x MIR604 x TC1507 x 5307 and b) Bt11 x MIR162 x MIR604 x TC1507 x 5307. 4 . If 5307 corn is registered, it will be the fourth CRW
- protected Bt corn product on the market. The availability

of multiple CRW

- protected corn products will increase gr ower choice and price competition, likely resulting in lower seed prices for consumers and higher adoption rates. 5. Data provided b y S yn genta from a three

- year study showed that 5307 corn provided significantly greater crop yi eld (bushels/acre) than non

- Bt corn with or without insecticide use. Sy ngenta reported a yi eld benefit of 63 bushels/acre compared to non

- Bt corn without insecticide and 25 bushels/acre relative to non

- Bt corn treated with Force 3G insecticide. Yield from 5307 was comparable (stat istically equivalent) to MIR604 corn (a separate CRW

- protected PIP registered by Sy ngenta). 6 . Registration of 5307 corn is expected to result in further reduction of chemical insecticide use b y growers. This is of special importance since many pesticides registered for CRW

- control are highly toxic to humans and the environment, while eCry3.1Ab

- expressing corn poses no foreseeable human health or environmental risks (refer to the Human Health Assessment and Environmental Assessment chapters in this BRAD do cument)
. Sy ngenta’s submission provided a summary of the conventional insecticides used for CRW control; many of these products are “restricted use” due to high mammalian toxicity and bear environmental hazards statements warning of dangers to birds, mamm als, fish, aquatic invertebrates, and bees. EPA previously conducted an assessment of these insecticides and their associated mammalian and environmental toxicities for Cry 3Bb1, a previously

- registered CRW PIP (see U.S. EPA 2010). This analy sis also demon strated the high toxicity and environmental concerns of many CRW insecticides used in field corn. In view of these minimal risks and the clear benefits related to the Bacillus thuringiensis eCry 3.1Ab insecticidal protein and the genetic material necessary for its production (via elements of vector PSYN12274) in 5307 Corn (SYN

-  53  7
- 1) , EPA believes that the use of the product s during the limited period of the conditional registration s will not cause an y unreasonable adverse effects. Although the data with respect to this particular new active ingredient are satisfactory , they are not sufficient to support an unconditional registration under FIFRA section 3(c)(5). Additional data are necessary to evaluate the risk posed by the continued use of th ese product s . Consequently , EPA is imposing the data requirements specified earlier in this chapter
. EPA has determined, as explained in section II( G ) of this Biopesticides Registration Action Document ( BRAD ) , that the third criterion f or a FIFRA section 3(c)(7)(C) conditional registration has been fulfilled because use of the Bacillus thuringiensis eCry 3.1Ab insecticidal protein and the genetic material necessary

for its production (via elements of vector PSYN12274) in 5307 Corn (SYN

-  5 3  7
- 1) under these registration s is in the public interest. The submitted data , in support of th ese registration s under FI FRA section 3(c)(7)(C) , have been reviewed and determined to be adequate. Studies mentioned above are included in the terms, conditi ons,Bacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

102 and limitations of these registrations. Th ese registration s will not cause unreasonable adverse effects to man or the environment and are in the public interest. The expiration date of the registrations is proposed as August 1, 2016. **C. Period of**  **Registration** In the 2001 Bt Corn reassessment, EPA determined that it was appropriate to amend the then

- existing registrations to extend the period of registration of those products to an expiration date of October 15, 2008. All of the products being as sessed at that time were efficacious against lepidopteran pests. EPA based this action on the finding that use of Cry 1Ab or Cry 1F expressed in corn will not significantly increase the risk of unreasonable adverse effects on the environment “ for the limited time period of 7 additional y ears (to October 15, 2008). ” These registrations were later amended to extend the period of registration to an expiration date of September 30, 2010. EPA subsequently granted time

- limited registrations to products efficacious against coleopteran corn rootworm pests. For example, EPA registered Cry 3Bb1 on February 24, 2003, to May 1, 2004, and extended that registration twice, to February 24, 2008, and September 30, 2010. As set forth elsewhere in this document, EPA ’ s primary c oncern for the Bt protected transgenic corn products is the possibility that target pests will develop resistance to one or more of the plant

incorp or ated protectant ( 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 potent ial development of resistance. Registrants similarly have been busil y developing various products, product mixes (i.e., so

- called “ py ramids ” and “ stacks ” ), and re sistance strategies, to maximize agronomic benefits and address resistance management iss ues. The result has been a vast array of product combinations and, occurring over the past couple of ye ars, a re - emergence of varying refuge requirements for different products. As discussed in the 2001 Bt PIP BRAD (at IID13), the earliest Bt corn registrations did not include mandatory

refuge requirements. There was a lack of scientific consensus as to what the appropriate refuge requirement should be, and, it was ass umed that the limited market penetration of these earl y crops would be so low as to guarantee that adequate natural refuges would be available from neighboring non

-Bt corn fields. From 1995 to 1997, Bt corn registrations included voluntary refuge requireme nts of 0% to 20% in the C orn B elt. In 1999, the Agricultural Biotechnology Stewardship Technical Committee ( ABSTC ) , in conjunction with the National Corn Growers Association, proposed uniform insect resistance management ( IRM ) requirements for Bt corn regi strations. 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 b e treated for insects, or a 50% treated refuge in cotton

- growing areas; all refuges to be planted within one

- half mile of the Bt corn field. These uniform requirements brought certaint y and consistency to the market after the initial period where man y B t corn products had different refuge requirements. Recentl y, however, as productBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

103 developers have begun to conceive of products with different combinations of “ py ramided ” products (i.e., products containing two or more toxins efficacious against the same pe st) and “ stacked ” products (i.e., products combining toxins efficacious against different pests), the refuge requirements hav e begun to vary
. For example, certain products require a 20% external refuge; some products permit a 5% external refuge; one produc t incorporates a 10% seed blend refuge; we have applications in process for products that propose to incorporate a 5% seed blend refuge; and other permutations are possible. Given the profusion of various toxin combinations and refuge options, we can no l onger proceed on the basis that, as concerns insect resistance management, all products are equal. Itwas a relatively simple proposition when the default requirement of a 20% spray ed refuge applied to almost all of the Bt corn crops in the market. Under those circumstances, the relative durability of products against the development of resistance was functionally equivalent, and, as a consequence, imposing functionally equivalent registr ation periods was appropriate. That is now no longer the case. As part of our continually evolving regulatory

approach to the continuall y evolving product mix wrought by

developers, we think it appropriate to revise our regulatory requirements in scientifically defensible way s to reflect the comparative level of risks posed by the products that we regulate. Here, for example, where we ’ ve determined that a particular product, or category of products, likely

will pose less risk of insect resistance developing to a particular PI P protein, we think it appropriate to grant that p articular product, or category of products, a registration for a period greater than that granted a corresponding product that poses a greater risk of insect resistance developing. This approach is reflectiv e of complementary principles: first, to ensure that we apply our limited resources to the products that pose greater risk of adverse effects to the environment; and, second, to conserve the resources that registrants and applicants must expend in amending the registrations of products that pose less ris k of adverse effects to the environment. The scheme that we are follow ing include s registration periods of five, eight, and twelve ye ars; a fifteen

ye ar registration period will also be available, if adequatel y supported b y our science assessment . In this scheme, (i) a product with a single PIP toxin, and a 20% external refuge, qualifies for a five

- ye ar registration; (ii) a product with py ramided PIP toxins (i.e., two or more toxins with distinct, non

- cross reacting modes of action), that are non

- high dose (the definition for a high dose product remains unchanged), with either a seed blend or external refuge, qualifies for an eight

- ye ar registration; (iii) a product with py

ramided PIP toxins (i.e., two or more toxins with distinct, non

- cross reacti ng modes of action), that are high

- dose , with either a seed blend or external refuge, qualifies for a twelve

- ye ar registration; (iv) a product with py ramided PIP toxins (i.e., two or more toxins with distinct non

- cross reacting modes of actions), with eith er a seed blend or external refuge, that has been determined b y EPA ’ s science assessment to be 150% as durable as the baseline single toxin product with a 20% external refu ge, would qualify for a fifteen

- year registration. Products determined by EPA ’ s scie nce assessment to be less than 100% as durable as the baseline single toxin product with a 20% external refuge wo uld not qualify

for a five

- ye ar registration and the registration period for such products will be determined on a case

- by

- case basis consistent with the level of risk they pose. Similarly , instances where other risk issues may

arise, or where novel resistance concerns may be present, would also beBacillus thuringiensis eCry3.1Ab Corn

Biopesticides Registration Action Document (BRAD) Draft June 2012

104 determined on a case

- by

- case basis , as will novel refuge configurations that may present unique dur ability profiles
.