*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 Characterization 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). The 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:
 

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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 expressed in DAS-59122-7, and/or Cry1Ab protein 

expressed in MON810 corn events, when combined through traditional plant breeding. Protein 

expression analyses also demonstrated similar 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 known or putative toxin or allergen protein 

sequences were identified. 

Laboratory assays using sensitive target pest species 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 the 

environmental 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 material 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 support 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 using 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 PIP 
 

Page 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 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 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 Document 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 assessment for Cry1Ab corn is found in the Agency’s Bt

Crops Reassessment (EPA, 2001a). 

Pioneer is bridging all previously reviewed product 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 expression 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), and PAT (TC1507 and DAS-59122-7) protein 

sequences against known toxin or allergen proteins 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 cry1F gene from 

TC1507 corn, the cry35Ab1 and cry35Ab1 genes from DAS-59122-7 

corn, and the cry1Ab gene from MON810 corn. These data demonstrated 

equivalence among events because the transgenic inserts were stably 

integrated and retained when the parental lines are crossed to create 

1507×59122×MON810 corn. In addition, event-specific PCR methods 

detected and confirmed the presence of each expected transgenic DNA 

insert in the combination PIP corn event. 

Classification: ACCEPTABLE

476778-01 
 

Page 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 MON810 (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 proteins in the combination PIP was not 

affected by the presence of the other protein. 

Classification: ACCEPTABLE

476778-02 
 

Page 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 showed no significant amino acid sequence 

homologies using the Cry1Ab protein sequence as a query with any 

proteins known to be toxins. Therefore, 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 Sequence 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 

477864-05 
 

Page 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. Therefore, 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

477864-04 
 

Page 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 the 

Cry34Ab1 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 October 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.