*THIS REVIEW DOES NOT CONTAIN CONFIDENTIAL BUSINESS INFORMATION* **UNITED STATES ENVIRONMENTAL PROTECTION AGENCY**
WASHINGTON, D.C. 20460

OFFICE OF PESTICIDES AND TOXIC

SUBSTANCES February 19, 2010

MEMORANDUM

SUBJECT: Review of Human Health and Product Ch aracterization Data for Registration of Bacillus thuringiensis ( Bt ) Cry1F, Cry34Ab1, Cry35Ab1, and Cry1Ab Proteins and the Genetic Material Necessary for their Production in the Combination Plant-Incorporated Protectant (PIP) products: 1507 x MON810 [EPA Reg. No. 29964-T], 59122 x MON810 [EPA Reg. No. 29964-O], and 1507 x 59122 x MON810 [EPA Reg. No. 29964-I]; submitted by Pioneer Hi-Bred International,

Inc.

FROM: Annabel Waggoner, Environmental Protection Specialist [ signed ] Microbial Pesticides Branch, Biopesticides and Pollution Prevention Division (7511P)

THROUGH: John L. Kough, Ph.D., Biologist [ signed ] Microbial Pesticides Branch, Biopesticides and Pollution Prevention Division (7511P)

TO: Ann Sibold, Regulatory Action Leader Microbial Pesticides Branch, Biopesticides and Pollution Prevention Division (7511P)

ACTION REQUESTED : To review human health and product characterization data submitted by Pioneer in support of Sec. 3 Registration of the following combination PIP corn products: 1507 x MON810 (OECD Unique Identifier: DAS-01507-1 x MON-00810-6), 59122 x MON810 (OECD Unique Identifier: DAS-59122-7 x MON-00810-6), and 1507 x 59122 x MON810 (OECD Unique

Identifier: DAS-01507-1 x DAS-59122-7 x MON-00810-6). Th e combination PIP products were created via traditional breeding methods from crossing the following PIP events: TC1507 corn (OECD Unique Identifier: DAS-Ø15Ø7-1) expressing Bt Cry1F protein and the genetic material necessary (vector PHP8999) for its production; DAS-59122-7 corn (OECD Unique Identifier: DAS-59122-7) expressing Bt Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary (vector PHP17662) for their production; and/or MON810 corn (OECD Unique Identifier: MONØØ81Ø-6) expressing Bt Cry1Ab protein and the genetic material necessary (vector PV-ZMCT01) for its production.

CONCLUSION:Page 2 of 9 The molecular characterization of Bt Cry1F, Cry34Ab1, Cry35Ab1, and/or Cry1Ab insect control proteins and the genetic material necessary for their production in 1507 x MON810, 59122 x MON810, and 1507 x 59122 x MON810 combination PIP corn products demonstrated the stability and integrity of the intended transgenic DNA inserts of Cry1F protein expressed in TC1507, Cry34Ab1 and Cry35Ab1 proteins expresse d in DAS-59122-7, and/or Cry1Ab protein expressed in MON810 corn events, when combined through traditional plant breeding. Protein expression analyses also demonstrated sim ilar levels of Cry1F, Cry34Ab1, Cry35Ab1, and/or Cry1Ab protein concentrations of each respective parental event when compared to 1507 x MON810, 59122 x MON810, and 1507 x 59122 x MON810 combination PIP corn events.

The amino acid homology toxin assessments for the individual PIP events TC1507 and MON810 were submitted in conjunction with these data to support the registration of the combination PIP products and to complete the data matrices for events TC1507 and MON810 corn. Pioneer submitted updated acid homology assessments against toxins and allergens for the individual parental PIP events to reflect the current bioinformatic assessment in support of the human health data for the combination PIP products. No amino acid homology of the Cry1F, Cry34Ab1, Cry35Ab1, and Cry1Ab protein sequences to any know n or putative toxin or allergen protein sequences were identified.

Laboratory assays using sensitive target pest sp ecies found no antagonistic or synergistic effects among the Cry proteins expressed in each combination PIP product. This determination demonstrates functional equivalence among the single parental events and the combination PIP products and the results and conclusions of the non-target organism studies (conducted for each single parental event) can be bridged (EPA , 2001a, 2001b, and 2005). Therefore, no unreasonable adverse effects on non-target organisms and the environment are expected from the combination PIP products.

Collectively, these data justify bridging the existing findings and conclusions from theenvironmental and human health assessments conducted for the single PIP events TC1507, DAS59122-7, and MON810 to the combination PIP products: 1507 x MON810, 59122 x MON810, and 1507 x 59122 x MON810 corn (EPA, 2001a, 2001b, and 2005). These data support the finding that there is reasonable certainty of no harm to exposure of Cry1F, Cry34Ab1, Cry35Ab1, and/or Cry1Ab proteins and the genetic mate rial necessary for their production in 1507 x MON810, 59122 x MON810, and 1507 x 59122 x MON810 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.

In conclusion, all of the submitted data are classified as ACCEPTABLE and are sufficient to support Sec. 3 Registration of 1507 x MON810, 59122 x MON810, and 1507 x 59122 x MON810 combination PIP corn products. Furthermore, the individual parental event data for Cry1F, Cry34Ab1, Cry35Ab1, and/or Cry1Ab proteins suppor t the inclusion of the Cry1F [40 CFR § 174.504], Cry34Ab1 and Cry35Ab1 [40 CFR § 174.506], and/or Cry1Ab [40 CFR § 174.511] proteins expressed in the combination PIP products into the existing exemptions from the requirement of a tolerance for residues on food, since the mode of action for these proteins does not suggest a synergistic activity in combination for mammalian species.Page 3 of 9 RECOMENDATION : These data are sufficient to support registration for the combination PIP corn products: 1507 x MON810 [EPA Reg. No. 29964-T], 59122 x MON810 [EPA Reg. No. 29964-O], and 1507 x 59122 x MON810 [EPA Reg. No. 29964-I]; with the caveat that the registrant submit/cite any data required to support the PIP registrations of the individual parental events: TC1507, DAS59122-7, and MON810 corn, as well as the combination PIP product TC1507 x DAS-59122-7, within the timeframes required by the terms and conditions of EPA Registration Numbers 29964- 3, 29964-4, 524-489 and 29964-5, respectively.

DATA REVIEW RECORD:

Active Ingredients: Bacillus thuringiensis Cry1F, Cry34Ab1, Cry35Ab1, and/or Cry1Ab insecticidal proteins and the genetic material necessary for their production in

corn Product Names: 1507 x MON810, 59122 x MON810, and 1507 x 59122 x MON810 corn Company Name: Pioneer Hi-Bred International, Inc.

ID No: 29964-O, -T, and -I

Chemical Number: 006481, 006490, and 006430

Decision Number: 406162, 406793, and 406161

DP Barcode : 367436, 367438, and 367408

MRID No.:

476778-01 Molecular characterization of maize combined trait product DAS01507-1

×DAS-59122-7×MON-00810-6×MON-00603-6 usi ng Southern blot analysis and event-specific polymerase chain reaction

476778-02 Laboratory Characterization of Key Lepidopteran Pest Response to Pyramided Events

478800-01 Additional Addendum to expressed trait protein concentration of maize lines containing events DAS-01507-1, DAS-59122-7, MON-00810-6, and combined trait products DAS-01507-1×MON-00810-6, DAS-59122-

7×MON-00810-6 and DAS-01507- 1× DAS-59122-7×MON-00810-6

477864-05 Evaluation of the amino acid sequence similarity of the Cry1F protein to the NCBI Protein Sequence datasets

477864-06 Evaluation of the amino acid sequence similarity of the Cry1Ab protein from MON810 to the NCBI Protein Sequence datasets

BACKGROUND:

Pioneer has submitted data in support for Sec. 3 registration of the following combination PIPPage 4 of 9 products: 1507 x MON810 [EPA Reg. No. 29964-T], 59122 x MON810 [EPA Reg. No. 29964-O], and 1507 x 59122 x MON810 [EPA Reg. No. 29964-I]. These combinations are hybrids created by crossing the individual corn events: TC1507, DAS-59122-7, and MON810 corn, via traditional breeding methods. The following sections summarize the regulatory information for each event present in the combination PIP corn products:

TC1507 (Herculex 1TM) produces the Bt var. aizawai Cry1F protein to selectively control larvae of European corn borer ( Ostrinia nubilalis, ECB) and other lepidopteran insect pests. In addition, TC1507 also produces the phosphinothricin acet yl transferase (PAT) protein from Streptomyces hygroscopicus , to confer tolerance to glufosinate-ammonium, the active ingredient in Liberty ®
herbicide. The Agency previously established a tolerance exemption for Cry1F [40 CFR §174.520] in corn and Event TC1507 is currently registered as a PIP [EPA Reg. No. 29964-3]. A summary of the Agency’s risk and benefits assessment for Cry1F corn is found in the Agency’s Bt Crops Reassessment (EPA, 2001a) and updated in the 2001 Biopesticides Registration Action Document for Cry1F (EPA, 2001b).

DAS-59122-7 (Herculex RWTM) produces the Bt strain PS149B1 Cry34Ab1 and Cry35Ab1 proteins to protect against western corn rootworm ( Diabrotica virgifera virgifera , WCRM) and other coleopteran pests. In addition, DAS-59122-7 produces the phosphinothricin acetyl transferase (PAT) protein from Streptomyces hygroscopicus , to confer tolerance to glufosinate-ammonium, the active ingredient in Liberty ® herbicide. The Agency previously established a tolerance exemption for Cry34Ab1 and Cry35Ab1 [40 CFR §174.506] in corn and Event DAS-59122-7 is currently registered as a PIP [EPA Reg. No.29964-4]. The Agency’s risk and benefits assessment is found in the 2005 Biopesticides Registration Action Do cument for Cry34Ab1 and Cry35Ab1 (EPA, 2005).

MON810 (YieldGardTM) produces Bt subsp. kurstaki strain HD-1 Cry1Ab protein to selectively control larvae of ECB and other lepidopteran insect pests. In 1996, EPA granted a registration for the Cry1Ab protein and the genetic material necessary for its production in MON810 corn. In 2001, EPA granted the re-registration of MON810 corn based on a reassessment of the human health and other data collected over the first five years of registration. The Agency previously established an exemption from the requirement for a food tolerance for Cry1Ab [40 CFR §180.1173] and MON810 corn is currently registered as a PIP [EPA Reg. No. 524-489]. A summary of the Agency’s risk and benefits asse ssment for Cry1Ab corn is found in the Agency’s Bt Crops Reassessment (EPA, 2001a).

Pioneer is bridging all previously reviewed pr oduct characterization and human health data found acceptable for the registration of the individual parental events to support the registration of 1507 x MON810, 59122 x MON810, and 1507 x 59122 x MON810 corn. These data can be bridged if the submitted data for the combination PIP events are demonstrated as similarly and functionally equivalent to their respective parental events. The data reviewed in this memorandum include: molecular characterization data, protein expressi on analyses, and a synergism study. In addition, amino acid toxin homology assessments were submitted to complete the data matrices for events

TC1507 and MON810 corn.Page 5 of 9 Pioneer also submitted new amino acid homology assessments for Cry1F (TC1507), Cry34Ab1 and Cry35Ab1 (DAS-59122-7), Cry1Ab (MON810), a nd PAT (TC1507 and DAS-59122-7) protein sequences against known toxin or allergen protei ns to reflect the most updated bioinformatic assessment. Prior amino acid homology assessments were conducted as part of the original registrations of the single parental TC1507, DAS -59122-7, and MON810 corn events. Therefore, this memorandum also includes updated amino acid homology assessments to verify that there were no changes in the results and conclusions of the previously submitted data for the single parental corn events in order to support of the human health data for the combination PIP products.

SUMMARY OF SUBMITTED DATA:

The Agency has reviewed the data submitted by Pioneer and their study type/titles, conclusions, and respective MRID numbers are provided in the Table 1 for the submitted product characterization data and Table 2 for human health data in support for the 1507 x MON810, 59122 x MON810, and 1507 x 59122 x MON810 combination PIP corn products. The human health data were also submitted to complete the data matrices for events TC1507 and MON810 corn. The updated human health data are provided in Table 3 and contain the study type/titles, conclusions, and respective MRID numbers for PIP events TC1507, DAS-59122-7, and MON810 corn. The data summarized in Table 3 were also submitted to support the registration of 1507 x MON810, 59122 x MON810, and 1507 x 59122 x MON810 combination PIP corn products.

TABLE 1. Product Characterization Data submitted for 1507 x MON810, 59122 x MON810,

and 1507 x 59122 x MON810 Combination PIP Corn Products

Study Type/Title Summary MRID #

Product

Characterization/

Molecular

characterization of

maize combined trait product DAS-01507-

1×DAS-59122- 7×MON-00810-6 using Southern blot analysis and Event-specific Polymerase Chain

reaction Molecular analyses (restriction enzyme digests and Southern blots) were performed to compare the integrity of the transgenic inserts in events TC1507, DAS-59122-7, and MON810 corn with the transgenic DNA inserts in the combination PIP 1507×59122×MON810 corn product, produced by conventional plant breeding. The Southern blot data showed the predicted DNA hybridization patterns of the cry 1F gene from TC1507 corn, the cry 35Ab1 and cry 35Ab1 genes from DAS-59122-7 corn, and the cry 1Ab gene from MON810 corn. These data demonstrated equivalence among events because the transgenic inserts were stably integrated and retained when the pa rental lines are crossed to create 1507×59122×MON810 corn. In addition, event-specific PCR methods detected and confirmed the presen ce of each expected transgenic DNA

insert in the combination PIP corn event.

Classification: ACCEPTABLE 476778-01Page 6 of 9 TABLE 1. Product Characterization Data submitted for 1507 x MON810, 59122 x MON810,

and 1507 x 59122 x MON810 Combination PIP Corn Products

Study Type/Title Summary MRID #

Protein Expression

Levels / Additional Addendum to expressed trait protein

concentration of maize lines containing events TC1507, DAS-59122-7, MON810, and

combination PIP 1507 x MON810, 59122 x MON810, and 1507 x

59122 x MON810 corn product A field study was conducted using quantitative enzyme-linked immunosorbent assay (ELISA) methods to statistically compare the level of expression for Cry1F, Cry34Ab1, Cry35Ab1, Cry1Ab and PAT proteins expressed in 1507× 59122 × MON810, 1507 × MON810 and 59122 × MON810 combination PIP corn events to the individual parental events. No statistically significant differences were found for Cry1F, Cry34Ab1, Cry35Ab1, and Cry1Ab protein expression between 1507 × 59122 × MON810 and the parental corn events 1507, 59122, or MON810. No statistical differences were observed in any plant tissue for Cry1F, PAT, and Cry1Ab protein expression when 1507 × MON810 was compared to events 1507 and MON810 or when event 59122 was compared to event MON810. Based on the results, the protein concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and Cry1Ab expressed in the combination PIP events 1507 × 59122 × MON810, 1507 × MON810, and 59122 × MON810 are comparable to the respective protein concentrations of their respective single parental events.

Classification: ACCEPTABLE 478800-01

Note: This study supersedes

MRID No.

47510904

and

478654-01

Synergism study / Laboratory Characterization of Key Lepidopteran Pest Response to Pyramided

Events A seven-day laboratory sensitive insect bioassay was conducted to determine if the combination PIP product 1507 x 59122 x MON810 has a synergistic effect in comparison to the individual parental events TC1507 (expressing Cry1F protein) and M ON810 (expressing Cry1Ab protein) on target lepidopteran pests. The pests used in the bioassay were European corn borer (ECB, Ostrinia nubilalis ), southwestern corn borer (SWCB, Diatraea grandiosella ), fall armyworm (FAW, Spodoptera furgiperda ), and corn earworm (CEW, Helicoverpa zea ). The observed and expected larval mortality in the 1507 x 59122 x MON810 group were similar and mean larval weight of the survivors exposed to 1507 x 59122 x MON810 leaf tissue was not significantly different from the single parental events or the negative control of non- Bt maize. These results indicate that the Cry1F and Cry1Ab proteins act independently and do not have a synergistic or antagonistic effect on the target pests, other than an additive effect. Quantitative ELISA results also confirmed that the expression of each of the prot eins in the combination PIP was not
affected by the presence of the other protein

.

Classification: ACCEPTABLE 476778-02Page 7 of 9 TABLE 2. Human Health Data submitted for 1507 x MON810, 59122 x MON810,

and 1507 x 59122 x MON810 Combination PIP Corn Products

Study Type/Title Summary MRID #

Toxicity Study/

Evaluation of the amino acid sequence similarity of the Cry1Ab protein

from MON810 to the NCBI Protein Sequence

datasets The 818 amino acid sequence of event MON810 corn expressing Cry1Ab protein was sequentially searched and compared for protein sequence homology to known toxins contained in the NCBI Protein Sequence

datasets. The BLASTP search show ed no significant amino acid sequence homologies using the Cry1Ab protein sequence as a query with any proteins known to be toxins. Theref ore, the Cry1Ab protein does not share any amino acid sequence homology with any proteins known to be toxins.

Classification: ACCEPTABLE 477864-06

Toxicity Study/

Evaluation of the amino acid sequence similarity of the Cry1F protein

from TC1507 to the NCBI Protein Sequence

datasets The 605 amino acid sequence of event TC1507 corn expressing Cry1F protein was sequentially searched for comparisons to known toxins contained in the NCBI Protein Seque nce datasets. The BLASTP search showed no significant amino acid sequence homologies using the Cry1Ab protein sequence as a query with any proteins known to be toxins. Therefore, the Cry1F protein does not share any amino acid sequence homology with any proteins known to be toxins.

Classification: ACCEPTABLE 477864-05Page 8 of 9 TABLE 3. Updated Human Health Data submitted for PIP events TC1507, DAS-59122-7, and MON810 Corn

Study Type/Title Summary MRID #

Allergenicity Study/ Comparison of the Amino Acid Sequence Identity Between the Cry1F Protein and Known Protein Allergens No identity matches of ~ 35% over ~ 80 amino acid residues were observed for the Cry1F protein sequence against the protein sequences of known allergens. In addition, there were no 8 or greater contiguous identical amino acid matches observed with the Cry1F protein sequence. Ther efore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted allergen homology assessment for the Cry1F protein expressed in event TC1507 corn in (see MRID No. 449717-01).

Classification: ACCEPTABLE 477864-01

Allergenicity Study/ Comparison of the Amino Acid Sequence Identity Between the Cry34Ab1 and Cry35Ab1

Proteins and Known Protein Allergens No identity matches of ~ 35% over ~ 80 amino acid residues were observed for the Cry34Ab1 and Cry35Ab1 protein sequence against protein sequences of known allergens. In addition, there were no 8 or greater contiguous identical amino acid matches observed with the Cry34Ab1 and Cry35Ab1 protein sequence. Therefore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted allergen homology assessment for the Cry34Ab1 and Cry35Ab1 proteins expressed in event DAS-59122-7 corn (see MRID No. 452422-05).

Classification: ACCEPTABLE 477864-02

Allergenicity Study/ Comparison of the Amino Acid Sequence Identity Between the

PAT Protein and Known Protein Allergens No identity matches of ~ 35% over ~ 80 amino acid residues were observed for the PAT protein sequence against the protein sequences of known allergens. In addition, there were no 8 or greater contiguous identical amino acid matches observed with the PAT protein sequence. Therefore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted allergen homology assessment for the PAT protein (see MRID No. 449717-01).

Classification: ACCEPTABLE 477864-03

Allergenicity Study/ Comparison of the Amino Acid Sequence Identity Between the Cry1Ab Protein from Event

MON810 and Known Protein Allergens No identity matches of ~ 35% over ~ 80 amino acid residues were observed for the Cry1Ab protein sequence against the protein sequences of known allergens. In addition, there were no 8 or greater contiguous identical amino acid matches observed with the Cry1Ab protein sequence. Therefore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted allergen homology assessment for the Cry1Ab protein expressed in event MON810 corn (see MRID No. 458789-02).

Classification: ACCEPTABLE 477864-04Page 9 of 9 TABLE 3. Updated Human Health Data submitted for PIP events TC1507, DAS-59122-7, and MON810 Corn

Study Type/Title Summary MRID #

Toxicity Study/ Evaluation of the Amino Acid Sequence Similarity of the PAT Protein to the NCBI Protein Sequence

Datasets No significant amino acid sequence homologies were identified using the PAT protein sequence as a query with any proteins known to be toxins contained within the NCBI Protein Sequence datasets. Therefore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted toxin homology assessment for the PAT protein (see MRID No. 465847-01).

Classification: ACCEPTABLE 477864-07

Toxicity Study/ Evaluation of the Amino Acid Sequence Similarities of the Cry34Ab1 and Cry35Ab1 Proteins to the NCBI Protein Sequence

Datasets No significant amino acid sequence homologies were identified using the Cry34Ab1 and Cry35Ab1 protein sequences as a query with any proteins known to be toxins contained within the NCBI Protein Sequence datasets. Therefore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted toxin homology assessment for theCry34Ab1 and Cry35Ab1 proteins expressed in event DAS59122-7 corn (see MRID No. 465847-01).

Classification: ACCEPTABLE 477864-08

REFERENCES :

EPA (2001a). Biopesticides Registration Action Document for Bacillus thuringiensis ( Bt ) Plant- Incorporated Protectants, dated Oc tober 21, 2001. US Environmental Protection Agency, Washington, DC.

EPA (2001b). Biopesticides Registration Action Document for Bacillus thuringiensis ( Bt ) Cry1F Plant-Incorporated Protectants, updated August 2005. US Environmental Protection Agency, Washington, D.C.

EPA (2005). Biopesticides Registration Action Document for Bacillus thuringiensis ( Bt ) Cry34Ab1 and Cry35Ab2 Plant-Incorporated Protectants, dated October 2005. US Environmental Protection Agency, Washington, D.C.