BIOPESTICIDES REGISTRATION ACTION DOCUMENT

PLANT-INCORPORATED PROTECTANTS:

Bacillus thuringiensis Cry1Ac Protein and the Genetic Material

Necessary for its Production [PC Code 006527]

and

Bacillus thuringiensis Cry1F Protein and the Genetic Material

Necessary for its Production [PC Code 006528]

as expressed in

Event DAS-81419-2 Soybean (PIP product) 

[OECD Unique Identifier: DAS-81419-2]

U.S. Environmental Protection Agency

Office of Pesticide Programs

Biopesticides and Pollution Prevention Division

January 9, 2014
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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Bacillus thuringiensis Cry1Ac and Cry1F as DAS-81419-2 Soybean

Table of Contents

BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM................................................................................3

GLOSSARY OF ACRONYMS AND ABBREVIATIONS ....................................................................................................4

EXECUTIVE SUMMARY .......................................................................................................................................................5

I. ACTIVE INGREDIENT OVERVIEW ...........................................................................................................................7

II. REGULATORY BACKGROUND ..................................................................................................................................8

III. RISK ASSESSMENT SUMMARIES..............................................................................................................................9

A. Product Characterization ................................................................................................................................................9

B. Human Health Assessment ............................................................................................................................................10

C. Environmental Effects Assessment..................................................................................................................................16

D. Insect Resistance Management.......................................................................................................................................19

IV. ENVIRONMENTAL JUSTICE .................................................................................................................................20

V. RISK MANAGEMENT AND PROPOSED REGISTRATION DECISION..........................................................21

VI. TERMS AND CONDITIONS OF THE REGISTRATION(S).................................................................................21

APPENDIX A: Product characterization and human health effects – Bacillus thuringiensis Cry 1Ac and 

Cry1F proteins expressed in Event DAS-81419-2 Soybean plant-incorporated protectant..............................................23

APPENDIX B: Ecological and environmental effects – Bacillus thuringiensis Cry1Ac and Cry1F proteins

expressed in Event DAS-81419-2 Soybean plant-incorporated protectant.....................................................................34

APPENDIX C: Insect Resistance Management....................................................................................................................62

APPENDIX D: References......................................................................................................................................................69

APPENDIX E: Bibliography of data submitted for registration of Bacillus thuringiensis Cry1Ac and Cry1F 

proteins expressed in Event DAS-81419-2 Soybean plant-incorporated protectant.................................76
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM

Microbial Pesticides Branch

Product Characterization and Human Health

John Kough, Ph.D.

Chris Wozniak, Ph.D.

Environmental Fate and Effects

Shannon Borges, M.S.

Annabel Waggoner, B.S.

Insect Resistance Management

Alan Reynolds, M.S.

Office of General Counsel

Angela Huskey

Chris Kaczmarek

Biopesticides Registration Action Document Regulations

Denise Greenway, B.S.

Kimberly Nesci, M.S.

Alan Reynolds, M.S. 
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GLOSSARY OF ACRONYMS AND ABBREVIATIONS 

APHIS Animal and Plant Health Inspection Service (USDA)

bp Base pairs

BPPD Biopesticides and Pollution Prevention Division

BRAD Biopesticides Registration Action Document

b.w. body weight

CAS Chemical Abstracts Service

40 CFR Title 40 of the Code of Federal Regulations

cDNA Copied (or Copy) DNA

cfu colony forming units

dsRNA Double-Stranded RNA

cos Cosmid

oC Temperature in Centigrade or Celsius Degrees

DER Data Evaluation Record

DNA Deoxyribonucleic Acid

EPA Environmental Protection Agency (the “Agency”)

ELISA Enzyme-Linked Immunosorbent Assay

EPA Reg. No. EPA Registration Number

FFDCA Federal Food, Drug, and Cosmetic Act

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

FQPA Food Quality Protection Act

FR Federal Register

g Gram

IU International Units

IRM Insect Resistance Management

kg kilogram

L Liter

MRID No. Master Record Identification Number

mg Milligram

mL Milliliter

μg Microgram

MP Manufacturing-Use Product

mRNA Messenger RNA

NE No Effect

ng nanogram (10-9gram)

NIOSH National Institute for Occupational Safety and Health

OPP Office of Pesticide Programs

OCSPP Office of Chemical Safety and Pollution Prevention

OECD Organisation for Economic and Community Development

PC Pesticide Chemical

PCR Polymerase Chain Reaction 

PIP Plant-Incorporated Protectant

pg picogram (10-12 gram)

ppb parts per trillion

ppm parts per million

ppt parts per trillion

PTGS Post-Transcriptional Gene Silencing

RNA Ribonucleic Acid

rRNA Ribosomal RNA

T-DNA transfer DNA from Agrobacterium

TGAI Technical Grade of the Active Ingredient

USDA United States Department of Agriculture

U.S. EPA (EPA) United States Environmental Protection Agency

U.S. FDA United States Food and Drug Administration
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EXECUTIVE SUMMARY

The United States Environmental Protection Agency (U.S. EPA) proposes to register two new

Bacillus thuringiensis (Bt) soybean plant-incorporated protectant (PIP) active ingredients as 

described in this Biopesticide Registration Action Document (BRAD).

On July 24, 2012, Dow AgroSciences LLC (Dow) submitted an application for a breeding/seed 

increase registration of a new transgenic soybean (Glycine max) PIP, Event DAS-81419-2

Soybean, EPA File Symbol 68467-EN. The proposed PIP is a new transformation event that 

expresses lepidopteran-active insecticidal proteins (also known as “Cry” proteins) derived from 

the soil bacterium Bacillus thuringiensis (Bt). The proposed new PIP expresses both the Cry1Ac 

and Cry1F proteins in soybean. Different events of Cry1Ac have been previously registered in 

corn, cotton, and soybean. Similarly, separate events containing Cry1F have been registered in 

corn and cotton. However, the expression of Cry1F and Cry1Ac proteins in Event DAS-81419-2 

Soybean represents their first use together in soybean. Dow concurrently filed a petition for a 

permanent exemption from the requirement of a tolerance for Cry1F protein residues in or on 

soybean food and feed commodities.

On October 1, 2009, the EPA announced a new policy to provide a more meaningful opportunity

for the public to participate in major registration decisions before they occur. According to this 

policy, the EPA intends to provide a public comment period prior to making a registration 

decision for, at minimum, the following types of applications: new active ingredients; first food 

uses; first outdoor uses; first residential uses; or any other registration actions for which the 

Agency believes there may be significant public interest.

 

Consistent with the policy of making registration actions more transparent, the proposed Bt

Plant-Incorporated Protectant (PIP) product, DAS-81419-2 Soybean (EPA File Symbol 68467-

EN), expressing both the Cry1Ac and Cry1F proteins, is subject to a 15-day comment period as a 

new active ingredient. The docket identification number associated with these registration 

actions, and accessed through either http://www.regulations.gov or 

http://www.epa.gov/pesticides/regulating/registration-status.html, is EPA-HQ-OPP-2013-0703. 

The following documents are available for comment in EPA-HQ-OPP-2013-0703: 

(1) This draft Biopesticides Registration Action Document (BRAD); 

(2) The December 30, 2013, Environmental Risk Assessment for PIP Event DAS-81419-2;

(3) The November 7, 2013, Review of Insect Resistance Management (IRM) Considerations for 

Bt soybean event DAS-84149-2

(4) The November 22, 2013, Review of Human Health and Product Characterization Data for 

Registration of B. thuringiensis Cry1Ac and Cry1F Proteins and the Genetic Material Necessary

for their Production in DAS-81419-2 Soybean.

and

(5) a draft label for the PIP (EPA File Symbol 68467-EN)
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While a final decision on registration is contingent upon review and consideration of public 

comments, EPA believes that, based upon the data and information submitted in support of the 

proposed Bt soybean Plant-incorporated Protectant (PIP) pesticide product, DAS-81419-2

Soybean, it is appropriate to issue the registration. The basis for this preliminary decision can be 

found in the risk assessments for the proposed Bt soybean Plant-Incorporated Protectant (PIP) 

pesticide product, DAS-81419-2 Soybean, which are characterized throughout this BRAD. 

This BRAD presents EPA’s evaluations of the data and information submitted by Dow for the 

proposed registration of: 

1. EPA File Symbol 68467-EN: Event DAS-81419-2 Soybean expressing Bt Cry1Ac and 

Bt Cry1F insecticidal proteins. 

The genes that are responsible for the Bt Cry1Ac and Bt Cry1F proteins, cry1Ac and cry1F, have

been modified for their expression in plants. These proteins are referred to as Cry1Ac and Cry1F, 

respectively, in this BRAD. The event also includes the pat gene that produces Phosphinothricin 

Acetyltransferase (PAT) enzyme, an inert ingredient in the proposed product that confers 

tolerance of the soybean plant to the herbicide, glufosinate. 

DAS-81419-2 was developed by Agrobacterium-mediated transformation of soybean using the 

2T-DNA plasmid vector pDAB9582 and produces Bacillus thuringiensis protein δ-endotoxins, 

Cry1Ac and Cry1F. A single T-DNA insert containing each of the intact plant transcription units 

(PTUs) for the cry1Fv3, cry1Ac(synpro), and pat genes was integrated into soybean to create 

DAS-81419-2. This protein is intended to provide protection from feeding damage caused by a 

number of lepidopteran pests. 

Dow applied for pesticide registration of Cry1Ac and Cry1F proteins as co-expressed in Event

DAS-81419-2 soybean as a new PIP product, DAS-81419-2 Soybean, for use as a PIP in 2014, 

under section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). Dow

concurrently filed a petition for a permanent exemption from the requirement of a tolerance for 

Cry1F protein residues in or on soybean food and feed commodities. The EPA believes that, 

based upon its assessment of the data and information submitted by Dow, it is in the interest of 

the public and the environment to issue the registration proposed by Dow.
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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I. ACTIVE INGREDIENT OVERVIEW

Active 

Ingredients

and Office of 

Pesticide 

Programs (OPP) 

Chemical Code 

and File Symbol:

Bt Cry1Ac insecticidal protein and the genetic material necessary for its

production in DAS-81419-2 Soybean PIP product: 006527 

Bt Cry1F insecticidal protein and the genetic material necessary for its

production in DAS-81419-2 Soybean PIP product: 006528 

EPA File Symbol 68467-EN.

OECD Unique Identifier: DAS-81419-2

Applicant/

Manufacturer:

Dow AgroSciences LLC (Dow) 

9330 Zionsville Road

Indianapolis, In 46268

Type of Pesticide: Plant-incorporated Protectant (PIP)

Use: Insecticide

Target Pests: Larval stage lepidopteran pests of soybean, such as soybean looper 

(Pseudoplusia includens), velvetbean caterpillar (Anticarsia gemmatalis), 

beet armyworm (Spodoptera exigua), cotton bollworm/corn earworm

(Helicoverpa zea), green cloverworm (Plathypena scabra), and cutworms 

(Agrotis spp.)

Mode of action:

Insecticides formulated with microbial Bt have been in use worldwide since 1961. These 

microbial active ingredients are the source of the genes that express the Cry1Ac and Cry1F

proteins in the proposed Bt Soybean PIP. There is no mechanistic evidence that Bt proteins pose 

risks for humans or mammals during their use as pesticides or as PIPs (OECD, 2007). When Cry

proteins are ingested by an insect, they are dissolved under the alkaline conditions specific to the 

insect’s gut, and then activated upon cleavage by the insect’s midgut enzymes (proteases). These 

activated Cry proteins must bind specifically to glycoprotein or glycolipid receptors on 

microvillar membrane of the insect’s midgut to initiate the pore-forming process that ultimately

kills the insect. The fragment of the protein that is toxic to insects then interacts with specific 

high-affinity receptors on the microvilli of the target insect’s midgut epithelium (stomach), in 

particular brush border membrane vesicles (BBMV) (Ferré and Van Rie, 2002). Such receptors 

are not present in vertebrate species. Cry proteins must enter the cell membrane and form a pore. 

Although receptor binding appears to be essential for the insecticidal activity of the crystal 

proteins, binding by itself may not lead to toxicity. It appears that the initial protein-receptor 

interaction or binding is a reversible process, while the irreversible insertion of at least part of the 

Cry protein (the fragment that is toxic to insects) into the cell membrane is responsible for

forming the pore. Consequently, the ion gradient across the membrane is compromised, and the 

columnar cells of the midgut swell and lyse osmotically. Lysis leads to disruption of the gut 

epithelium, resulting in starvation, and insect death (Ferré and Van Rie, 2002). 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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II. REGULATORY BACKGROUND 

Applications for use of Cry1Ac and Cry1F expressed in DAS-81419-2 Soybean PIP

A. Cry 1Ac and Cry1AF proteins

1. Registration Application and Permanent Tolerance Exemption

a. Cry1Ac and Cry1F PIP FIFRA Section 3 Registration Application

On November 22, 2013, EPA published a Notice of Receipt in the Federal Register (78 FR 

70043), announcing that Dow submitted an application to register a new active ingredient, Bt

Cry1Ac and Cry1F insecticidal proteins expressed in soybean (DAS-81419-2 Soybean; EPA File 

Symbol 68467-EN), not included in any currently registered pesticide products. No public 

comments were received in response to this publication.

b. Cry1Ac protein - Permanent Tolerance Exemption

Residues of Cry1Ac protein are exempt from the requirement of a tolerance when used as PIPs in 

all food commodities (40 CFR § 174.510). EPA granted an exemption from tolerance for

Cry1Ac protein in all food and feed commodities on April 11, 1997 (62 FR 17722). The 

tolerance exemption was published in the Code of Federal Regulations (40 CFR §180.1155). In 

October 2001, EPA completed a reassessment of this tolerance exemption considering all of the 

existing data, public literature, and public comments. The reassessment determined that the 

tolerance exemption met all the required scientific and regulatory standards. This tolerance 

exemption for the Cry1Ac protein is not event-specific and applies to all Cry1Ac protein and any

other event producing the Cry1Ac protein that might be found in the food supply. On April 25, 

2007, the tolerance exemption for Cry1Ac protein was reassigned to 40 CFR § 174.510 (72 FR 

20435). Based on this existing tolerance exemption, a petition to establish a tolerance or 

tolerance exemption was not required for the Cry1Ac protein expressed in this soybean PIP. 

c. Cry1F protein - Permanent Tolerance Exemption

Concurrently with the FIFRA section application, Dow submitted a petition (PP2F8066) to 

establish a permanent exemption from the requirement of a tolerance for residues of Bacillus 

thuringiensis Cry1F protein in or on soybean. EPA published a Notice of Filing of the petition in 

the Federal Register on November 22, 2013 (78 FR 70007) and the public was given a 30-day

comment period. No public comments were received in response to this publication.

Event DAS-81419-2 Soybean also expresses the PAT enzyme that is exempt from the 

requirement of a tolerance when used as a PIP inert ingredient in all food commodities (40 CFR 

§174.522).
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III. RISK ASSESSMENT SUMMARIES

EPA assessed the data submitted by Dow in 2012 for the proposed PIP soybean registration and 

is presenting summaries of the product characterization and human health risk assessments in

Appendix A (Cry1Ac and Cry1F expressed in soybean DAS-81419-2 Soybean). Summaries of 

ecological and environmental risk assessments and Insect Resistance Management (IRM) for the 

PIP product are presented in Appendices B and C, respectively. In its assessment, the EPA relied 

upon data and other information submitted by the applicant and general knowledge of best 

available scientific technology. 

The classifications that are found for each data submission are assigned by Agency science 

reviewers and are an indication of the usefulness of the information contained in the documents 

for risk assessment. A rating of “ACCEPTABLE” indicates the study is scientifically sound and 

is useful for risk assessment. A “SUPPLEMENTAL” rating indicates the data provide some 

information that can be useful for risk assessment. The studies may have certain aspects 

determined not to be scientifically acceptable (“SUPPLEMENTAL: UPGRADABLE”). If a 

study is rated as “SUPPLEMENTAL: UPGRADABLE,” the Agency always provides an 

indication of what is lacking or what can be provided to change the rating to “ACCEPTABLE.” 

If there is simply a “SUPPLEMENTAL” rating, the reviewer will often state that the study is not 

required by 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 new data need to be submitted.

For the acute toxicity data requirements, toxicity categories are assigned based on the 

hazard(s) identified from studies and/or other information submitted to the Agency in support of 

a pesticide registration. The active ingredient or particular product is classified into Toxicity

Category I, II, III, or IV, where Toxicity Category I indicates the highest toxicity and Toxicity

Category IV indicates the lowest toxicity. 

A. Product Characterization 

All product characterization data requirements for the Bt soybean PIP containing the Cry1Ac and

Cry1F proteins and their co-expressed traits in DAS-81419-2 Soybean have been satisfied. The 

soybean event developed by Dow to express these PIPs is:

Event DAS-81419-2 Soybean: co-expresses Cry1Ac and Cry1F proteins, derived 

from Bacillus thuringiensis (Bt). It was developed by Agrobacterium-mediated 

transformation of soybean using the 2T-DNA plasmid vector pDAB9582 and 

produces Bacillus thuringiensis protein δ-endotoxins, Cry1Ac and Cry1F. A single TDNA insert containing each of the intact plant transcription units (PTUs) for the 

cry1Fv3, cry1Ac(synpro), and pat genes was integrated into soybean to create 

DAS-81419-2. 

DAS-81419-2 Soybean co-expresses Cry1Ac and Cry1F proteins to provide protection against 

feeding damage by lepidopteran insect larvae. 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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For summaries of the data evaluated for product characterization for these PIPs, see Appendix A.

The summaries include discussions of the following: 

 Product Characterization 

 Transformation System and Genetic Elements

 Protein Characterization 

 Analytical Detection Methods

 Protein Expression

As discussed in the summaries, Cry1Ac and Cry1F proteins are expressed at very low parts per 

million (ppm) levels in various parts of the transgenic soybean plant and are shown to be 

equivalent to the microbially expressed proteins

B. Human Health Assessment 

1. Toxicological Profile

EPA reviewed the available scientific data and other relevant information submitted in support of 

this action to register Cry1Ac and Cry1F as the PIP DAS-81419-2, 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. 

All toxicology data requirements for the Bt soybean PIP containing Cry1Ac and Cry1F proteins 

co-expressed in DAS-81419-2 Soybean have been satisfied. Acceptable Tier I mammalian 

toxicology data/information support the proposed registration of the active ingredients.

Furthermore, Tier II and Tier III studies were not required for either Cry1Ac or Cry1F proteins, 

based on the lack of acute toxicity or pathogenicity in the Tier I studies. Refer to Tables in 

Appendix A for brief summaries of data evaluated to satisfy toxicity and allergenicity data 

requirements for these PIPs.

Acute toxicity

Cry1Ac and Cry1F proteins

The following summarizes the final determinations regarding acute toxicology data requirements 

for Cry1Ac and Cry1F proteins. See Appendix A in this BRAD for more detailed summaries. 

Cry1Ac

Previously submitted acute oral toxicity data demonstrated a lack of mammalian toxicity at high 

levels of exposure (ingestion) to the pure Cry1Ac protein. These data demonstrate the safety of 

this protein at a level well above maximum possible consumption levels that are reasonably 

anticipated as expressed in the crop. Basing this conclusion on acute oral toxicity data without 

requiring further toxicity testing and residue data is similar to the Agency position regarding 

toxicity testing and the requirement of residue data for the microbial Bacillus thuringiensis 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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products from which this plant-incorporated protectant was derived (See 40 CFR Sec. 

158.740(b)(2)(i)). For microbial products, further toxicity testing (Tiers II & III) and residue data

are triggered by significant adverse acute effects in studies, such as the acute oral toxicity study, 

to verify the observed adverse effects and clarify the source of these effects. 

An acute oral toxicity study in mice (MRID No. 45542313) indicated that Cry1Ac is non-toxic to 

humans and other mammals. Cry1Ac protein is a δ-endotoxin from B. thuringiensis that has been 

used extensively in both microbial and plant-incorporated protectants as a means of insect pest 

management. An existing exemption from the requirement of a tolerance for Cry1Ac (CFR 40 

Section 174.510; 72 FR 20435, April 25, 1997) in all food and feed commodities precluded the 

need for a separate tolerance action in conjunction with this review of product characterization 

and human health assessment.

Cry1F

An acute oral toxicity study in mice (MRID No. 45542314) indicated that Cry1F is non-toxic to 

humans and other mammals. Cry1F protein is a δ-endotoxin from B. thuringiensis that has been 

used extensively in plant-incorporated protectants as a means of insect pest management. An 

existing exemption from the requirement of a tolerance for Cry1F (CFR 40 Section 174.504; 72 

FR 20434, Apr. 25, 2007) in cotton and corn (CFR 40 Section 174.520; 72 FR 20435 Apr. 25, 

2007), precluded the need for a separate tolerance action in conjunction with this review of 

product characterization and human health assessment. Cry1F was previously granted an 

exemption from the requirement of a tolerance for expression in cotton (CFR 40 Section 

174.504) and maize (CFR Section 174.520); a separate petition for an exemption from the 

requirement of a tolerance for Cry1F as expressed in soybean (PP2F8066) is included with the 

submission from Dow for registration of DAS-81419-2 soybean. 

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 by Cry1Ac and 

Cry1F; even at relatively high dose levels, the Cry1Ac and Cry1F proteins are not considered 

toxic to humans and other mammals. Further, amino acid sequence comparisons showed no 

similarities that would raise a safety concern between the Cry1Ac and Cry1F proteins and known 

toxic proteins in protein databases

Hypersensitivity Incidents: Reporting is required when incidents occur. No hypersensitivity

incidents, including immediate-type or delayed-type reactions in humans or animals, occurred 

during the manufacture of Bt Cry1Ac and Cry1F proteins as DAS-81419-2 Soybean PIP. Should 

any future hypersensitivity incidents occur, they must be reported to the Agency.

Allergenicity Assessment

Since Cry1Ac is a protein, allergenic potential was also considered. Currently, no definitive tests 

for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight-ofevidence approach where the following factors are considered: source of the trait; amino acid 

sequence comparison with known allergens; and biochemical properties of the protein, including

in vitro digestibility in simulated gastric fluid (SGF) followed by simulated intestinal fluid (SIF), 

and glycosylation. This approach is consistent with the approach outlined in the Annex to the 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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Codex Alimentarius “Guideline for the Conduct of Food Safety Assessment of Foods Derived 

from Recombinant-DNA Plants.” The allergenicity assessment for Cry1Ac follows:

1. Source of the trait. Bacillus thuringiensis is not considered to be a source of allergenic 

proteins. 

2. Amino acid sequence. A comparison of the amino acid sequence of Cry1Ac with known 

allergens showed no significant overall sequence similarity or identity at the level of 

eight contiguous amino acid residues.

3. Digestibility. (MRID No. 45542319) The Cry1Ac protein was digested rapidly in 

simulated gastric fluid containing pepsin. Small peptides remaining following gastric 

simulated digestion were completely degraded to amino acid residues in SIF upon contact 

(US EPA 2005, 2010a).

4. Glycosylation. Cry1Ac expressed in soybean was shown not to be glycosylated.

5. Conclusion: Considering all of the available information, EPA has concluded that the 

potential for Cry1Ac to be a food allergen is minimal.

Since Cry1F is a protein, allergenic potential was also considered. Currently, no definitive tests 

for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight-ofevidence approach where the following factors are considered: source of the trait; amino acid 

sequence comparison with known allergens; and biochemical properties of the protein, including

in vitro digestibility in simulated gastric fluid (SGF) followed by simulated intestinal fluid (SIF), 

and glycosylation. This approach is consistent with the approach outlined in the Annex to the 

Codex Alimentarius “Guideline for the Conduct of Food Safety Assessment of Foods Derived 

from Recombinant-DNA Plants.” The allergenicity assessment for Cry1F follows:

1. Source of the trait. Bacillus thuringiensis is not considered to be a source of allergenic 

proteins. 

2. Amino acid sequence. A comparison of the amino acid sequence of Cry1F with known 

allergens showed no significant overall sequence similarity or identity at the level of 

eight contiguous amino acid residues.

3. Digestibility. (MRID No. 45542318) The Cry1F protein was digested rapidly (< 5 min) 

in simulated gastric fluid containing pepsin. Small peptides remaining following gastric 

simulated digestion were completely degraded to amino acid residues in SIF upon contact 

(US EPA 2005, 2010b).

4. Glycosylation. Cry1F expressed in soybean was shown not to be glycosylated.

5. Conclusion: Considering all of the available information, EPA has concluded that the 

potential for Cry1F to be a food allergen is minimal.

Cry1Ac and Cry1F were shown not to be glycosylated in extracts of DAS-81419-2 soybean; it is 

unlikely to be glycosylated in any other crops because in order for a protein to be glycoslyated, it 

needs to contain specific recognition sites for the enzymes involved in glycosylation, and the 

mechanisms of protein glycosylation are similar in different plants (Lerouge et al., 1998).

Considering all of the available information, EPA has concluded that the potential for Cry1Ac

and Cry1F to be food allergens is minimal. 

Tolerance Exemptions
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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Permanent tolerance exemptions have been established for residues of Cry1Ac and PAT, as 

discussed in Chapter II under REGULATORY BACKGROUND, above. 

EPA’s reviews of the toxicology data support granting a permanent exemption from tolerance for 

residues of Cry1F protein in/on soybean food/feed commodities under section 408 of the Federal 

Food, Drug, and Cosmetic Act (FFDCA).

On the basis of evaluations of all relevant data submissions for Cry1F, EPA has determined that 

the data support amendment of 40 CFR §174 to establish a permanent tolerance exemption for 

residues of Cry1F in/on soybean food and feed commodities.

2. Endocrine Disruptors

As required under FFDCA section 408(p), the Agency has developed the Endocrine Disruptor 

Screening Program (EDSP) to determine whether certain substances (including pesticide active 

and other ingredients) may have an effect in humans or wildlife similar to an effect produced by

a “naturally occurring estrogen, or other such endocrine effects as the Administrator may

designate.” The EDSP employs a two-tiered approach to making the statutorily required 

determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a 

chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal 

systems. Chemicals that go through Tier 1 screening and are found to have the potential to 

interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP where the 

Agency will determine which, if any, of the Tier 2 tests are necessary based on the available 

data. Tier 2 testing is designed to identify any adverse endocrine-related effects caused by the 

substance, and establish a quantitative relationship between the dose and the E, A, or T effect.

Between October 2009 and February 2010, the Agency issued test orders/data call-ins for the 

first group of 67 chemicals, which contains 58 pesticide active ingredients and 9 inert 

ingredients. This list of chemicals was selected based on the potential for human exposure 

through pathways such as food and water, residential activity, and certain post-application 

agricultural scenarios. This list should not be construed as a list of known or likely endocrine 

disruptors.

Cry1Ac and Cry1F proteins are not among the group of 58 pesticide active ingredients on the 

initial list to be screened under the EDSP. Under FFDCA section 408(p), the Agency must 

screen all pesticide chemicals. Accordingly, the Agency anticipates issuing future EDSP 

orders/data call-ins for all pesticide active ingredients. 

For further information on the status of the EDSP, the policies and procedures, the list of 67 

chemicals, the test guidelines and the Tier 1 screening battery, please visit our website: 

http://www.epa.gov/endo/.

3. Food Quality Protection Act (FQPA) Considerations

a. Aggregate Exposures 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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In examining aggregate exposure, section 408 of FFDCA directs EPA to consider available 

information concerning exposures from the pesticide residue in food and all other nonoccupational 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).

EPA considered available information on the aggregate exposure levels of consumers (including 

major identifiable subgroups of consumers) to the PIP residue and to other related substances. 

These considerations include dietary exposure under the tolerance exemption and all other 

tolerances or exemptions in effect for the PIP residue, and exposure from non-occupational 

sources. 

As previously discussed, data indicated that Cry1F protein is non-toxic to humans and other 

mammals, and Cry1F protein was shown to be rapidly digested in vitro. Exemptions from the 

requirement of a tolerance already have been established for Bacillus thuringiensis Cry1F protein 

in cotton (40 CFR § 174.504) and corn (40 CFR § 174.520). The EPA has considered dietary

exposure under the tolerance exemption and all other exemptions in effect for the PIP residue 

and exposure from non-occupational sources. When Cry1F protein is used as a PIP in soybean, it 

is expressed at very low levels in the plant. Humans may be exposed to extremely low levels in 

the diet. There is also a very remote possibility that Cry1F protein can get in the water supply the 

same way that other proteins in crop debris can migrate into the ground, and, possibly, drinking 

water. Because such potential dietary exposure from soybean or drinking water is expected to be 

several orders of magnitude lower than the amounts of these proteins shown to have no toxicity

in mammalian tests, EPA concludes that negligible exposure via food and drinking water

would present no harm, based on the lack of mammalian toxicity and allergenicity potential, and 

the rapid digestibility demonstrated in SGF for the PIP.

 

Non-occupational dermal and inhalation exposure is not expected, since the PIP is expressed and 

contained within soybean plant cells, which essentially eliminates these exposure routes or 

reduces these exposure routes to negligible. The use sites of this PIP are all agricultural, for 

insect control, so there would be no exposure to infants and children from residential, school or 

lawn use. The amino acid sequence homology of known aeroallergens was included in the amino 

acid comparison of Cry1F protein with known food allergens, and the results indicated that no 

respiratory allergenicity would be expected if Cry1F protein were inhaled. The amino acid 

sequence results are discussed in Section II.B., above. Additionally, soybean flowers in a selfpollinating fashion wherein the anthers generally release pollen within a closed or covered floral 

tube. Pollen movement from soybean is minimal and highly localized when it happens (i.e., the 

pollen does not move readily on wind currents).

Taking all these data and information into consideration, EPA concludes that even if negligible 

aggregate exposure should occur it would present no harm to the U.S. human population due to 

the lack of mammalian toxicity and the rapid digestibility demonstrated for the Cry1F protein.

b. Cumulative Effects from Substances with a Common Mechanism of Toxicity
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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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 substances that have a common mechanism 

of toxicity.”

Cry1F protein is not considered toxic. It does not share a common mechanism of toxicity with 

any other substances, nor does it appear to produce a toxic metabolite produced by other 

substances. For the purposes of this assessment, therefore, EPA has assumed that Cry1F protein 

does not have a common mechanism of toxicity with other substances. Thus, EPA concludes that 

there are no cumulative effects associated with Cry1F protein 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

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

To evaluate human risk, EPA considered the validity, completeness, and reliability of the 

available data from the studies cited in Unit III regarding potential health effects for Cry1F 

protein. This evaluation included the low levels of expression of Cry1F protein in soybean, as 

well as the lack of acute oral toxicity at high dose levels, heat stability, and in vitro digestibility

of this protein. EPA also considered the minimal potential for allergenicity and the non-toxic 

source of the protein. Because of this lack of demonstrated mammalian toxicity, no protein 

residue chemistry data for Cry 1F were required for a human health effects assessment. 

Finally, and specifically with 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 with a 

common mechanism of toxicity. In addition, FFDCA section 408(b)(2)(C) provides that EPA 

shall apply an additional tenfold margin of safety for infants and children in the case of threshold 

effects to account for prenatal and postnatal toxicity and the 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 this 

BRAD, EPA concluded that there are no threshold effects of concern and, as a result, that an 

additional margin of safety for infants and children is unnecessary in this instance.

4. International Residue Limits

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 (MRLs) 

established by the Codex Alimentarius Commission (Codex), as required by 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 safety 

standards-setting organization 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 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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408(b)(4) requires that EPA explain the reasons for departing from the Codex level. No Codex 

maximum residue level exists for Bacillus thuringiensis Cry1Ac and Cry1F proteins as expressed 

in Event DAS-81419-2 Soybean.

C. Environmental Effects Assessment 

Ecological Effects Data for Cry1F and Cry1Ac Insecticidal Proteins Expressed in 

Event DAS-81419-2 Soybean

All ecological and environmental effects data requirements for the Bt soybean PIPs containing 

Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean have been satisfied. The 

submission of results of a confirmatory soil degradation study, however, is needed to evaluate 

the degradation rate of the Cry1Ac and Cry1F proteins sampled specifically from representative 

soybean soil systems. The completed soil degradation study must be received by the EPA within 

18 months of the DAS-81419-2 Soybean Notice of Pesticide Registration date. For a more 

comprehensive discussion of the Agency’s assessment of the data and information submitted 

concerning ecological and environmental risks for the registration of these PIPs (Cry1Ac, Cry1F, 

and their combination in DAS-81419-2 Soybean) refer to Appendix B. The tiered approach EPA

uses to evaluate ecological and environmental effects, and summaries of the data evaluated, are

included.

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. Limit dose 

testing on representative organisms from several taxa was performed in support of Section 3 

FIFRA registration of event DAS-81419-2 expressing Bt-derived Cry1F and Cry1Ac insecticidal 

proteins. As stated above, BPPD’s risk assessments focus more greatly on beneficial nontarget 

invertebrates, since they are most closely related to organisms susceptible to the insecticidal 

action of Bt toxins. The Cry1F and Cry1Ac proteins are meant to target species within the order 

Lepidoptera (e.g. moths). Bt toxins are known typically to have a limited host range, however, to 

address any unforeseen change in activity spectrum as a result of laboratory protein synthesis and 

to fulfill the published registration data requirements EPA requires that test species used for 

nontarget insect evaluations should include several invertebrate species that are not related to the 

target pests. Earthworm studies are also recommended. 

The applicant has requested to bridge to the Agency database of previously reviewed toxicity

studies of select nontarget organisms (NTOs) that supported the currently registered 

WideStrike PIP cotton product (EPA Reg. No. 68467-3). The WideStrike PIP cotton line 

was produced by conventional cross breeding of cotton lines DAS-21023-5 (also described as 

3006-210-23) expressing Cry1F protein and DAS-24236-5 (also described as 281-24-236) 

expressing Cry1Ac protein. The environmental risk assessment for the insecticidal proteins 

(Cry1Ac and Cry1F) expressed in WideStrike cotton was previously evaluated for adverse 

impacts on nontarget organisms and have been shown to pose negligible risk (US EPA 2005). 

Since exposure may also occur to other nontarget organisms, EPA has received a number of 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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studies and data waiver rationales to comply with the Agency’s published data requirements on 

other nontarget organisms. 

The toxicity of Cry1F and Cry1Ac proteins has been previously evaluated on several species of 

invertebrates including: lady beetle (Hippodamia convergens), green lacewing (Chrysoperla 

rufilabris), honey bee (Apis mellifera), collembola (Folsomia candida), parasitic wasp (Nasonia 

vitripennis) and earthworm (Eisenia fetida). Two new dietary toxicity studies were submitted in 

support for event DAS-81419-2 soybean, which includes Northern Bobwhite (Colinus 

viginianus) and Rainbow Trout, (Oncorhynchus Mykiss). The individual results for the cited and 

newly submitted nontarget organism studies in support for Cry1F and Cry1Ac are summarized in 

Table 1. The studies are described in more detail below, and full reviews of each study can be 

found in the individual Data Evaluation Records. 

To support data bridging from the previously conducted environmental risk assessment for 

Cry1F and Cry1Ac insecticidal proteins co-expressed in WideStrike PIP cotton line, an 

environmental risk assessment was conducted using a “weight-of-evidence” approach based on 

the following lines of evidence to support the registration of event DAS-81419-2 soybean:

1) Confirmation of biological equivalency of Cry1F and Cry1Ac proteins expressed in 

event DAS-81419-2 soybean plant and previously characterized microbial-derived 

Cry1F and Cry1Ac protein test substances; 

2) No significant increase in exposure to NTOs based on calculated margins of exposure 

for protein concentrations in event DAS-81419-2 plant tissue matrices relative to the 

dose concentrations used in the NTO laboratory toxicity tests conducted on Cry1Ac and 

Cry1F; and 

3) No adverse effects on nontarget arthropods on a population and community level 

observed from a field monitoring study and other supporting supplemental information 

(e.g. meta-analysis, target feeding studies, and biological specificity to the target pest). 

Exposure estimates were compared with endpoints reported from Tier I toxicity studies 

previously conducted on various representative nontarget test species to determine a Margin of 

Exposure (MOE), the ratio of the endpoint value to the EEC. The MOEs for representative NTOs 

were recalculated based on high-end exposure estimates (HEEEs) (US EPA, 2009) derived from 

Cry1Ac and Cry1F protein expression levels quantified from various plant tissues of event DAS81419-2 soybean in a field study. A MOE of 50% mortality at 10X the estimated environmental 

exposure is regarded as sufficient to demonstrate negligible risk and was also the threshold for 

consideration of uncertainties in the risk assessment (US EPA 1998; US EPA 2007). If 

subsequent expression characterization in the commercial event leads to a safety margin of 

<10X, then the consequences are considered in terms of uncertainties in the ecological risk 

assessment below (US EPA 2007). 

Endangered Species

Because of the selectivity of Cry1F and Cry1Ac insecticidal crystal proteins for lepidopteran 

species and lack of evidence of effects on other nontarget species, the Agency has investigated 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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concerns for Federally listed threatened and endangered insect species in the order Lepidoptera. 

Because soybean pollen is not expected to move beyond the planted soybean field and its 

immediate margins, as discussed above, any exposure to lepidopterans would be expected to 

occur within those areas. Exposure could occur via direct consumption of PIP event DAS-81419-

2 soybean plants or consumption of DAS-81419-2 pollen that falls on non-soybean plants within 

the soybean field and its immediate margins. However, oral exposure to significant amounts of 

Cry1F and Cry1Ac via pollen consumption is not likely. Little pollen is expected to be released 

from soybean flowers, since soybean plants are predominantly self-pollinated and anthers usually

dehisce and release pollen before flowers open. Airborne pollen concentrations have been 

measured at very low levels within soybean fields (mean of 0.18 grains/cm2/day) (Yoshimura et 

al. 2006). Based on this analysis, BPPD concludes that exposure resulting from pollen falling on 

potential non-soybean food plants in the field and immediate margins is not sufficient to cause 

effects in listed lepidopterans. Therefore, any significant exposure would have to occur through 

consumption of the DAS-81419-2 soybean plants on the field or via tritrophic effects, in which 

predatory beneficial arthropod species consume soybean pest species feeding on PIP event DAS81419-2 soybean plant tissues. 

A search of EPA’s LOCATES database indicates that three species of listed lepidopterans are 

present in U.S. counties in which soybeans are grown. These are the Karner blue butterfly 

(Lycaeides melissa samuelis), St. Francis’s Satyr Butterfly (Neonympha mitchellii fransisci), and 

Mitchell’s Satyr Butterfly (Neonympha mitchellii mitchellii). The potential effects of Bt PIPs in 

corn on the Karner blue butterfly was extensively analyzed in BPPD’s Bt Crops Reassessment 

(USEPA 2001b, 2004b and 2010a and b) and in the endangered species assessment section in the 

BRAD for event MON87701 soybean expressing Cry1Ac protein (see US EPA 2010c). As 

previously discussed, soybean pollen is not expected to be deposited on plants in or around 

soybean fields in amounts sufficient to cause effects in sensitive lepidopterans. Ecology and life 

history information demonstrated that habitat requirements for larvae and adults of two of the 

identified species (Mitchell's Satyr butterfly and Saint Francis' Satyr butterfly) do not overlap 

with commercial soybean acreage, and are therefore not expected to be impacted by PIP event 

DAS-81419-2 cultivation (Bartel and Sexton 2009; Barton and Bach 2005; US FWS 1997). 

There is a possible overlap between the geographic range for Karner blue butterfly and soybean 

use sites. EPA has previously reviewed information on the proximity of Karner blue habitats, and 

none are known to exist immediately adjacent to agricultural fields (“adjacent to” was defined as 

0-3 m for corn fields, which is reasonably applied to soybean fields) (US EPA 2001b). Karner 

blue larvae feed only on lupines (Lupinus spp.) and soybean is neither identified as a larval food 

source, nor is soybean open-pollinated. Thus, pollen from event DAS-81419-2 soybean is highly

unlikely to be deposited on lupine leaves (NatureServe 2013). Furthermore, soybean is not 

identified as a nectaring source for Karner blue adults. Therefore, the risk posed to the Karner 

blue butterfly was considered to be negligible. 

Based on the above analysis, EPA determines that there will be no direct effect to listed 

lepidopteran species as a result of the cultivation of DAS-81419-2 soybeans as proposed. 

Obligate relationships between insectivorous listed species with lepidopterans that are expected 

to be found in soybean fields, especially pest species that feed on DAS-81419-2 plants, are not 

currently known. Since the Cry1F and Cry1Ac in DAS-81419-2 soybeans targets only 

lepidopteran insects, loss of the pest insects as a result of DAS-81419-2 are expected to be offset 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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by the presence of other insects that could act as food sources for listed species, including 

beneficial insects that are known not to be affected by Cry1F and Cry1Ac. Effects on species 

other than insects have also been determined to be very unlikely because of the specificity of 

Cry1F and Cry1Ac.

Lastly, since the Agency previously determined Cry1F and Cry1Ac proteins are not expected to 

have adverse effects on mammals, birds, plants, freshwater and estuarine/marine fish and 

invertebrates, nontarget insects and other invertebrate species at the EEC, a “No Effect” 

determination is made for direct and indirect effects to Federally listed threatened and 

endangered species and their designated Critical Habitats. In addition, EPA does not expect that 

any threatened or endangered plant species will be affected by outcrossing to wild relatives or by 

competition with such entities. Hybrid soybean does not exist in the wild in the United States, 

nor do any wild plants that can interbreed with soybean in the United States.

D. Insect Resistance Management

The goal of insect resistance management (IRM) is to mitigate the potential risk of resistance in 

Bt crops, including Bt soybean. IRM typically involves two phases: 1) data submitted to assess 

the risk of resistance for a certain Bt toxin and crop including information and data on pest 

biology, dose expression of the toxins in the PIP, simulation modeling, cross resistance potential 

and baseline susceptibility of the target pests to the toxin(s); and 2) a post-registration 

stewardship program to implement the resistance mitigation measures which includes resistance 

monitoring, remedial action (should resistance occur), grower compliance with IRM 

requirements, and grower education. 

Dow submitted IRM considerations with their application for registration of DAS-81419-2 

Soybean (MRID No. 48828415 and its replacement, MRID No. 49024110). BPPD concluded 

that Dow’s IRM submissions satisfied the IRM requirements for DAS-81419-2 Soybean when 

used for breeding/seed increase.

BPPD’s major IRM conclusions from its assessment of DAS-81419-2 Soybean are detailed 

below. 

Conclusions and Requirements

1) Because DAS-81419-2 Soybean is intended for breeding/seed increase, Dow did not propose a 

species-specific IRM plan for the product. Rather, the company proposed to mitigate resistance 

risk by acreage limitations on a county (1,000, 10,000, or 25,000 acres) and national (250,000 

acres) scale. BPPD agrees that given the low acreage projected by Dow, it is unlikely that there 

will be a significant risk of resistance to the main soybean lepidopteran pests in the United 

States. These insects include soybean looper, velvetbean caterpillar, beet armyworm, cotton 

bollworm, green cloverworm, tobacco budworm, and cutworms (Agrotis spp.). This conclusion 

is further supported by the biology of the target insects, which are highly polyphagous (feeding 

on a number of wild hosts and cultivated crops) and (for some species) have limited 

overwintering in non-tropical areas.
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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2) Should a full commercial registration or expanded acreage be sought in the future, Dow must

submit a complete, species-specific IRM assessment for DAS-81419-2 Soybean. Data needed to 

support such a review should include additional information on pest biology, dose, simulation 

modeling, cross resistance, resistance monitoring, and potential impacts on the natural refuge 

strategy for Bt cotton.

3) The use of DAS-81419-2 Soybean on a limited basis for seed increase purposes should not 

significantly impact the natural refuge strategy in place for Bt cotton PIPs. Soybean is one of the 

non-cotton crops that have been considered as part of natural refuge for tobacco budworm and 

cotton bollworm. While it can be expected that plantings of DAS-81419-2 soybean will remove 

some of the currently-available natural refuge for Bt cotton, this potential reduction will likely be 

small relative to the total amount of natural refuge present in southern cotton regions. Dow has 

proposed to further mitigate the impact on natural refuge by limiting DAS-81419-2 to 10,000 

acres in counties with > 25,000 total acres of soybean and 1,000 acres in counties with < 25,000 

total acres of soybean. 

4) As a term of registration, Dow must submit a resistance monitoring and remedial action plan 

for DAS-81419-2 Soybean. A resistance monitoring plan limited to investigations of unexpected 

pest damage is appropriate for a capped acreage registration. BPPD notes that monitoring of two 

potential target insects, tobacco budworm and cotton bollworm, is already being conducted as 

part of the WideStrike Bt cotton monitoring program. Dow must also compile annual sales and 

acreage data for each state and provide a DAS-81419-2 Soybean-specific report to EPA if 

requested. Since DAS-81419-2 Soybean will be deployed without a structured refuge 

requirement, a compliance assurance plan and grower education program is not necessary.

Overall, BPPD concluded that Dow has submitted adequate resistance management data and 

information to support breeding/seed increase use of the product. There are no data gaps, though 

Dow must submit additional information, described above, to support any future request for 

either expanded acreage or commercial registration. 

A full discussion of BPPD’s IRM review of the supporting data for the Cry1Ac and Cry1F 

proteins expressed as DAS-81419-2 Soybean PIP, including technical references, is available as 

Appendix C of this BRAD.

IV. ENVIRONMENTAL JUSTICE

EPA seeks to achieve environmental justice, the fair treatment and meaningful involvement of all 

people, regardless of race, color, national origin, or income, in the development, implementation, 

and enforcement of environmental laws, regulations, and policies. To help address potential 

environmental justice issues, the Agency seeks information on any groups or segments of the 

population who, as a result of their location, cultural practices, or other factors, may have 

atypical, unusually high exposure to the Bt proteins Cry1Ac and Cry1F as expressed in PIP 

Event DAS-81419-2 Soybean compared to the general population. 

For additional information regarding environmental justice issues, please visit EPA’s web site at 

http://www.epa.gov/compliance/environmentaljustice/index.html.
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V. RISK MANAGEMENT AND PROPOSED REGISTRATION 

DECISION

Section 3(c)(5) of FIFRA provides for the registration of a new active ingredient if it is 

determined that (A) its composition is such as to warrant the proposed claims for it; (B) its 

labeling and other materials required to be submitted comply with the requirements of FIFRA; 

(C) it will perform its intended function without unreasonable adverse effects on the 

environment; and (D) when used in accordance with widespread and commonly recognized 

practice, it will not generally cause unreasonable adverse effects on the environment. 

The four criteria of the Eligibility Determination for Pesticidal Active Ingredients are satisfied by 

the science assessments supporting the PIPs containing Bacillus thuringiensis proteins Cry1Ac

and Cry1F as expressed in Event DAS-81419-2 Soybean. These PIPs are not expected to cause 

unreasonable adverse effects and are likely to provide protection as claimed when used 

according to label instructions. The EPA believes, therefore, that DAS-81419-2 Soybean is 

eligible for registration for the proposed uses. 

VI. TERMS AND CONDITIONS OF THE REGISTRATION(S)

As a term of registration, the applicant must submit or provide the following:

Expression in a Terrestrial Environment: OCSPP 885.5200 (40 CFR § 158.2150)

Although the registrant submitted a sufficient rationale of rapid degradation of Cry1F and 

Cry1Ac proteins in other crop soil systems, a soil degradation study has not been evaluated in 

soybean for this specific PIP event. A previous EPA FIFRA SAP noted that in soil degradation 

and field persistence studies, toxin degradation rates may vary with the crop, the toxin produced 

by the transformation event, the microbial community in the recipient soil microcosm, the 

statistical characterization of toxin persistence, and/or the initial dose of toxin in the experiment 

(US EPA 2001a). These differences in Cry toxin concentration may be due to the fact that 

different genetic constructs were used and/or inserted at different locations in the plant genome 

(Yu et al. 2012). Plant genotype and parental backgrounds have also been recognized as 

important factors influencing protein expression (Kranthi et al. 2005; Adamczyk and Sumerford 

2001; and Torres et al. 2006). Other important factors that affect degradation of Cry toxins 

include environmental conditions such as temperature, light, drought, and soil properties (e.g. 

pH, texture, clay content) (Adamczyk et al. 2004; Chen et al. 2005; Jiang et al. 2006; Rochester 

2006; Dong et al. 2008; Hallikeri et al. 2009; Addison and Rogers 2010; Chen et al. 2011; 

Ranjithkumar et al. 2011). To reduce this uncertainty, a soil fate study to determine the 

degradation rate (DT50) of Cry1F and Cry1Ac proteins sampled from representative soils of 

soybean growing regions is to be conducted as confirmatory data. The completed soil 

degradation study must be received by the EPA within 18 months of the DAS-81419-2 Soybean 

Notice of Pesticide Registration date. 

Insect Resistance Management

Dow must submit a resistance monitoring and remedial action plan for DAS-81419-2 Soybean. 

Although a full resistance monitoring plan with pest sampling and detection bioassays is not 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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warranted for this seed increase registration, an approach based on investigations of unexpected 

pest damage reports is appropriate. Monitoring for the subject proteins is conducted as part of the 

WideStrike cotton (EPA Registration Number 68467-3) monitoring program, and a remedial 

action plan (should resistance be documented) aligned with the plans in place for Bt corn or 

cotton (e.g., a “stop sale” if resistance develops) can be adapted for DAS-81419-2 Soybean. 

Additionally, annual sales and total acreage data reports for each state must be compiled by Dow 

for submission to the EPA (upon request). 

The subject registration is limited to seed increase and to a total of 250,000 acres per year with 

no more than 20,000 acres per county (in non-cotton growing regions); 10,000 acres per county

(in cotton-growing counties with at least 25,000 acres of soybean); or 1,000 acres (in cottongrowing counties with less than 25,000 acres of soybean) per year

A. Adverse Effects

Reports of all incidents of adverse effects to the environment must be submitted to the EPA

under the provisions stated in FIFRA Section 6(a)(2).

B. Hypersensitivity Incidents

All incidents of hypersensitivity (including both suspected and confirmed incidents) must be 

reported to the Agency under the provisions of 40 CFR §158.2140(d).
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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APPENDIX A: Product characterization and human health effects –

Bacillus thuringiensis Cry1Ac and Cry1F Proteins expressed in Event 

DAS-81419-2 Soybean plant-incorporated protectant.

A.1. BACKGROUND

Event DAS-81419-2 expresses the δ-endotoxins Cry1Ac and Cry1F derived from Bacillus 

thuringiensis. Product characterization and human health data submitted for seed increase 

registration of DAS-81419-2 are summarized below. 

A.2. PRODUCT CHARACTERIZATION 

Event DAS-81419-2 was developed by Agrobacterium-mediated transformation of soybean 

using the 2T-DNA plasmid vector pDAB9582 and produces Bacillus thuringiensis protein δendotoxins, Cry1Ac and Cry1F. A single T-DNA insert containing each of the intact plant 

transcription units (PTUs) for the cry1Fv3, cry1Ac(synpro), and pat genes was integrated into 

soybean to create DAS-81419-2. This protein is intended to provide protection from feeding 

damage caused by a number of lepidopteran pests. 

a. Transformation System and Genetic Elements

Molecular characterization of DAS-81419-2 soybean by Southern blot analysis confirmed that a 

single T-DNA insert containing each of the intact plant transcription units (PTUs) for the 

cry1Fv3, cry1Ac(synpro), and pat genes from plasmid pDAB9582, was integrated into DAS81419-2 soybean. In addition, a minor (<100 bp) fragment of the cry1Ac(synpro) gene was 

identified on the 5′ end of the T-DNA insert. Data were generated to evaluate the integration and 

integrity of the cry1Fv3, cry1Ac(synpro), and pat genes inserted into the soybean genome. 

Characterization of the integration of non-coding regions (designed to regulate the coding 

regions), such as promoters and terminators was also performed. The inserted DNA was stably

inherited across the five generations (T1, T2, T3, T4, and F2) evaluated. No transformation 

plasmid backbone sequence was found in DAS-81419-2 soybean as demonstrated by Southern 

blot analysis using probes covering the entire region of the plasmid flanking the T-DNA insert.

b. Protein Characterization

Protein characterization data demonstrate that the plant-produced Cry1Ac and Cry1F have 

biochemical and functional activities that are similar to those of the Pseudomonas fluorescensproduced proteins that were used in several toxicity studies. The following techniques were used 

to characterize and compare the plant-produced and the Pseudomonas fluorescens-produced 

proteins: sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot 

analysis, densitometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) 

/mass spectrometry, glycosylation analysis, N-terminal amino acid sequencing, and insecticidal 

activity bioassays. Glycoslyation analysis indicated that the protein is not glycoslyated. These 

analyses demonstrated the structural and functional similarity between the plant-produced and 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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the Pseudomonas fluorescens-produced Cry1Ac and Cry1F proteins and justified the use of 

Pseudomonas fluorescens-produced protein in toxicity studies. 

c. Analytical Detection Methods

An exemption from the requirement of a food tolerance is already in existence (since 1997) for 

Cry1Ac protein for all food commodities (40 CFR 174.510) and an appropriate analytical 

detection method was previously reviewed for Cry1Ac. An exemption from the requirement of a 

food tolerance exists for Cry1F, however, the exemption is limited to expression in maize (40

CFR 174.520) and cotton (40 CFR 174.504). A petition for an exemption from the requirement 

of a food tolerance for Cry1F in soybean was included with the application from Dow. 

Dow AgroSciences LLC analytical method 110674, "Method Validation for the Determination 

Cry1Ac Protein in Soybean Tissues by Enzyme-Linked Immunosorbent Assay (ELISA)", was 

demonstrated to be suitable for its intended purpose. The method was validated over the 

concentration range of 0.1 to 2 ng Cry1Ac/mg dry weight (DW) and has a validated limit of 

quantitation (LOQ) in soybean tissues of 0.2 ng/mg DW and a limit of detection (LOD) of 0.1 

ng/mg DW. The Cry1Ac protein was recovered at acceptable levels from soybean tissues. The 

Cry1Ac protein was efficiently extracted from all tested soybean tissues. The assay was shown to 

have acceptable accuracy, precision and ruggedness, and no false positive or false negative 

results were seen below the target LOD. It is concluded that this Cry1Ac ELISA method has 

been demonstrated to be suitable for quantitative measurements of the Cry1Ac protein in 

soybean tissue. 

Dow AgroSciences LLC analytical method 110675, "Determination of Cry1F Protein in Soybean 

Tissues Using an Enzyme-Linked Immunosorbent Assay (ELISA)", was demonstrated to be 

suitable for its intended purpose. The method was validated over the concentration range of from 

0.05 ng/mg to 1 ng/mg DW for root R3, from 0.15 ng/mg to 1.5 ng/mg DW for forage R3, from 

0.2 ng/mg to 2 ng/mg DW for grain, and from 0.25 ng/mg DW to 2.5 ng/mg DW for leaf V5 and 

leaf V10-12. The method has a validated limit of quantitation (LOQ) of 0.1 ng/mg DW for root 

R3, 0.3 ng/mg DW for forage R3, 0.4 ng/mg DW for grain, and 0.5 ng/mg DW for leaf V5 and 

leaf V10-12, and a limit of detection (LOD) of 0.05 ng/mg DW for root R3, 0.15 ng/mg DW for 

forage R3, 0.20 ng/mg DW for grain, and 0.25 ng/mg DW for leaf V5 and leaf V10-12. Cry1F 

protein was recovered at acceptable levels from soybean tissues. This Cry1F ELISA method has 

been demonstrated to be suitable for quantitative measurements of the Cry1F protein in soybean 

tissue.

d. Protein Expression

Expression level data were provided for Cry1Ac in different plant tissues and at different growth 

stages. Cry1Ac protein is expressed at relatively low levels in event DAS-81419-2 soybean. The 

data were produced using ELISA methods for this protein. Summary results are provided below 

in Table A.1. Table A.2. provides summaries of the product characterization studies and data 

provided.

Table A.1. Protein Expression in DAS-81419-2 soybean
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 25 of 78

Summary of Cry1Ac Protein Expression

Test Cry1Ac ng/mg Tissue Dry Weight

Matrix Entry Description Overall 

Mean

Std. Dev. 

(n=10)

Min/Max 

Range STMRa HAFTb

V5 Leaf 7 DAS-81419-2 25.44 6.61 12.10 - 40.20 26.90 35.25

V10-12 Leaf 7 DAS-81419-2 23.16 6.17 10.70 - 37.45 22.15 34.61

Forage 7 DAS-81419-2 5.54 2.54 1.38 - 11.83 5.44 10.28

Root 7 DAS-81419-2 0.39 0.24 [0.12] - 1.12 0.32 0.97

Grain 7 DAS-81419-2 1.04 0.10 0.79 - 1.40 1.04 1.20

a

Supervised Trials Mean Residue b Highest Average Field Trial

Summary of Cry1F Protein Expression

Test Cry1F ng/mg Tissue Dry Weight

Matrix Entry Description Overall 

Mean

Std. Dev. 

(n=10)

Min/Max 

Range STMRa HAFTb

V5 Leaf 7 DAS-81419-2 56.75 15.03 24.60 - 99.50 56.30 76.05

V10-12 Leaf 7 DAS-81419-2 39.07 16.60 12.75 - 76.71 38.70 59.98

Forage 7 DAS-81419-2 20.28 11.29 5.34 - 44.62 20.64 40.23

Root 7 DAS-81419-2 5.23 3.74 1.09 - 16.08 4.12 14.21

Grain 7 DAS-81419-2 13.80 1.24 10.41 - 16.95 13.71 16.21

a

Supervised Trials Mean Residue b Highest Average Field Trial

Summary of PAT Protein Expression

Test PAT ng/mg Tissue Dry Weight

Matrix Entry Description Overall 

Mean

Std. Dev. 

(n=10)

Min/Max 

Range STMRa HAFTb

V5 Leaf 7 DAS-81419-2 5.23 0.88 3.25 - 7.35 5.30 6.93

V10-12 Leaf 7 DAS-81419-2 5.60 1.14 2.55 - 7.56 5.76 7.32

Forage 7 DAS-81419-2 4.06 1.30 1.24 - 6.12 4.02 5.69

Root 7 DAS-81419-2 0.63 0.12 0.44 - 1.05 0.63 0.85

Grain 7 DAS-81419-2 0.86 0.13 0.63 - 1.12 0.83 1.06

a

Supervised Trials Mean Residue b Highest Average Field Trial
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 26 of 78

Table A.2. Summary of Product Characterization Data for PIP Event DAS-81419-2 

soybean (EPA Reg. No. 68467-EN) 

Study Type/Title Summary MRID No.

Molecular 

Characterization of 

DAS-81419-2 

Soybean

Data from multiple Southern blots probed with various fragments of 

the cry and pat genes, as well as promoter and 3’ UTR sequences, 

indicate that a single insertion of T-DNA is present in DAS-81419-2 

Soybean. Samples from five generations of DAS-81419-2 soybean 

yielded identical patterns of hybridization indicating that the insertion 

is stably introduced into the plant genome. Further, the absence of 

pDAB9582 backbone sequences was demonstrated with probing of 

Southern blots using 4 different sequences (i.e., Specr, Backbone, 1, 2 

and 3) outside of the T-DNA region of the plasmid. All blots 

included genomic DNA from a non-transgenic soybean, Maverick, 

and a Maverick DNA sample spiked with pDAB9582 DNA as

negative and positive controls, respectively. 

Classification: ACCEPTABLE

48828401

Protein Expression of 

a Transformed 

Soybean Cultivar 

Containing Cry1Ac, 

Cry1F, and 

Phosphinothricin 

Acetyltransferase 

(PAT) - Event DAS81419-2

Measures of protein accumulation at different growth stages and in 

different organs of DAS-81419-2 Soybean plants were obtained 

using appropriate methodology. The authors of this study quantified 

Cry1Ac, Cry1F and PAT proteins in leaf, forage (aerial portions of 

plant without pods), root and seed of soybean cultivated in 10 

different localities typical for this crop. The ELISA-based methods 

were typical for this technique and the findings were statistically 

acceptable (i.e., variance is within acceptable limits). Sample 

handling and processing were also performed appropriately.

Classification: ACCEPTABLE

48828402

Biological 

Equivalency of 

(DAS-81419-2) and 

microbe-produced 

Cry1Ac and Cry1F 

Proteins

With respect to biological activity of the plant-produced and 

microbe-produced Cry1Ac and Cry1F proteins, LC50 data for the 

target pest Chrysodeixis includens (soybean looper) indicate that the 

plant- and microbe-derived Cry proteins retain similar activity 

against this insect. LC50 values vary by less than 15%, which is an 

acceptable level of variance for such a biological system. 

Classification: ACCEPTABLE

48828403

Method Validation 

for the Determination 

of Cry1Ac Protein in 

Soybean Tissues by 

Enzyme-Linked 

Immunosorbent 

Assay (ELISA)

Analytical method 110674 was validated to perform robustly in the 

quantitation of Cry1Ac protein in soybean organs in the range of 0.2 

ng/mg DW to 2.9 ng/mg DW using an ELISA-based technique with 

two monoclonal antibodies raised against Cry1Ac. The method 

performed with acceptable level of accuracy in the quantitation of 

Cry1Ac in soybean organ samples, including leaf, root, forage (aerial 

portions of plant without pods), and seed. Given some matrix effects, 

a 2X dilution of samples is recommended for soybean samples to 

more accurately quantify Cry1Ac. Variability in results was within 

48828404
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 27 of 78

acceptable limits as the Coefficient of Variation (CV) values were all 

less than 20%. No cross reactivity with a variety of nontarget proteins 

was detected and extraction efficiencies from soybean samples were 

at acceptable levels. 

Classification: ACCEPTABLE

Method Validation 

for the Determination 

of Cry1F Protein in 

Soybean Tissues 

Using an EnzymeLinked 

Immunosorbent 

Assay (ELISA)

The ELISA-based double antibody sandwich method employed by 

the study authors accurately quantified target protein Cry1F in 

soybean organs sampled (root, forage, leaves, seed). The analytical

method 110675 was validated over a range of protein concentration 

values appropriate for the levels as found in cultivated soybean 

spiked with known quantities of Cry1F protein. The range is, 

however, on the lower end of most transgenic soy samples as 

evidenced in other volumes of this submission as some leaf samples 

contained concentrations of Cry proteins in the 25 to 56 ng/mg DW 

range. 

Matrix effects were adequately resolved and indicate that in practice, 

dilution of root, leaf, seed and forage samples will need to be 

performed at 1X, 5X, 4X, and 3X respectively to minimize these 

effects on quantification of Cry1F in soybean. The variance recorded 

among samples and between different analysts was less than 20%, an 

acceptable amount when working with complex plant organ extracts, 

with the exception of root samples spiked to yield quantities of 

Cry1F protein at or near the LOD (0.1 ng/mg DW). Higher 

variability at or near an LOD is not unexpected. The ELISA-based 

method 110675 was not confounded by false positives or negatives 

and only minimal cross reaction with Cry1Ac (0.114 %) was 

observed while other nontarget proteins were similarly non-cross 

reactive. The method appears robust enough for practical use in the 

detection of Cry1F in soybean.

Classification: ACCEPTABLE

48828405

Characterization of 

the Cry1Ac Protein 

Derived from 

Transgenic Soybean 

Event DAS-81419-2

The study authors employed several techniques to adequately 

demonstrate that the Cry1Ac protein from soybean event DAS81419-2 is biochemically equivalent to the Cry1Ac protein as 

expressed in Pseudomonas fluorescens. Lateral test flow strips (an 

ELISA-based method), immunoprecipitation, detection of glycosyl 

residues, Western blot, MALDI-TOF / MS, MALDI-TOF/TOF, 

MS/MS, and SDS-PAGE analyses all support the contention that the 

two proteins are biochemically equivalent. Samples were processed, 

stored and handled in an appropriate manner to maintain integrity of 

these protein samples. 

Classification: ACCEPTABLE

48828416

Characterization of 

the Cry1F Protein 

Derived from 

Transgenic Soybean 

Event DAS-81419-2

The study authors employed several techniques to adequately 

demonstrate that the Cry1F protein from soybean event DAS-81419-

2 is biochemically equivalent to the Cry1F protein as expressed in 

Pseudomonas fluorescens. Lateral test flow strips (an ELISA-based 

method), immunoprecipitation, detection of glycosyl residues, 

48828417
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 28 of 78

Western blot, MALDI-TOF / MS, MALDI-TOF/TOF, MS/MS, and 

SDS-PAGE analyses all support the contention that the two proteins 

are biochemically equivalent. Samples were processed, stored and 

handled in an appropriate manner to maintain integrity of these 

protein samples. 

Classification: ACCEPTABLE

Amended Response 

to EPA Comment on 

Characterization of 

the Cry1F Protein 

Derived from 

Transgenic Soybean 

Event DAS-81419-2 

Study – Address 

amino acid sequence 

similarity between 

Dow’s Cry1F 

microbial protein and 

soybean produced 

Cry1F: Related to 

MRID 488284-17

The synthetic Cry1F protein expressed in DAS-81419-02 Soybean 

and the Cry1F protein expressed in Pseudomonas fluorescens strain 

MR872 differ at 4 amino acid positions in the full length proteins. 

The variations or alterations in amino acid residues for these 4 

positions (residues 604, 608, 624, 629) do not affect the biochemical 

or biological properties evaluated in this risk assessment. For the 

purposes of testing, as performed, the proteins are considered 

equivalent. 

Classification: ACCEPTABLE

49081501

A. 3. HUMAN HEALTH ASSESSMENT 

Section 408(c)(2)(A)(i) of the Federal Food, Drug, and Cosmetic Act (FFDCA) allows EPA to 

establish an exemption from the requirement for a tolerance (the legal limit for a pesticide 

chemical residue in or on a food) only if EPA determines that the exemption is “safe.” Section 

408(c)(2)(A)(ii) of the FFDCA defines “safe” to mean that “there is a reasonable 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 and in residential settings, but does not include 

occupational exposure. Pursuant to section 408(c)(2)(B), in establishing or maintaining in effect 

an exemption from the requirement of a tolerance, EPA must take into account the factors set 

forth in section 408(b)(2)(C), which require EPA to give special consideration to exposure of 

infants and children to the pesticide chemical residue in establishing a tolerance and to “ensure 

that there is a reasonable certainty that no harm will result to infants and children from aggregate 

exposure to the pesticide chemical residue... .” 

Additionally, section 408(b)(2)(D) of the FFDCA requires that the Agency consider “available 

information concerning the cumulative effects of a particular pesticide’s residues” and “other 

substances that have a common mechanism of toxicity.” EPA performs a number of analyses to 

determine the risks from aggregate exposure to pesticide residues. First, EPA determines the 

toxicity of pesticides. Second, EPA examines exposure to the pesticide through food, drinking 

water, and through other exposures that occur as a result of pesticide use in residential settings. 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 29 of 78

Dow submitted a pesticide tolerance petition (PP2F8066) under the FFDCA, requesting 40 CFR 

Part 174 be amended by establishing an exemption from the requirement of a tolerance for 

residues of Bacillus thuringiensis (Bt) Cry1F insect control protein and the genetic material 

necessary for its production in food commodities of soybean.

Consistent with section 408(b)(2)(D) of the FFDCA, EPA has reviewed the available scientific 

data and other relevant information in support of this action and considered its validity, 

completeness and reliability, and the relationship of this information to human risk. EPA has also 

considered available information concerning the variability of the sensitivities of major 

identifiable subgroups of consumers, including infants and children.

a. Toxicological Profile

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 health risk. No indication of toxicity toward mammals was noted in the 

review of the two Cry proteins expressed in soybean. 

It should be noted that the allergenicity of Cry1Ac protein was evaluated previously (Wozniak, 

2010) as evidenced in MRID No. 47841709 (US EPA, 2010). Based upon sequence comparisons 

to known allergens and digestibility assays (MRID No. 47841708), Cry1Ac was considered to be 

of little potential to function as an allergen. 

An exemption from the requirement of a food tolerance was established previously for 

phosphinothricin acetyltransferase (PAT; 40 CFR 174.522) as expressed as part of a plantincorporated protectant, so no further toxicological review of this protein is contained herein. 

The human health studies submitted for Cry1Ac and Cry1F are summarized in Table A.3. below.

Table A.3. Summary of Human Health Data conducted for PIP Event DAS-81419-2 

soybean (EPA Reg. No. 68467-EN) 

Study Type/Title Summary MRID No.

Sequence Similarity 

Assessment of Cry1F 

Protein to Known 

Allergens by 

Bioinformatics 

Analysis

The study authors have utilized appropriate search methods in 

comparing the sequence of their Cry1F protein with the 

‘allergenonline’ database of known allergens. The default parameters 

applied to the search are sufficient to identify sequences with > 35% 

homology over any 80 amino acid sequence and to detect any 

individual 8 amino acid sequences which may represent epitopes of 

allergens. Given the exhaustive analysis of Cry proteins, including 

Cry1F and closely related δ-endotoxins, over many years for the 

presence of sequences representing potential allergenic epitopes and 

the general lack of demonstrated IgE-mediated immune responses to 

those exposed to preparations of biopesticidal Bacillus thuringiensis, 

48828406
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 30 of 78

the findings are not surprising or unexpected. 

It should be noted that the allergenicity of Cry1F protein was 

evaluated previously (Wozniak, 2002; Matten 2005) as evidenced in 

MRID No. 44971701 and discussed in a Biopesticide Registration 

Action Document (US EPA, 2005; ibid 2010). Based upon sequence 

comparisons to known allergens and digestibility assays (MRID No. 

45542312), Cry1F was considered to be of little potential to function 

as an allergen. 

Classification: ACCEPTABLE

Sequence Similarity 

Assessment of Cry1F 

to Known Toxins by 

Bioinformatics 

Analysis

The study authors queried appropriate databases containing 

sequences of proteins, including those with potential toxicity to 

vertebrates. Parameters chosen for the search were selected to detect 

proteins with minimal homology in sequence such that even those 

with insufficient similarity to trigger a toxic reaction may be 

uncovered. Of the 672 proteins or hypothetical protein sequences 

returned from the search E-value < 1.0, including 646 with E-value

<0.01, 618 were clearly related to δ-endotoxins from Bacillus 

thuringiensis and two were related to proteins from other Bacillus

species (e.g, B. cereus). 

Of the remaining 28 protein hits from the databases, some of the 

sequences were from parasporal proteins of related species (e.g., 

Lysinibacillus sphaericus, Paenibacillus lentimorbus) and also from 

hypothetical proteins (of no known function) from eubacteria, slime 

molds / Mycetozoa and a fungal species (Fusarium oxysporum). 

These proteins and sequences of hypothetical proteins are not known 

to represent toxic moieties with respect to vertebrates. 

As is typical with previously characterized Cry proteins of B. 

thuringiensis, no toxicity to vertebrates, including man, is expected 

from consumption or contact with these proteins. 

Classification: ACCEPTABLE

48828407

Sequence Similarity 

Assessment of 

Cry1Ac Protein to 

Known Allergens by 

Bioinformatics 

Analysis

The study authors have utilized appropriate search methods in 

comparing the sequence of their Cry1Ac protein with the 

‘allergenonline’ database of known allergens. The default parameters 

applied to the search are sufficient to identify sequences with > 35% 

homology over any 80 amino acid sequence and to detect any 

individual 8 amino acid sequences which may represent epitopes of 

allergens. Given the exhaustive analysis of Cry proteins, including 

Cry1Ac and closely related δ-endotoxins, over many years for the 

presence of sequences representing potential allergenic epitopes and 

the general lack of demonstrated IgE-mediated immune responses to 

those exposed to preparations of biopesticidal Bacillus thuringiensis, 

the findings are not surprising or unexpected. 

It should be noted that the allergenicity of Cry1Ac protein was 

evaluated previously as evidenced in MRID No. 45542319 (US EPA, 

2005). Based upon sequence comparisons to known allergens and 

48828408
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 31 of 78

digestibility assays (MRID No. 47841708), Cry1Ac was considered 

to be of little potential to function as an allergen. 

REFERENCES: 

US Environmental Protection Agency (2010) BIOPESTICIDE 

REGISTRATION ACTION DOCUMENT, Bacillus thuringiensis 

Cry1Ac Protein and the Genetic Material (Vector PV-GMIR9) 

Necessary for Its Production in MON 87701 (OECD Unique 

Identifier: MON 877Ø1-2) Soybean [PC Code 006532] 

http://www.epa.gov/pesticides/biopesticides/pips/bt-cry1acprotien.pdf

Classification: ACCEPTABLE

Similarity Assessment 

of Cry1Ac Protein to 

Known Toxins by 

Bioinformatics 

Analysis

The study authors queried appropriate databases containing 

sequences of proteins, including those with potential toxicity to 

vertebrates. Parameters chosen for the search were selected to detect 

proteins with minimal homology in sequence such that even those 

with insufficient similarity to trigger a toxic reaction may be 

uncovered. Of the 684 proteins or hypothetical protein sequences 

returned from the search, 662 hits were clearly related (E-value

<0.01) to δ-endotoxins from Bacillus thuringiensis and two were 

related to proteins from other Bacillus species. 

Of the remaining 52 protein hits from the databases, some of the 

sequences were from parasporal proteins of related species (e.g., 

Bacillus cereus, Paenibacillus lentimorbus) and also from 

hypothetical proteins (of no known function) from eubacteria, slime 

molds / Amoebozoa and fungal species. These proteins and 

sequences of hypothetical proteins are not known to represent toxic 

moieties with respect to vertebrates. 

As is typical with previously characterized Cry proteins of B. 

thuringiensis, no toxicity to vertebrates, including man, is expected 

from consumption or contact with these proteins. 

Classification: ACCEPTABLE

48828409

b. Mammalian Toxicity and Allergenicity Assessment

Previously submitted acute oral toxicity data demonstrated a lack of mammalian toxicity at high 

levels of exposure (ingestion) to the pure Cry1Ac protein. These data demonstrate the safety of 

this protein at a level well above maximum possible consumption levels that are reasonably 

anticipated as expressed in the crop. Basing this conclusion on acute oral toxicity data without 

requiring further toxicity 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 Sec. 

158.740(b)(2)(i)). For microbial products, further toxicity testing (Tiers II & III) and residue data 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 32 of 78

are triggered by significant adverse acute effects in studies, such as the acute oral toxicity study, 

to verify the observed adverse effects and clarify the source of these effects. 

An acute oral toxicity study in mice (MRID No. 45542313) indicated that Cry1Ac is non-toxic to 

humans and other mammals. Cry1Ac protein is a δ-endotoxin from B. thuringiensis that has been 

used extensively in both microbial and plant-incorporated protectants as a means of insect pest 

management. An existing exemption from the requirement of a tolerance for Cry1Ac (CFR 40 

Section 174.510; 72 FR 20435, April 25, 1997) in all food commodities precluded the need for a 

separate tolerance action in conjunction with this review of product characterization and human 

health assessment.

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 by Cry1Ac, 

even at relatively high dose levels, the Cry1Ac protein is not considered toxic. Further, amino 

acid sequence comparisons showed no similarities that would raise a safety concern between the 

Cry1Ac protein and known toxic proteins in protein databases. 

Since Cry1Ac is a protein, allergenic potential was also considered. Currently, no definitive tests 

for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight-ofevidence approach where the following factors are considered: source of the trait; amino acid 

sequence comparison with known allergens; and biochemical properties of the protein, including

in vitro digestibility in simulated gastric fluid (SGF) followed by simulated intestinal fluid (SIF), 

and glycosylation. This approach is consistent with the approach outlined in the Annex to the

Codex Alimentarius “Guideline for the Conduct of Food Safety Assessment of Foods Derived 

from Recombinant-DNA Plants.” The allergenicity assessment for Cry1Ac follows:

1. Source of the trait. Bacillus thuringiensis is not considered to be a source of allergenic 

proteins. 

2. Amino acid sequence. A comparison of the amino acid sequence of Cry1Ac with known 

allergens showed no significant overall sequence similarity or identity at the level of 

eight contiguous amino acid residues.

3. Digestibility. (MRID No. 45542319 ) The Cry1Ac protein was digested rapidly in 

simulated gastric fluid containing pepsin. Small peptides remaining following gastric 

simulated digestion were completely degraded to amino acid residues in SIF upon contact 

(US EPA 2005; 2010a).

4. Glycosylation. Cry1Ac expressed in soybean was shown not to be glycosylated.

5. Conclusion: Considering all of the available information, EPA has concluded that the 

potential for Cry1Ac to be a food allergen is minimal.

An acute oral toxicity study in mice (MRID No. 45542314) indicated that Cry1F is non-toxic to 

humans and other mammals. Cry1F protein is a δ-endotoxin from B. thuringiensis that has been 

used extensively in plant-incorporated protectants as a means of insect pest management. 

Existing exemptions from the requirement of a tolerance for Cry1F in cotton (CFR 40 Section 

174.504; 72 FR 20434, April 25, 2007) and corn (CFR 40 Section 174.520; 72 FR 20435, April

25, 2007) precluded the need for a separate tolerance action in conjunction with this review of 

product characterization and human health assessment. Cry1F was previously granted an 

exemption from the requirement of a tolerance for expression in cotton (CFR 40 Section 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 33 of 78

174.504) and maize (CFR Section 174.520); a separate petition (PP2F8066) for an exemption 

from the requirement of a tolerance for Cry1F as expressed in soybean is included with the 

submission from Dow for registration of DAS-81419-2 Soybean. 

Since Cry1F is a protein, allergenic potential was also considered. Currently, no definitive tests 

for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight-ofevidence approach where the following factors are considered: source of the trait; amino acid 

sequence comparison with known allergens; and biochemical properties of the protein, including

in vitro digestibility in simulated gastric fluid (SGF) followed by simulated intestinal fluid (SIF), 

and glycosylation. This approach is consistent with the approach outlined in the Annex to the

Codex Alimentarius “Guideline for the Conduct of Food Safety Assessment of Foods Derived 

from Recombinant-DNA Plants.” The allergenicity assessment for Cry1F follows:

1. Source of the trait. Bacillus thuringiensis is not considered to be a source of allergenic 

proteins. 

2. Amino acid sequence. A comparison of the amino acid sequence of Cry1F with known 

allergens showed no significant overall sequence similarity or identity at the level of 

eight contiguous amino acid residues.

3. Digestibility. (MRID No. 45542318 ) The Cry1F protein was digested rapidly (< 5 min) 

in simulated gastric fluid containing pepsin. Small peptides remaining following gastric 

simulated digestion were completely degraded to amino acid residues in SIF upon contact 

(US EPA 2005; 2010b).

4. Glycosylation. Cry1F expressed in soybean was shown not to be glycosylated.

5. Conclusion: Considering all of the available information, EPA has concluded that the 

potential for Cry1F to be a food allergen is minimal.

Cry1Ac and Cry1F were shown not to be glycosylated in extracts of DAS-81419-2 soybean; it is 

unlikely to be glycosylated in any other crops because in order for a protein to be glycoslyated, it 

needs to contain specific recognition sites for the enzymes involved in glycosylation, and the 

mechanisms of protein glycosylation are similar in different plants (Lerouge et al., 1998).

c. Overall Safety Conclusions

The continued use of Cry1Ac and Cry1F proteins in plant expression systems (plant-incorporated 

protectants) and in microbial biopesticides is fully supported by the information presented in this 

registration submission relative to human and animal health concerns. The lack of mammalian 

toxicity and allergenicity effects following thorough examination of pertinent information, as 

well as the efficacy noted in the insect bioassays, indicates that the specificity of the δendotoxins Cry1Ac and Cry1F as an insect management mechanism is safe as proposed for use 

in soybean intended for cultivation, and human and animal consumption. 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 34 of 78

APPENDIX B: Ecological and environmental effects – Bacillus thuringiensis

Cry1Ac and Cry1F proteins expressed in Event DAS-81419-2 Soybean plantincorporated protectant

Dow submitted data in support for Sec. 3 Seed-Increase Registration of event DAS-81419-2

soybean line [EPA Reg. No. 68467-EN; OECD Unique Identifier: DAS-81419-2] for use as a

Plant-Incorporated Protectant (PIP). The proposed PIP is a new transformation event that 

expresses lepidopteran-active Cry1F and Cry1Ac proteins in soybean. The insecticidal proteins 

Cry1F and Cry1Ac (encoded by the cry1F gene and cry1Ac genes, respectively) are co-expressed 

in WideStrike cotton (OECD Unique Identifier: DAS-21Ø23-5 x DAS-24236-5), which is 

currently registered for use as a PIP cotton product (US EPA 2005). The cotton line 

WideStrike was produced by crossing cotton lines 281-24-236 (OECD Unique Identifier: 

DAS-24236-5) with 3006-210-23 (OECD Unique Identifier: DAS-21Ø23-5) via conventional 

breeding methods. Since these proteins are identical to those expressed in the proposed PIP 

product, a “weight-of-evidence” approach was used to bridge data from WideStrike cotton in

support for the environmental risk assessment of event DAS-81419-2 soybean. Thus, the 

findings of the environmental risk assessment for event DAS-81419-2 soybean include the

extrapolation of previously submitted nontarget organism (NTO) toxicity studies and results 

from field studies currently found in the Agency database for the registration of WideStrike® 

expressing Cry1F and Cry1Ac proteins for use as a PIP in cotton. The biological equivalency of 

Cry1F and Cry1Ac proteins expressed in event DAS-81419-2 soybean plant was confirmed with 

the previously characterized microbial-derived Cry1F and Cry1Ac protein test substances. No 

significant increase in exposure to NTOs was demonstrated based on calculated margins of 

exposure for protein concentrations in event DAS-81419-2 plant tissue matrices relative to the 

dose concentrations used in the NTO laboratory toxicity tests conducted on Cry1Ac and Cry1F. 

Lastly, a field monitoring study showing no differences on nontarget arthropods on a population 

and community level and other supporting supplemental information (e.g. meta-analysis, target 

feeding studies, and biological specificity to the target pest) provided additional certainty to 

support the “weight-of-evidence” that there is negligible risk to NTOs representing diverse 

taxonomic groups and exposure scenarios from the cultivation of DAS-81419-2 soybean.

Therefore, the submitted data supports the applicant’s request and the findings from the 

environmental risk assessment conducted for WideStrike® cotton can be bridged to support the 

Sec. 3 Registration of PIP event DAS-81419-2 (US EPA 2005). Based on extrapolating from 

previous studies on Bt proteins, which tend to be very species specific in their activity, the 

Agency concludes that the cultivation of event DAS-81419-2 soybean will not result in adverse 

effects to nontarget organisms, are not likely to persist in the soil, and pose no risk of gene flow 

and development of weediness in wild relatives, and additionally will have no effect, direct or 

indirect, on Federally listed threatened and endangered species and their designated Critical 

Habitats.

BACKGROUND

Dow has developed event DAS-81419-2 soybean line [EPA Reg. No. 68467-EN; OECD Unique 

Identifier: DAS-81419-2] for use as a plant-incorporated protectant (PIP). Event DAS-81419-2 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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soybean was produced by Agrobacterium-mediated transformation with plasmid pDAB9582. 

The inserted T-DNA from this plasmid contains three transgenes: 1) synthetic truncated cry1F

gene from Bacillus thuringiensis (Bt) var. aizawai; 2) cry1Ac gene isolated from Bt var. kurstaki; 

and 3) a version of the phosphinothricin acetyl transferase (pat) gene from Streptomyces 

viridochromogenes that has been optimized for expression in soybean. The Cry1F and Cry1Ac 

proteins, (encoded by the cry1F and cry1Ac genes, respectively) confer resistance to certain 

lepidopteran pests. The PAT enzyme encoded by the pat gene provides the plant with tolerance 

to herbicide applications containing glufosinate-ammonium by acetylating the herbicide 

glufosinate-ammonium and making it unable to bind glutamine synthase. The proposed PIP is 

intended to provide protection against several lepidopteran pests of soybean, including soybean 

looper (Chrysodeixis includens, formerly Pseudoplusia includens), velvetbean caterpillar 

(Anticarsia gemmatalis), fall armyworm (Spodoptera frugiperda) and tobacco budworm 

(Heliothis virescens). The Cry1F and Cry1Ac proteins are co-expressed in the currently

registered PIP event WideStrike® cotton (EPA Reg. No. 68467-3). Dow has requested to bridge 

several environmental fate and effects studies previously reviewed for the registration of 

WideStrike® cotton (US EPA 2005) to support the registration of event DAS-81419-2 soybean 

PIP product. 

A “weight-of-evidence” approach has been used to determine if the supporting data submitted by

Dow is acceptable for bridging to the Agency’s database on the Cry1F and Cry1Ac proteins 

expressed in WideStrike® cotton and whether extrapolation of those studies and other 

supplemental information support the environmental risk assessment of event DAS-81419-2 

soybean.

I. Environmental Risk Assessment Process and Rationale for PIPs under EPA

The paragraphs below describe the process and rationale developed by the U.S. EPA’s 

Biopesticides and Pollution Prevention Division (BPPD) within the Office of Pesticide Programs 

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” exposure 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 early in an 

investigation tend to be broad in scope but relatively simple in design, and can be used to 

demonstrate acceptable risk under most conceivable conditions. When screening studies suggest 

potentially unacceptable risk additional studies are designed to assess risk under more realistic 

field exposure conditions. These later tests are more complex than earlier screening studies. Use 

of this “tiered” testing framework saves valuable time and resources by organizing the studies in 

a cohesive and coherent manner and eliminating unnecessary lines of investigation. Lower tier, 

high dose screening studies also allow tighter control over experimental variables and exposure 

conditions, resulting in a greater ability to produce statistically reliable results at relatively low 

cost1. 

 

1

Nontarget 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 allowed an 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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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) nontarget organism hazard tests are conducted at exposure 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 system to assess the toxicity of a PIP to representative 

nontarget 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 (nontarget) organism. The screening tests evaluate single species in a 

laboratory setting with mortality as the end point. Tiers II – IV generally 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.

EPA’s Office of Chemical Safety and Pollution Prevention (OCSPP) has published a series of 

test guidelines that contain testing methods and standards for conducting, evaluating, and 

reporting of data needed to support the registration of a pesticide under FIFRA. The testing 

methods, which utilize the tiered approach, are published in the OCSPP Harmonized Test 

Guidelines, Series 850 and 885.2 These guidelines, as defined in 40 CFR § 158.70(c), apply to 

microbes and microbial toxins when used as biological pesticides, including those that are 

naturally occurring, and those that are strain-improved, either by natural selection or by

deliberate genetic manipulation. Therefore, PIPs containing microbial toxins are also covered by

these testing guidelines. Further, most registered PIP products express insecticidal proteins 

derived from Bacillus thuringiensis (Bt), a common soil bacterium that is also found in several 

"reduced risk" microbial pesticides and currently available commercially to prevent the damage 

caused by insect pests. Microbial pesticides containing Bt proteins have a long history of safe use 

and undergone extensive toxicity testing showing no adverse effects to human or animal health 

(US EPA 2000 and 2001a; McClintock et al. 1995; Mendelsohn et al. 2003).

The Tier I screening maximum hazard dose (MHD) approach to environmental hazard 

assessment is based on some factor (whenever possible >10) times the Estimated Environmental 

 

endpoint of 50% mortality to be used as a trigger for additional 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 30 individuals is sufficient to allow a treatment effect of 30% to be differentiated from a 

50% effect with 95% confidence using a one-sided Z test. A one-sided test is appropriate because only effects of 

less than 50% indicate that further experiments are not needed to evaluate risk. 

2

OCSPP Harmonized Test Guidelines, Series 850 - Ecological Effects. Available at the EPA website: 

 http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series850.htm

OCSPP Harmonized Test Guidelines, Series 885 - Microbial Pesticide. Available at the EPA website: 

http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series885.htm
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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Concentration3(EEC), which is the maximum amount of active ingredient expected to be 

available to terrestrial and aquatic nontarget organisms in the environment. 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 hypotheses. Elevated test doses, therefore, add certainty 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 days. Some test species, notably nontarget insects, may 

be difficult to culture and the suggested test duration has been adjusted accordingly. Control and 

treated insects should be observed for at least 30 days or in cases where an insect species cannot 

be cultured for 30 days, 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.4 A less 

than 50% mortality 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 any effect might still be 

detected at more realistic field [1X EEC] concentrations and routes of exposure.5 

When screening tests indicate a need for additional data, the OCSPP Harmonized Guidelines call 

for testing at incrementally lower doses in order to establish a definitive LD50 and to quantify the 

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

sufficient to determine an LD50 value and, when necessary, the Lowest Observed Effect 

Concentration (LOEC), No Observed Adverse Effect Level (NOAEL) , or reproductive and 

behavioral effects such as feeding inhibition, weight loss, etc. In the final analysis, a risk 

assessment is made by comparing the LOAEC to the EEC; when the EEC is lower than the 

LOAEC, a no risk conclusion is made. These tests offer greater environmental realism, but they

may have lower statistical power. Appropriate statistical methods, and appropriate statistical 

power, must be employed to evaluate the data from the definitive tests. Higher levels of 

 

3

The dose margin can be less than 10x where uncertainty in the system is low or where high concentrations of test 

material are not possible to achieve due to test organism feeding habits or other factors. High dose testing also may 

not be necessary where many species are tested or tests are very sensitive, although the test concentration used must 

exceed 1X EEC.

4

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 is not 

‘safety factor testing’. In a “10X safety factor” test any adverse effect noted is a “level of concern”, whereas in the 

EPA environmental risk assessment scenario any adverse effect is viewed as a concern only at 1X the field 

exposure. 

5

The 1X EEC test dose is based on plant tissue content and is considered a high worst case dose (sometimes 

referred to as HEEC). This 1X EEC is still much greater than any amount which any given nontarget organism may 

be ingesting in the field because most nontarget organisms do not ingest plant tissue.
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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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. LC50, ED50, 

or LD50 >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 highly conservative factor. The published EPA Level of Concern [LOC] is 

50% mortality at 5X EEC 6(US EPA 1998). 

Validation: The tiered testing approach was developed for the EPA by the American Institute of 

Biological Sciences (AIBS) and confirmed in 1996 as an acceptable method of environmental 

hazard assessment by a FIFRA Scientific Advisory Panel (SAP) on microbial pesticides and 

microbial toxins. The December 9, 1999 SAP agreed that the Tiered approach was suitable for use 

with Plant-Incorporated Protectants (PIPs); however, this panel recommended that, for PIPs with 

insecticidal properties, additional testing of beneficial invertebrates closely related to target species 

and/or likely to be present in GM crop fields should be conducted. Testing of Bt Cry proteins on 

species not closely related to the target insect pest was not recommended, although it is still 

performed to fulfill the published EPA nontarget species data requirements. In October 2000, 

another SAP also recommended that field testing should be used to evaluate population-level 

effects on nontarget organisms. The August 2002 SAP, and some public comments, generally 

agreed with this approach, with the additional recommendation that test surrogate organisms 

should be selected on the basis of potential for field exposure to the subject protein (US EPA 2000, 

2001a, 2002 and 2004a). A White Paper developed by the U.S. States Department of Agriculture 

(USDA) and EPA discussed the tiered testing approach and provided additional guidance for 

conducting nontarget invertebrate ecological risk assessments specifically for crops expressing 

insecticidal proteins (US EPA 2007).

Chronic studies: Additional higher-tier (e.g. Tier II, III, and IV) testing for chronic effects on 

nontarget organisms are only conducted when they are triggered (i.e. toxicity studies that show 

50% mortality when tested at least 5X EEC). Since delayed adverse effects and/or accumulation 

of toxins through the food chain are not expected to result from exposure to Bt PIP proteins, 

these protein toxins are not routinely tested for chronic effects on nontarget organisms. However, 

the 30 day test duration requirement does amount to subchronic testing when performed at field 

exposure test doses. 

Conclusion: The tiered testing approach to test guidelines ensures, to the greatest extent possible, 

that the Agency requires the minimum amount of data needed to make scientifically sound 

regulatory decisions. The EPA believes the Tier I screening maximum hazard dose (MHD) 

approach presents a reasonable approach for evaluating environmental hazards related to the use 

of biological pesticides and for identifying negative results with a high degree of confidence (US 

EPA 2007). The Agency expects that Tier I testing for short-term hazard assessment will be 

sufficient for most studies submitted in support of PIP registrations (US EPA 2007). However, if 

 

6

The established peer and EPA Science Board reviewed guidance on screening test levels of concern is 50% 

mortality at 5X the environmental concentration. The appropriate endpoints in high dose limit/screening testing are 

based on mortality of the treated, as compared to the untreated (control) nontarget organisms. A single group of 30 

test animals may be tested at the maximum hazard dose.
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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long range adverse effects must be ascertained, then higher-tier longer-term field testing will be 

required. As noted above, the October 2000 SAP and the National Academy of Sciences (NAS 

2000) recommended testing nontarget organisms directly in the field. This approach, with an 

emphasis on testing invertebrates found in Bt cotton fields, was also recommended by the August 

2002 SAP and was supported by several public comments. Based on these recommendations, the 

Agency has required large-scale, long term field studies as a condition of registration on: 1) 

monitoring nontarget invertebrate populations (e.g. community level effects); and 2) determining 

the bioavailability/persistence of Cry proteins remaining in soils representative of different 

growing regions of Bt crops. These studies were conducted as a precautionary measure 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 in 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 

term effects of cultivation of these Cry proteins on the invertebrate community structure in Bt

crop fields has since been adequately addressed. Specifically, a meta-analysis7of the data 

collected from 42 field studies indicated that nontarget invertebrates are generally more abundant 

in fields of Bt crops than in non-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 

effects 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 crops have not revealed any unexpected nontarget 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 result in reductions in the abundance of the pests as prey. 

Proteins are not known to persist in the environment due to the ubiquitous nature of proteases in 

microbes. The biological nature of proteins makes them readily susceptible to metabolic, 

microbial, and abiotic degradation once they are ingested or excreted into the environment. 

Sprayable microbial pesticide formulations containing Bt have a long history of safe use. Several 

laboratory studies have demonstrated rapid degradation of Cry proteins in various soils sampled 

from Bt crop growing regions. Long term field studies have been conducted showing no 

accumulation (i.e. persistence) of Cry proteins as a result of continuous planting of Bt crops 

(Head et al. 2002; Herman et al. 2002; Shan et al. 2008). Thus, the issue of long term effects of 

Cry proteins has also been addressed by several field studies showing a lack of persistence in 

various soil types sampled from representative Bt crop growing region. While there are reports 

that some proteins (Cry proteins) binding to soil particles, it has also been shown that these 

proteins are degraded rapidly by soil microbial flora upon elution from soil particles (Icoz and 

Stozky 2008).

 

7 This research was funded by Environmental Protection Agency grant CR-832147-01. The Bt crop nontarget 

effects database can be found on the National Center for Ecological Analysis and Synthesis (NCEAS). Website. 

(http://delphi.nceas.ucsb.edu/btcrops/).
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As a result, the Agency has determined no additional long-term field studies are required for 

monitoring nontarget invertebrate populations or soil protein persistence for Bt crops that contain 

similar Cry proteins expressed in previously registered PIP events with the exception of Tier I 

laboratory studies that “trigger” additional testing or if the proposed registration is a novel PIP. 

Overall, the Agency is in agreement with the conclusions of these studies and collectively, these 

results provide extensive data to support that Bt crops registered to date have not caused long 

term adverse effects on a population level to organisms not targeted by Bt proteins. Moreover, 

these field studies further substantiate the “tiered testing approach” by confirming that the 

absence of hazard in the Tier I toxicity studies is predictive of an absence of ecological harm in 

the field. These results are also consistent with earlier studies of Bt strains used as microbial 

insecticides, which demonstrated no significant detrimental impacts on populations of the 

nontarget organisms that were studied. Based on these considerations, the “tiered testing 

approach” has been demonstrated as an appropriate method for assessing environmental risk of 

Bt crops while ensuring, to the greatest extent possible, that the Agency requires the minimum 

amount of data needed to make scientifically sound regulatory decisions.

A. Environmental Exposure Assessment

Two separate FIFRA Scientific Advisory Panel reports (US EPA SAP 2000 and 2002) 

recommended that nontarget 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, 

the EPA determined that nontarget organisms with the greatest exposure potential to Cry protein 

in transgenic soybean fields are beneficial insects that feed on soybean leaf foliage and pods, 

particularly lepidopteran insects, and soil invertebrates. While EPA’s risk assessments of Bt

soybean have focused primarily 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 field exposure rates, which are 

typically based on protein expression levels within the plant for PIPs. Although it is 

recommended that nontarget testing be conducted at a test dose 10X the EEC whenever possible, 

the test dose margin can be less than 10X where uncertainty in the system is low or where high 

concentrations of test material are not possible to achieve due to test organism feeding habits. 

BPPD may also allow for testing at lower doses in cases where many species are tested or tests 

are very sensitive, although the concentration used must exceed the EEC. For the purposes of 

the nontarget organism studies submitted in support of Cry1F and Cry1Ac expressed in event 

DAS-81419-2 soybean, the test material dose levels were based on an estimated concentration of 

Cry1F and Cry1Ac proteins expressed in the tissue(s) with the highest concentration calculated 

from a field study quantifying protein expression levels in several soybean plant tissue parts 

(MRID No. 48828402). 

B. Ecological Effects Data for Cry1F and Cry1Ac Insecticidal Proteins Expressed in 

Event DAS-81419-2 Soybean

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 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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requirements include testing on birds, mammals, nontarget insects, honey bee, plants, and 

aquatic species, and information has been submitted to address these requirements. Limit dose 

testing on representative organisms from several taxa was performed in support of Section 3 

FIFRA registration of event DAS-81419-2 expressing Bt-derived Cry1F and Cry1Ac insecticidal 

proteins. As stated above, BPPD’s risk assessments focus more greatly on beneficial nontarget 

invertebrates, since they are most closely related to organisms susceptible to the insecticidal 

action of Bt toxins. The Cry1F and Cry1Ac proteins are meant to target species within the order 

Lepidoptera (e.g. moths). Bt toxins are known typically to have a limited host range, however, to 

address any unforeseen change in activity spectrum as a result of laboratory protein synthesis and 

to fulfill the published registration data requirements EPA requires that test species used for 

nontarget insect evaluations should include several invertebrate species that are not related to the 

target pests. Earthworm studies are also recommended. 

The applicant has requested to bridge to the Agency database of previously reviewed toxicity

studies of select nontarget organisms (NTOs) that supported the currently registered 

WideStrike PIP cotton product (EPA Reg. No. 68467-3). The WideStrike PIP cotton line 

was produced by conventional cross breeding of cotton lines DAS-21023-5 (also described as 

3006-210-23) expressing Cry1F protein and DAS-24236-5 (also described as 281-24-236) 

expressing Cry1Ac protein. The environmental risk assessment for the insecticidal proteins 

(Cry1Ac and Cry1F) expressed in WideStrike cotton was previously evaluated for adverse 

impacts on nontarget organisms and have been shown to pose negligible risk (US EPA 2005). 

Since exposure may also occur to other nontarget organisms, EPA has received a number of 

studies and data waiver rationales to comply with the Agency’s published data requirements on 

other nontarget organisms. 

The toxicity of Cry1F and Cry1Ac proteins has been previously evaluated on several species of 

invertebrates including: lady beetle (Hippodamia convergens), green lacewing (Chrysoperla 

rufilabris), honey bee (Apis mellifera), collembola (Folsomia candida), parasitic wasp (Nasonia 

vitripennis) and earthworm (Eisenia fetida). Two new dietary toxicity studies were submitted in 

support for event DAS-81419-2 soybean, which includes Northern Bobwhite (Colinus 

viginianus) and Rainbow Trout, (Oncorhynchus Mykiss). The individual results for the cited and 

newly submitted nontarget organism studies in support for Cry1F and Cry1Ac are summarized in 

Table 1. The studies are described in more detail below, and full reviews of each study can be 

found in the individual Data Evaluation Records. 

To support data bridging from the previously conducted environmental risk assessment for 

Cry1F and Cry1Ac insecticidal proteins co-expressed in WideStrike PIP cotton line, an 

environmental risk assessment was conducted using a “weight-of-evidence” approach based on 

the following lines of evidence to support the registration of event DAS-81419-2 soybean:

1) Confirmation of biological equivalency of Cry1F and Cry1Ac proteins expressed in 

event DAS-81419-2 soybean plant and previously characterized microbial-derived 

Cry1F and Cry1Ac protein test substances; 

2) No significant increase in exposure to NTOs based on calculated margins of exposure 

for protein concentrations in event DAS-81419-2 plant tissue matrices relative to the 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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dose concentrations used in the NTO laboratory toxicity tests conducted on Cry1Ac and 

Cry1F; and 

3) No adverse effects on nontarget arthropods on a population and community level 

observed from a field monitoring study and other supporting supplemental information 

(e.g. meta-analysis, target feeding studies, and biological specificity to the target pest). 

Exposure estimates were compared with endpoints reported from Tier I toxicity studies 

previously conducted on various representative nontarget test species to determine a Margin of 

Exposure (MOE), the ratio of the endpoint value to the EEC. The MOEs for representative NTOs 

were recalculated based on high-end exposure estimates (HEEEs) (US EPA, 2009) derived from 

Cry1Ac and Cry1F protein expression levels quantified from various plant tissues of event DAS81419-2 soybean in a field study. A MOE of 50% mortality at 10X the estimated environmental 

exposure is regarded as sufficient to demonstrate negligible risk and was also the threshold for 

consideration of uncertainties in the risk assessment (US EPA 1998; US EPA 2007). If 

subsequent expression characterization in the commercial event leads to a safety margin of 

<10X, then the consequences are considered in terms of uncertainties in the ecological risk 

assessment below (US EPA 2007). 

Table B.1. Summary of Environmental Effects Studies conducted for PIP Event DAS-81419-

2 soybean (EPA Reg. No. 68467-EN) and Scientific Rationales in support for bridging 

Cry1F and Cry1Ac data from the Agency database on WideStrike® PIP cotton (EPA Reg. 

No. 68467-3)

OCSPP

Guideline Study Type SUMMARY OF RESULTS AND CLASSIFICATION MRID No.

WideStrike® PIP cotton data (EPA Reg. No. 68467-3)

885.4150 Wild Mammal 

Testing, Tier I 

Not required; Mammalian wildlife exposure to Cry1F and Cry1Ac 

proteins is considered likely; however, the results from two acute oral 

toxicity studies indicated no adverse effects on mice from microbialderived Cry1F and Cry1Ac proteins administered via oral gavage at the 

maximum hazard dose. The NOAEL exceeded 600 mg/kg body weight for 

Cry1F (MRID No. 45542312) and 700 mg/kg bw for Cry1Ac (MRID No. 

45542313). These data are sufficiently representative of toxicity that 

would be expected in wild mammals, and based on the results of this 

study, risk to wild mammals resulting from exposure to Cry1F and 

Cry1Ac proteins is not expected.

Classification: ACCEPTABLE for data bridging

45542312

and

45542313

850.1010 Aquatic 

Invertebrate 

Acute Toxicity 

Test, 

Daphnia magna

In a 48-hour static test with freshwater daphnids (Daphnia magna) there 

were no observed adverse effects with Cry1F and Cry1Ac in combination 

at respective concentrations of 510 and 2,500 μg/L. No immobility or 

other adverse effects were seen during the study. The 24-hour and 48hour 

EC50s for daphnia exposed to the Cry1F + Cry1Ac mixture were >510 μg 

Cry1F/L and >2500 μg Cry1Ac/L (represents a worst-case exposure of 

one kg of transgenic cotton pollen per liter of pond water). This rate of 

fortification represents 298X and >23,000X the anticipated EEC for 

Cry1F and Cry1Ac protein in surface water. Therefore, no hazard to 

aquatic invertebrates is expected from incidental exposure to WideStrike 

cotton pollen. 

45808412
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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OCSPP

Guideline Study Type SUMMARY OF RESULTS AND CLASSIFICATION MRID No.

Classification: ACCEPTABLE for WideStrike Cotton; 

SUPPLEMENTAL for DAS-81419-2 soybean.

885.4280 Estuarine and 

Marine Animal 

testing, Tier I 

Not required. Exposure in marine and/or estuarine environments is not 

expected to be significant.

N/A

885.4300 Nontarget Plant 

Studies, Tier I 

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

885.4380 Honey Bee 

Larva Testing, 

Tier I 

(Apis mellifera)

At 1.98 μg Cry1F + 11.94 μg Cry1Ac per mL sugar water no effect on 

survival of larvae to adult emergence was seen. The LC50 is >4X pollen 

expression. Therefore no hazard to honey bee larvae and adult bee 

emergence is anticipated. 

Classification: ACCEPTABLE for data bridging

45542316

885.4340 Nontarget 

Insect Testing, 

Tier I

(Nasonia 

vitripennis)

At 5.2 μg Cry1F + 46.8 μg Cry1Ac per mL sugar water at 10 days, no 

effect of limit dose with LC50 > 13X pollen expression was seen on 

parasitic hymenoptera larva. Minimal exposure and no hazard to parasitic 

Hymenoptera from Cry1F and Cry1Ac proteins are expected. Testing of a 

species more common to cotton fields is recommended. 

Classification: ACCEPTABLE for data bridging

45808411

885.4340 Nontarget 

Insect Testing, 

Tier I 

(Chryosperla 

camea)

A dietary toxicity study on green lacewing (Chrysoperla rufilabris) larvae 

was conducted by presenting the green lacewings with a moth egg 

(Sitotroga sp.) diet incorporated with Bt Cry1F and Cry1Ac proteins. The 

dietary LC50 was >48.2 μg/g diet for Cry1Ac and >5.2 μg/g diet for 

Cry1F, which represented at least at 14X the concentration found in 

pollen. No effect was noted at the Cry1F and Cry1Ac protein levels 

expressed in pollen that would be encountered by green lacewings in the 

field. Because of questionable ingestion of the test material another 

species (e.g, minute pirate bug), which is more likely to be exposed, 

should be tested. 

Classification: ACCEPTABLE for data bridging

45808410

885.4340 Adult Lady 

Beetle Testing, 

Tier I 

(Hippodamia 

convergens)

At 300 μg Cry1F + 22.5 μg Cry1Ac per mL sugar water, no effect of limit 

dose with LC50> 780X Cry1F pollen expression and > 8X Cry1Ac pollen 

expression on adult lady beetles. Based on these results, no hazard to H. 

convergens is expected when feeding on WideStrike® cotton pollen in the 

field. 

Classification: ACCEPTABLE for data bridging

45542315

885.4340 Collembola 

Chronic Dietary 

Toxicity Study, 

Tier I 

(Folsomia 

candida)

The combination of 709 μg Cry1F + 22.6 μg Cry1Ac per g diet and cotton 

leaf tissue showed no effect on adult survival and reproduction at up to 

10X the anticipated field level of expression. Therefore, no hazard to 

decomposers represented by collembola is expected from exposure to 

WideStrike cotton in the field.

Classification: ACCEPTABLE for data bridging

45808409
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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OCSPP

Guideline Study Type SUMMARY OF RESULTS AND CLASSIFICATION MRID No.

OECD 

Guideline 

207 

Earthworm 

Toxicity Study, 

(Eisenia 

foetida)

A 14-day study on earthworms exposed to soils treated with microbialproduced Cry1Ac and Cry1F, individually and in combination, was 

performed. The 14-day LC50s were >247 mg a.i./kg for Cry1F; >107 mg 

a.i./kg for Cry1Ac, and > 247 mg a.i./kg Cry1F + >107 mg a.i./kg Cry1Ac 

in the test with the two proteins combined. There were no overt signs of 

toxicity to earthworms exposed to soils containing nominal concentrations 

of Cry1F and Cry1Ac at 50X the expected worst case EEC [this represents 

concentrations which are 792X and 5479X higher than the expected EEC 

for incorporation of defoliated cotton plants into the top 15 cm of soil].

Classification: SUPPLEMENTAL

45580701

885.5200 Expression in a 

terrestrial 

Environment 

Tier II 

The soil half-life of the plant expressing Cry1F and Cry1Ac was estimated 

as 1.3 days in a laboratory study with a representative soil from a cotton 

growing region. The Cry proteins were not detectable after 14 days. These 

results verify that the Cry1F and Cry1Ac proteins degrade rapidly in 

cotton soil.

Classification: ACCEPTABLE for WideStrike Cotton; 

SUPPLEMENTAL for DAS-81419-2 Soybean. 

45556801

DAS-81419-2 Soybean data (EPA Reg. No. 68467-EN)

885.4050 Avian Dietary 

Toxicity Study, 

Tier I

(Colinus 

viginianus)

In a 14-day acute dietary toxicity study, Northern bobwhites (Colinus 

virginianus) were exposed to avian diet fortified with a 7.9:1.0 mixture 

of Cry1F and Cry1Ac proteins with a test substance concentration of 128 

mg /kg body weight (24.52 mg a.i./kg body weight based on percent a.i.) 

by oral gavage. Results showed no apparent effects on mortality, sublethal 

observations, body weight, and food consumption at this treatment level.

The 14 day acute oral LD50 for the test substance was > 128 mg /kg bw

(24.52 mg a.i./kg body weight based on percent a.i.) and the 14 day NOEL 

for the test substance was > 128 mg /kg bw (24.52 mg a.i./kg body weight 

based on percent a.i.). Monogastric animals like birds cannot safely 

consume raw soybean seed since it contains endogenous antinutrients 

(Friedman et al., 1991). The estimated feed consumption per bird was 33 

grams/day. Therefore, in relation to the daily diet, Cry1F was dosed at 

0.660 mg AI/gm (660 ppm) and Cry1Ac was dosed at 0.083 mg AI/gm 

(83 ppm). The HEEE concentration of Cry1F in raw DAS-81419-2 

soybean seed is 14.34 ng/mg dw (ppm) and 1.08 ng/mg dw (ppm) for 

Cry1Ac (MRID No. 48828413). The MOEs was calculated for Cry1F and 

Cry1Ac in the quail study relative to raw soybean seed consumed as 100% 

of the diet was 46X (660 ppm/14.34 ppm) and 77X (83 ppm/1.08 ppm), 

respectively, which provides a large safety margin even if bird could 

safely ingest raw soybean as their sole source of food.

Thus, no adverse effects on avian wildlife are expected from incidental 

field exposure to DAS-81419-2 soybean expressing Bt Cry1F and 

Cry1Ac insecticidal proteins.

Classification: ACCEPTABLE

48828410

885.4200 Freshwater Fish 

Testing, Tier I

(Onchorynchus 

mykiss) 

An acute oral dietary toxicity study on rainbow trout (Onchorynchus 

mykiss) was conducted to assess the effects of a basal fish diet fortified 

with a mixture of microbial-derived Cry1F and Cry1Ac proteins for eight 

days. No fish mortality and no sublethal effects were observed in any of 

the control or test treatment organisms during this study. Results show the 

8-day LD50 value with rainbow trout was > 100 mg a.i./kg diet wet weight 

48828411
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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OCSPP

Guideline Study Type SUMMARY OF RESULTS AND CLASSIFICATION MRID No.

(>200 mg a.i./kg diet dry weight). The NOEC was reported as > 100 mg 

a.i./kg diet weight (>200 mg a.i./kg diet dry weight) based on the lack of 

mortalities, sublethal effects, and apparent lack of effects on standard 

length and body weight. In addition, exposure to freshwater fish in aquatic 

environments is also considered to be limited . In view of the lack of 

toxicity and minimal aquatic exposure, no adverse effects to freshwater 

fish as a result of the cultivation of DAS-81419-2 soybeans are expected.

Classification: ACCEPTABLE

NonguidelineNontarget 

Arthropod 

Census Field 

Study

A field study was conducted to monitor nontarget arthropods in fields of 

DAS-81419-2 soybean in comparison to non-transgenic fields for any 

statistically significant differences in the types of taxa collected and their 

abundance for determining any adverse effects on a community level to 

nontarget arthropods. DAS-81419-2 soybean, a near-isogenic nontransgenic control (Maverick) and four non-transgenic reference lines 

(DSR 3590; IL 3505; Porter 75148; Williams 82) were monitored in 

replicated field trials at two locations in the USA, Richland, Iowa and 

York, Nebraska. Each trial site included the six soybean lines arranged in 

a randomized complete block design with four replicate blocks. Three 

sampling methods were used to monitor arthropod abundance: 1) pitfall 

trapping, 2) sticky card trapping and 3) vertical beat sheet sampling. A 

total of 74 taxonomic groupings were detected and assessed across field 

sites. To determine biological relevance of any statistical differences, 

results were interpreted in the context of the magnitude of differences, 

observations in reference plots, and trend consistency across sampling 

methods, sampling periods, and locations. Analysis of data found no 

statistically significant differences of biological relevance in the nontarget 

arthropod populations present in fields planted with DAS-81419-2 

soybean and the non-transgenic control soybean lines.

Classification: SUPPLEMENTAL

48828412

NonguidelineEnvironmental 

Risk 

Assessment 

Summary on 

Nontarget 

Arthropods 

An environmental risk assessment was separately prepared by the 

registrant to summarize the potential for adverse effects of cultivating PIP 

event DAS-81419-2 soybean on nontarget arthropods associated with 

soybean agroecosystems with a special emphasis on beneficial taxa and 

threatened or endangered species. Dow requested data bridging to the 

existing ecotoxicological studies on the effects of Bt Cry1F and Cry1Ac 

proteins expressed in WideStrike® PIP cotton on representative nontarget

insect species, including beneficials, pollinators, detritivores, and 

incidentals. Exposure estimates for Cry1F and Cry1Ac protein 

concentrations in PIP event DAS-81419-2 soybean were compared with 

endpoints reported from toxicity studies conducted for WideStrike

cotton on select test species to determine the MOE, the ratio of the 

endpoint value to the EEC. Most MOEs exceeded 10X the estimated 

environmental concentrations. None of the estimated exposure values 

exceeded NOECs for any of the tested species, indicating that the most 

sensitive individual in a population is not likely to be adversely affected. 

Other supplemental information (i.e. meta-analyses, biological specificity 

to the pest species, targeted toxicity feeding studies), the biology of the 

crop and lack of exposure (due to feeding habits/food preferences) support 

the conclusion of no adverse effects to NTO species from PIP event DAS81419-2 soybean. Other supporting data includes environmental fate data 

48828413
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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OCSPP

Guideline Study Type SUMMARY OF RESULTS AND CLASSIFICATION MRID No.

of the Cry1Ac and Cry1F proteins, results from a field survey showing no 

effects on the populations of nontarget arthropods associated with PIP 

event DAS-81419-2, and supplemental information from the FIFRA 

Endangered Species Task Force databases for identifying threatened and 

endangered species that may be exposed to PIP event DAS-81419-2 

soybean. 

Classification: SUPPLEMENTAL A soil fate study is needed to 

demonstrate rapid protein degradation of Cry1F and Cry1Ac in a variety 

of soil types that are common in soybean cultivation as confirmatory data.

NonguidelineSupporting 

document

A response to EPA Comments on Cry1F(synpro) ICP and 

Cry1Ac(synpro) insecticidal crystal protein was submitted for the 

acute oral toxicity study with the northern bobwhite (MRID No. 488284-

10). Protein identity and analytical assays confirmed the equivalence of 

and their respective certificates of analysis were submitted to demonstrate 

the equivalence of Dow’s test material #101811 to plant-produced protein 

and provide a justification of the test dosage. 

Classification: SUPPLEMENTAL to MRID No. 48828410

49044807

NonguidelineSupporting 

document

A response to EPA Comments on Cry1F(synpro) and Cry1Ac(synpro) 

insecticidal crystal proteins was submitted to demonstrate the equivalence 

of Dow’s test material #101811 to the plant-produced protein used in the

8-day dietary study with the rainbow trout (Oncorhynchus mykiss) 

WALBAUM (MRID No. 488284-11).

Classification: SUPPLEMENTAL to MRID No. 48828411

49044808

NonguidelineSupporting 

document

A response to EPA Comments on Cry1F(synpro) ICP and 

Cry1Ac(synpro) insecticidal crystal protein was submitted to address 

several data gaps in the registrant’s environmental risk summary (MRID 

No. 48828413) for PIP event DAS-81419-2 soybean. Additional data was 

needed to complete the risk assessment on the environmental fate of 

Cry1F and Cry1Ac proteins expressed in PIP event DAS-81419-2 

soybean. Supporting literature and data waiver rationales were presented 

for evaluating soil fate, gene flow and the potential for weediness.

Classification: SUPPLEMENTAL to MRID No. 48828413

49044809

Additional support for a complete environmental risk assessment is gained from an overview of 

the assessment conducted to support the WideStrike® cotton EPA Sec. 3 registration (EPA Reg. 

No. 68467-3). A brief summary of the EPA’s environmental risk assessment is provided in the 

following section:

C. WideStrike cotton (lepidopteran active) Environmental Risk Assessment 

Summary (EPA Reg. No. 68467-3)

Potential adverse effects to nontarget organisms by Cry1F and Cry1Ac proteins have been 

reviewed in Bt Cry1F/Cry1Ac WideStrike cotton BRAD (US EPA 2005). The following is a 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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summary of the environmental risk assessment for Cry1F and Cry1Ac. EPA performed risk 

assessments on plants, wild mammals, birds, fish, aquatic invertebrates, earthworms, terrestrial 

nontarget insects (including honey bee, parasitic wasps, green lacewings, ladybird beetle, 

springtails [Collembola toxicity/reproduction], and monarch butterflies), as well as field 

evaluations of the effects of Cry1F and Cry1Ac exposure on nontarget insects in cotton fields, 

soil degradation/persistence studies, and an endangered species impact assessment, particularly 

for Lepidoptera. In addition, gene flow and weediness assessments via pollen and Cry protein 

DNA uptake by plants were also performed. Cry1F and Cry1Ac protein in soil has been shown 

to degrade rapidly to very low levels. EPA concluded that there is sufficient information to 

believe that there is no risk from the uses of Cry1F and Cry1Ac cotton to nontarget wildlife, 

aquatic, and soil organisms. These studies were individually reviewed by the Agency and 

reported in Data Evaluation Reports (DERs). For detailed information of each study, refer to the 

DER according to its respective assigned MRID number in the associated environmental effects 

data summary tables for WideStrike cotton (reviewed in US EPA 2005).

At present, the Agency is aware of no identified significant adverse effects of Cry1F and 

Cry1Ac proteins on the abundance of nontarget organisms in any population in the aquatic or 

terrestrial field environment. Field testing and field census data submitted to the Agency show 

minimal to undetectable changes in the beneficial insect abundance or diversity. To date the 

available field test data show that compared to crops treated with conventional chemical 

pesticides, the transgenic crops have no detrimental effect on the abundance of nontarget 

invertebrate populations. In addition, no direct or indirect effects on Federally 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 Cry1F and Cry1Ac proteins by wild or weedy 

relatives of cotton 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 (US EPA 2005). 

In conclusion, the risk assessment found no unreasonable adverse effects on the environment 

from cultivation of WideStrike cotton expressing Cry1F and Cry1Ac proteins for use as a PIP.

D. Protein Equivalence

The October 2000 SAP recommended that while actual plant material is the preferred test 

material, microbial-derived protein is also a valid test substance, particularly 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 event DAS-81419-2 soybean 

registration, several of the nontarget toxicological studies used microbial-produced, purified 

Cry1F and Cry1Ac protein test substances that were originally developed for the Widestrike

PIP cotton registration; in addition to plant tissue (lypholized leaf tissue) from event DAS81419-2 soybean. 

In support of bridging data from previously reviewed NTO toxicological studies conducted for 

WideStrike cotton (US EPA 2005), the biochemical properties the microbial-derived Cry1F 

and Cry1Ac protein test substances were compared with plant-produced Cry1F and Cry1Ac 

proteins expressed in event DAS-81419-2 soybean to determine that the proteins from the two 

host sources were biologically equivalent. The proteins were characterized and equivalence was 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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evaluated based on a panel of analytical tests and assays. The results of these evaluations provide 

a detailed characterization of the Cry1F and Cry1Ac proteins isolated from event DAS-81419-2 

soybean and confirmed their equivalence to the microbial-derived Cry1F and Cry1Ac proteins 

(MRID Nos. 48828403 and 49044803; reviewed in US EPA 2013). Therefore, the results of the 

NTO toxicity studies that utilized the microbial-derived Cry1F and Cry1Ac test protein 

substances can be bridged to support the environmental risk assessment of event DAS-81419-2 

soybean. Furthermore, development of additional nontarget organism toxicity testing can be 

waived for PIP event DAS-81419-2 soybean and the results and conclusions from the 

environmental risk assessment conducted for the WideStrike cotton PIP line can be bridged to 

support event DAS-81419-2 soybean (US EPA 2005).

E. Nontarget Organism Effects Cry1F and Cry1Ac insecticidal proteins expressed 

in Event DAS-81419-2 Soybean (EPA Reg. No. 68467-EN)

1. Avian Wildlife

The primary routes of exposure of birds to Cry1F and Cry1Ac insecticidal proteins expressed in 

PIP event DAS-81419-2 soybean plants expected to occur through consumption of event DAS81419-2 plant material (e.g., leaf foliage and pods) and invertebrates that feed on event DAS81419-2 soybean plant material. Exposure via consumption of soybean seeds may be limited, 

however, by compounds that interfere with digestion and nutrient uptake, such as trypsin 

inhibitors. Other routes of exposure (e.g., inhalation) are not expected to be significant, since 

Cry1F and Cry1Ac proteins are contained primarily within the plant.

In a 14-day acute dietary toxicity study, Northern bobwhites (Colinus virginianus) were exposed 

to avian diet fortified with a 7.9:1.0 mixture of Cry1F and Cry1Ac proteins by oral gavage 

(MRID No. 48828410). Results showed no apparent effects on mortality, sublethal observations, 

body weight, and food consumption at this treatment level. The 14 day acute oral LD50 for the 

test substance was > 128 mg/kg bw (24.52 mg a.i./kg body weight based on percent a.i.) and the 

14 day NOEL for the test substance was > 128 mg /kg bw test substance (24.52 mg a.i./kg body

weight based on percent a.i.). 

BPPD estimates the exposure to birds in the field for soybeans is minimal. Monogastric animals 

like birds cannot safely consume raw soybean seed since it contains endogenous antinutrients 

(Friedman et al., 1991). The estimated feed consumption per bird was 33 grams/day. Therefore, 

in relation to the daily diet, Cry1F was dosed at 0.660 mg a.i./gm (660 ppm) and Cry1Ac was 

dosed at 0.083 mg a.i./gm (83 ppm). The HEEE concentration of Cry1F in raw DAS-81419-2 

soybean seed is 14.34 ng/mg dw (ppm) and 1.08 ng/mg dw (ppm) for Cry1Ac (MRID No. 

48828413). The MOE was calculated for Cry1F and Cry1Ac in the quail study relative to raw 

soybean seed consumed as 100% of the diet was 46X (660 ppm/14.34 ppm) and 77X (83 

ppm/1.08 ppm), respectively, which provides a large safety margin even if birds could safely 

ingest raw soybean as their sole source of food.

Furthermore, the Cry1F and Cry1Ac proteins have been registered as PIPs in several crops with 

no identified concerns for birds, and there is a history of use with no reported incidents involving 

birds in the field, including use in a crop that is typically ingested by birds (corn). Therefore, 

based on this history, current toxicity data, and the BPPD’s assumptions of the lack of avian 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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exposure, no adverse effects to birds are anticipated as a result of the registration of DAS-81419-

2 Soybean.

 2. Wild Mammals

Wild mammals could be exposed to Cry1F and Cry1Ac proteins produced by event DAS-81419-

2 soybean plants by consumption of plant material and invertebrates that feed on event DAS81419-2 soybean plant material. As with birds, these are expected to be the primary sources for 

exposure. 

Data are available with which to determine the risk of Cry1F and Cry1Ac proteins from DAS81419-2 soybeans to wild mammals. The results from the acute oral toxicity studies showed no 

significant toxicity to mice from acute oral testing with the Cry1F and Cry1Ac proteins, with a 

NOAEL that exceeds 600 mg/kg body weight for Cry1F (MRID No. 45542312) and 700 mg/kg 

for Cry1Ac (MRID No. 45542313). Based on the estimated expression level in leaf (V5) tissue 

(63.3 ng/mg dw) converted to dose (0.00153 mg/kg bw), adverse effects would not be expected 

at concentrations up to a MOE of >3.9 x 105X for Cry1F. Based on the estimated expression 

level in leaf (V5) tissue (28.3 ng/mg dw) converted to dose (0.000683 mg/kg bw), adverse 

effects would not be expected at concentrations up to a MOE of >1.0 x 106X for Cry1Ac. Thus, 

the MOEs greatly exceed the required 10X EEC showing no adverse effects when tested at the 

maximum hazard dose. These data are sufficiently representative of toxicity that would be 

expected in wild mammals, and based on the results of this study risk to 

wild mammals resulting from exposure to Cry1F and Cry1Ac proteins expressed in DAS-81419-

2 Soybean is not expected. 

 3. Freshwater Animals

The registrant submitted an acute oral dietary toxicity study on rainbow trout (Onchorynchus 

mykiss) to assess the effects of consuming a basal fish diet fortified with a mixture of microbialderived Cry1F and Cry1Ac proteins for eight days (MRID No. 48828411). No fish mortality and 

no sublethal effects were observed in any of the control or test treatment organisms during this 

study. Results show the 8-day LD50 value with rainbow trout was > 100 mg a.i./kg diet wet 

weight (>200 mg a.i./kg diet dry weight). The NOEC was reported as > 100 mg a.i./kg diet 

weight (>200 mg a.i./kg diet dry weight) based on the lack of mortalities, sublethal effects, and 

apparent lack of effects on standard length and body weight. In addition, exposure to freshwater 

fish in aquatic environments is also considered to be minimal.

For freshwater invertebrates, a 48-hour static toxicity study on daphnids (Daphnia magna) 

showed no adverse effects after exposure to WideStrike® cotton pollen expressing Cry1F and 

Cry1Ac in combination at respective concentrations of 510 and 2,500 μg/L (MRID No. 

45808412). Based on a worst-case exposure of one kg of transgenic cotton pollen per liter of 

pond water, the rate of fortification at these concentration are 298X and >23,000X the 

anticipated EEC for Cry1F and Cry1Ac protein in surface water. However, this study was 

determined of minimal value for evaluating the potential effects to nontartget freshwater 

invertebrates because the length of the study was not conducted at the Agency’s 

recommendation of at least 7-14 days, since the mode of action for Bt would likely take effect 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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after several days of exposure. In addition, EPA 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 (see US EPA 2010a) and that 

post-harvest crop residue is the most likely route of exposure to aquatic organisms (Carstens et 

al. 2011). However, in light of published studies showing reduced growth in caddis flies exposed 

to anti-lepidopteran Cry1A protein (Rosi-Marshall et al. 2007), concerns regarding the potential 

for exposure to shredder invertebrate species via consumption of Bt crop plant litter in aquatic 

environments. EPA determined exposure to shredder invertebrate species from Bt crops is likely

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). Nevertheless, EPA has required aquatic invertebrate testing lasting 7-10 days with 

Daphnia spp. to reduce uncertainty in past PIP registrations of Bt crops. A study with shredder 

species is even more appropriate since it would directly test the invertebrate species expected to 

be affected by Bt plant litter that may enter streams.

Specifically for soybean, the Agency has determined that the exposure of aquatic systems to 

Cry1F and Cry1Ac proteins produced in PIP event DAS-81419-2 soybean are negligible. 

Movement of Cry proteins from off-field pollen or post-harvest crop residues to aquatic habitats 

is not expected given that soybeans are primarily self-pollinated and cultivated soybean plants 

release pollen prior to flower opening (Abud et al. 2007; Caviness 1966). The Agency previously 

concluded that plant litter from both Bt corn and Bt cotton crops are not deposited in amounts 

high enough to result in adverse effects (US EPA 2001b). Since soybean produces less aboveground residue under cultivation (Green and Blackmer 1995), this conclusion is also made for 

the cultivation of DAS-81419-2 Soybean.

Based on the acute oral toxicity study on rainbow trout, adverse effects to freshwater fish are not 

anticipated as a result of the registration of Cry1F and Cry1Ac as expressed in event DAS81419-2 soybean. Risk posed to aquatic invertebrates (including the aquatic Lepidoptera) 

through DAS-81419-2 cultivation was concluded to be much reduced due to a low likelihood of 

exposure (Carstens et al. 2011) and no verified instances of sensitive species. In view of the lack 

of toxicity and minimal aquatic exposure, the rainbow trout study and the data waiver rationale 

for freshwater invertebrates are considered to be adequate to support the conclusion that 

deployment of event DAS-81419-2 for use as a PIP in soybean poses little risk to aquatic 

organisms.

 4. Estuarine and Marine Animals

BPPD typically waives the requirement of studies with estuarine/marine animals for Bt Cry

proteins because of an expected lack of exposure in these environments. Therefore, data were not 

required for Cry1F and Cry1Ac for the registration of DAS-81419-2 Soybean, and based on 

expected lack of exposure; adverse effects to these species are not anticipated.

5. Terrestrial and Aquatic Plant Species

BPPD typically waives nontarget plant testing for Bt Cry proteins, since the active ingredients 

are insect toxins (Bt δ-endotoxin) that have never shown any toxicity to plants. Therefore, BPPD 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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has concluded that adverse effects of Cry1F and Cry1Ac insecticidal proteins expressed in DAS81419-2 Soybean to terrestrial and aquatic plants are not anticipated.

The active ingredients Cry1Ac and Cry1F are part of a larger group of insect toxins produced a 

naturally-occurring Bacillus thuringiensis (Bt -endotoxins) that have not shown toxicity to 

plants. Effects to nontarget plants in the terrestrial and aquatic environments are not expected, 

and BPPD concludes that adverse effects to terrestrial and aquatic plants as a result of the 

registration and use of Cry1F and Cry1Ac insecticidal proteins expressed in event DAS-81419-2 

Soybean are not anticipated.

6. Invertebrate Species

BPPD assumes that nontarget insects receive exposure primarily through consumption of DAS81419-2 Soybean leaf foliage, pods, and occasionally other soybean plant tissues as well as 

indirect exposure through consumption of insects that feed on soybean plant tissue. The principal 

route of exposure to soil-dwelling invertebrates, such as collembola and earthworms, is assumed 

to be consumption of decomposing plant tissue, and also possibly plant exudates, in soil during 

feeding.

The registrant requested data bridging to the existing ecotoxicological studies on the effects of Bt 

Cry1F and Cry1Ac proteins expressed in WideStrike® PIP cotton on representative nontarget 

species, including nontarget beneficial insects, pollinators, detritivores, and incidentals (MRID 

No. 48828413). As previously discussed, the Cry1F and Cry1Ac proteins produced in DAS81419-2 soybeans were determined biologically equivalent with the microbial-derived Cry1F 

and Cry1Ac protein test substances used for producing toxicological data to support 

WideStrike® cotton (MRID No. 48828403 and 49044803; reviewed in US EPA 2013 ). The 

high-end exposure estimates (HEEEs) were derived from protein expression levels of Cry1F and 

Cry1Ac via Enzyme-linked immunosorbent assay (ELISA) analyses on various soybean plant 

tissue parts of PIP event DAS-81419-2. Exposure estimates for Cry1F and Cry1Ac protein 

concentrations in PIP event DAS-81419-2 soybean were compared with endpoints reported from 

toxicity studies previously conducted for WideStrike® cotton on various representative nontarget 

test species to determine a Margin of Exposure (MOE). The results of the MOE calculations are 

presented in Tables B.2. and B.3. 

A MOE of 50% mortality at 10X the estimated environmental exposure is regarded as sufficient 

to demonstrate negligible risk and was also the threshold for consideration of uncertainties in the 

risk assessment (US EPA 1998; US EPA 2007). If subsequent expression characterization in the 

commercial event leads to a safety margin of <10X, then the consequences are considered in 

terms of uncertainties in the ecological risk assessment below (US EPA 2007). The ratio of the 

endpoint value to the EEC and are presented in Tables B.2. and B.3. for Cry1F and Cry1Ac, 

respectively. The published EPA level of concern is 50% mortality at 5× MEEC (US EPA 1998). 

It should also be noted that the dose margin can be less than 10× where uncertainty in the test 

system is low. High dose testing also may not be necessary where many species are tested or 

tests are very sensitive, although the test concentration used must exceed 1× MEEC (US EPA 

1998; US EPA 2007). The registrant note that the concentrations tested were much higher than 

those encountered under field conditions. Since most MOEs exceeded 10X the estimated 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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environmental concentrations (Tables B.2. and B.3.), the results of the MOE calculations 

indicates the most sensitive individual in a population is not likely to be adversely affected. 

a. Beneficial Arthropods and Pollinators

Data from a dietary toxicity study on lady bird beetle previously conducted for WideStrike

cotton showed that adverse effects did not occur to the test insects at >300 μg/mL diet for Cry1F 

and >22.5 μg/mL diet for Cry1Ac assuming a maximum expected environmental concentration 

of 10.6 ng/mg fresh weight for Cry1F and 4.76 ng/mg fresh weight for Cry1Ac (see MRID No. 

45542315). Dow did not analyze protein expression levels for pollen or soybean floral nectar, as 

Cry1 protein expression in this secretion in four cotton lines is negligible (Wolt 2002; MRID No. 

45808420). Therefore, the MOE calculation for lady beetle relies on the highest level of protein 

expression found in the field expression study (V5 leaf). Based on the estimated expression level 

in leaf (V5) tissue, adverse effects would not be expected at concentrations up to 25.6X for 

Cry1F and 4.3X Cry1Ac.

The registrant requested data bridging to the dietary toxicity study on green lacewing 

(Chrysoperla rufilabris) larvae, which was conducted by presenting the green lacewings with a 

moth egg (Sitotroga sp.) diet incorporated with Bt Cry1F and Cry1Ac proteins (MRID No. 

45808410). Results showed that adverse effects did not occur to the test insects at >48.2 μg/g 

diet for Cry1Ac and >5.2 μg/g diet for Cry1F. However, this study was considered supplemental.

A previous EPA FIFRA Scientific Advisory Panel (SAP) determined that it is inappropriate to 

test the activity of insecticidal proteins by incorporating the Bt protein into a moth egg diet, since 

Bt protein may bind to the surface of the moth eggs, resulting in limited exposure to lacewings 

that feed with piercing sucking mouthparts (US EPA-SAP 2002). Furthermore, green lacewings 

do not consume much pollen in the field (US EPA-SAP 2002) and are not exposed to Bt proteins 

via consumption of aphids (Head et al. 2001). In the Agency’s environmental risk assessment of 

WideStrike cotton, this study was classified as supplemental and the Agency recommended 

other generalist predators (e.g. minute pirate bug) as a representative surrogate test organism for 

Tier I testing the effects of Cry1F and Cry1Ac proteins (US EPA 2005). Thus, the green 

lacewing study on the effects of Cry1Ac and Cry1F proteins expressed in DAS-81419-2 soybean 

can be bridged, but only offer supplemental information towards the environmental risk 

assessment of DAS-81419-2 soybean. Other information on the effects of Cry1Ac and Cry1F 

exist on green lacewing larvae concludes Cry1Ac and Cry1F toxins do not have a detrimental 

effect on the species when they are ingested either directly or through prey (Rodrigo-Simón 

2006; Tian et al. 2013). 

A dietary toxicity study that tested the effects of Cry1F and Cry1Ac expressed in DAS-81419-2 

soybean in honey bee (Apis mellifera) was also cited for data bridging. Results showed no effect 

on survival of larvae to adult emergence at >1.98 μg/mL sugar water diet for Cry1F and >11.9 

μg/mL sugar water diet for Cry1Ac (MRID No. 45542316). Pollen expression data for DAS81419-2 soybean was not available, however, the MOE previously calculated for WideStrike® 

cotton showed the LC50 was >4X the levels of pollen expressed. These data can be inferred to 

represent the MOE for event DAS-81419-2 soybean, since the same genetic construct that was 

used to transform WideStrike® cotton was used for event DAS-81419-2 soybean and the 

constitutive expression in pollen should be similar. In addition, other information from a metaanalysis (Duan et al. 2008) of several laboratory acute oral toxicity studies on honeybee is 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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available, which consistently demonstrated no biological activity in Hymenoptera order of 

insects from Cry proteins that target lepidopteran pests. Six Cry1F and Cry1Ac studies were 

included in the meta-analysis, suggesting that regardless of expression level in event DAS81419-2 soybean pollen the MOE will be acceptable. 

An acute toxicity study on parasitic wasp species (Nasonia vitripennis) was cited for data 

bridging. Results showed adverse effects did not occur to the test insects at >5.2 μg/g diet for 

Cry1F, respectively, and >46.8 μg/g diet for Cry1Ac, which represented at least at 14X the 

concentration found in WideStrike® cotton pollen (see MRID No. 45808411). Similar to the 

green lacewing and a honey bee study, the MOE was not determined for the parasitic wasp 

species due to the lack of pollen and nectar expression data, and some uncertainty remains. . 

However, the HEEE of Cry1F and Cry1Ac from V5 leaf sample (as opposed to pollen) relative 

to the dose concentrations used on honeybees and nontarget arthropods (likely to be exposed via 

pollen ingestion) in toxicity studies is considered highly conservative and the margins of 

exposure (see Tables B.2. and B.3.) are well above dose levels of Cry1Ac and Cry1F protein 

toxicity testing showing no adverse effects. 

The registrant noted that the concentrations tested were much higher than those encountered 

under field conditions. Since most MOEs exceeded 10X the estimated environmental 

concentrations (Tables B.2. and B.3.), the results of the MOE calculations indicates the most 

sensitive individual in a population is not likely to be adversely affected. 

Further, in regards to exposure of Cry1F and Cry1Ac proteins expressed in event DAS-81419-2 

soybean pollen, pollinators and other beneficial arthropods are anticipated to be less exposed 

based upon the cleistogamous nature of its flowers (in most cultivars). Soybean is predominantly 

self-pollinated and its pollen is essentially contained in the flower, since anthers usually dehisce 

and release pollen before flowers open (Caviness 1966; OECD 2000). Typical outcrossing rates 

are less than 1% (CPC 2004), so this suggests that contact of honeybees and other beneficial and 

predatory invertebrates to soybean pollen is minimal. So although the MOE to honeybees, green 

lacewings, and lady beetle were calculated from the HEEEs from Cry1F and Cry1Ac proteins in 

leaf tissue (V5) stage instead of pollen, the likelihood of actual field exposure to many nontarget 

invertebrates via pollen consumption or indirectly through consumption of pollen-feeding 

invertebrates is expected to be minimal. Since no pollen is expected to travel outside the field 

borders, off-field exposure to many nontarget invertebrates is further reduced. 

In any case, no adverse effects were found on previously conducted toxicity testing of Cry1F and 

Cry1Ac proteins on representative nontarget beneficial arthropods and pollinators species (e.g. 

honeybees, lady beetles, green lacewings) based on the majority of the MOEs exceeded 10X the 

estimated environmental concentrations (Tables B.2. and B.3.). The results of the MOE 

calculations indicate the most sensitive individual in a population is not likely to be adversely 

affected. Risk to honeybees and the nontarget arthropods is further reduced due to minimal 

potential for exposure via pollen. Further, published meta-analysis of laboratory and field studies 

showed no significant differences were noted on populations of nontarget invertebrates on a 

community level for Bt crops expressing Cry1F and/or Cry1Ac vs. non-transgenic crops 

(Marvier, et al 2007; Duan et al. 2008). Lastly, an independent field census study showed no 

differences in the population of nontarget arthropods (MRID No. 48828412) in field sites 

containing event DAS-81419-2 soybean vs. non-transgenic conventional soybean lines. Among 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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the taxa showing no significant population impacts were two that were also tested in the 

laboratory: Coccinellidae (lady beetles) and Parasitica (parasitoid wasps). In view of the lack of 

toxicity and limited exposure via pollen consumption, no adverse effects are anticipated on 

nontarget beneficial arthropods and pollinators from the cultivation of PIP event DAS-81419-2 

Soybean.

b. Soil Dwelling Invertebrates

An earthworm study conducted for WideStrike® cotton was also cited for data bridging. 

Earthworms (Eisenia foetida) were exposed to artificial soils treated with 247 mg/kg and 107 

mg/kg of microbial-produced Cry1Ac and Cry1F proteins, respectively, for 14 days (MRID No. 

45580701). The study showed no effects on mortality or burrowing time resulting from exposure 

to Cry1F and Cry1Ac present in artificial soil at approximately 716X for Cry1F and 1176X for 

Cry1Ac at the maximum EEC. The maximum EEC of the soil was 0.345 mg/kg for Cry1F and 

0.091 mg/kg for Cry1Ac based on soybean plant residue in the top 15cm of soil.

Data from a toxicity study on Collembolas (Folsomia candida) previously conducted for 

WideStrike cotton showed that adverse effects did not occur to the test insects at >702 mg/kg 

soil for Cry1F and >22.6 mg/kg soil for Cry1Ac (MRID No. 45808409). The MOE for Cry1Ac 

was calculated as >2035X and >248X for Cry1F, assuming a maximum EEC of 0.345 mg/kg 

for Cry1F and 0.091 mg/kg for Cry1Ac based on soybean plant residue in the top 15cm of soil. A 

lack of adverse effects following exposure to Cry1Ac protein has also been shown for 

collembolans (Folsomia candida) (Sims and Martin, 1997).

Based on the studies submitted for DAS-81419-2, as well as other information for Cry1F and 

Cry1Ac, risk to nontarget soil invertebrates resulting from the cultivation of PIP event DAS81419-2 Soybean is not expected.
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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Table B.2. Margins of Exposure calculated from Cry1F Protein Concentrations in PIP Event DAS-81419-2 

soybean plant tissues relative to Dose Concentrations used in the Nontarget Arthropod Ecotoxicological 

Studies previously conducted on WideStrike® PIP cotton plant- and microbial- derived Cry1F protein a

MRID No. Study Type Species Tissue 

sample HEEEb Tox. Endpoint MOE Comment

45542316

Honey Bee 

Larva 

Testing, 

Tier I

(885.4380)

Honeybee

(Apis 

mellifera)

Pollen ND >1.98 

μg/mL diet 

NOEC

ND No effects found 

for honey bee 

larvae and adults 

in published metaanalysis with 

Cry1F and/or 

Cry1Ac 

expressing Bt 

crops (Duan et al.

2008)

45808410

Nontarget 

Insect 

Testing, 

Tier I 

(885.4340)

Green 

lacewing

(Chryosperla 

camea)

Nectar, 

pollen

ND >5.2 μg/g 

diet acute 

oral LC50

ND No effects found 

in Cry1Ac and 

Cry1F protein lab 

study (RodrigoSimón et al. 2006) 

and tritrophic 

feeding study on 

green lacewings 

exposed to a diet 

of Cry1F or 

Cry1Ac resistant 

prey (Tian et al. 

2013)

45542315

Nontarget 

Insect 

Testing, 

Tier I 

(885.4340)

Lady beetle

(Hippodamia 

convergens)

Pollen 

(used V5 

Leaf)

10.6 

ng/mg fw

>300 μg/mL 

diet acute 

oral LC50

>25.6 Pollen expression 

data is not 

available; Highest 

expression in leaf 

converted to fw

for MOE 

calculation.

45808411

Nontarget 

Insect 

Testing, 

Tier I 

(885.4340)

Parasitic wasp

(Nasonia 

vitripennis)

Nectar ND >5.2 μg/mL 

diet acute 

oral LC50

ND No feeding in 

narrow soybean 

flowers

45808409

Nontarget 

Insect 

Testing, 

Tier I 

(885.4340)

Collembola

(Folsomia 

candida)

R3 

Forage 

soil 

residue

0.345 

mg/kg 

soil

>702 mg/kg 

soil NOEC

>2035 Plant residue in 

top 15 cm soil

45580701

Earthworm

Toxicity 

Study

Earthworm

(Eisenia 

foetida)

R3 

Forage 

soil 

residue

0.345 

mg/kg 

soil

247 mg/kg 

soil NOEC

716 Plant residue in 

top 15 cm soil

a Microbe-derived Cry1F protein used in laboratory studies was demonstrated to be equivalent with the Cry1F protein expressed in DAS-81419-2

b HEEE = mean expression level + (t0.1, upper tail, n-1 x std. dev.) / n1/2. HEEE values based on Cry1F expression level determined for DAS-81419-2 

plant tissue relevant to the potential exposure route for the organism of interest.

ND = Not Determined (data not available). 

Table B.2 reproduced from Table 3, page 26 from MRID No. 48828413
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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Table B. 3. Margins of Exposure calculated from Cry1Ac protein concentrations in DAS-81419-2 Soybean 

plant tissues relative to dose concentrations used in the Nontarget Arthropod Ecotoxicological Studies 

previously conducted on WideStrike® PIP cotton plant- and microbial- derived Cry1Ac protein a

MRID 

No.

Study Type 

and OCSPP 

Guideline 

No. 

Species

Tissue 

sample HEEEb Tox. 

Endpoint MOE Comment

45580701 Earthworm

Toxicity 

Study

Earthworm

(Eisenia 

foetida)

R3 

Forage 

soil 

residue

0.091 

mg/kg 

soil

107 mg/kg 

soil NOEC

1176 Plant residue in 

top 15 cm soil

45808409 Nontarget 

Insect 

Testing, Tier 

I (885.4340)

Collembola

(Folsomia 

candida)

R3 

Forage 

soil 

residue

0.091 

mg/kg 

soil

>22.6 mg/kg 

soil NOEC

>248 Plant residue in 

top 15 cm soil

45542316 Honey Bee 

Larva 

Testing, Tier 

I 

(885.4380)

Honeybee

(Apis 

mellifera)

Pollen ND >11.9μg/mL 

diet NOEC

ND No effects found

for honey bee 

larvae and adults

in published metaanalysis with 

Cry1F and/or 

Cry1Ac expressing 

Bt crops (Duan et 

al. 2008)

45808410 Nontarget 

Insect 

Testing, Tier 

I (885.4340)

Green 

lacewing

(Chryosperla 

camea)

Nectar, 

pollen

ND >46.8 μg/g 

diet acute oral 

LC50

ND No effects found 

in Cry protein lab 

study (RodrigoSimón et al. 2006) 

and tritrophic 

feeding studies on 

green lacewings 

exposed to a diet 

of Cry1F or 

Cry1Ac resistant 

prey (Tian et al. 

2013)

45542315 Nontarget 

Insect 

Testing, Tier 

I (885.4340)

Lady beetle

(Hippodamia 

convergens)

Pollen 

(used 

V5 

Leaf)

4.76 

ng/mg 

fw

>22.5 μg/mL 

diet acute oral 

LC50

>4.3 Highest expression 

in leaf converted 

to fw. No effect 

found in Cry1Ac 

feeding study in 

Coleomegilla 

maculata (Li et al. 

2011)

45808411 Nontarget 

Insect 

Testing, Tier 

I (885.4340)

Parasitic wasp

(Nasonia 

vitripennis)

Nectar ND >46.8 μg/mL 

diet acute oral 

LC50

ND No feeding in 

narrow soybean 

flowers

a Microbe-derived Cry1Ac protein used in laboratory studies have been demonstrated to be equivalent with the Cry1Ac protein expressed in 

DAS-81419-2

b HEEE = mean expression level + (t0.1, upper tail, n-1 x std. dev.) / n1/2. HEEE values based on Cry1Ac expression level determined for DAS-81419-2 

plant tissue relevant to the potential exposure route for the organism of interest.

ND = Not Determined (data not available). Table B.3 reproduced from Table 3, page 26 from MRID No. 488284-13
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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7. Supplemental Information

Where a lack of expression data prevented calculation of high-end exposure values, other 

supplemental information (i.e. meta-analyses, biological specificity to the pest species, targeted 

toxicity studies, or food preferences) was used as a supporting line of evidence to support the 

conclusion of no adverse effects on NTOs from Cry1F and Cry1Ac proteins expressed in DAS81419-2 soybean. To address any uncertainties in the risk assessment focused on taxonomic 

groups that were most likely to be exposed to Cry1F and Cry1Ac proteins expressed in PIP event 

DAS-81419-2 soybean in field settings, the potential for adverse effects of DAS-81419-2 on 

nontarget arthropod populations was assessed in a US field study (MRID No. 48828412). A 

nontarget arthropod field study with PIP event DAS-81419-2 soybean, a near-isogenic nontransgenic control (Maverick) and four non-transgenic reference lines (DSR 3590; IL 3505; 

Porter 75148; Williams 82) was conducted in 2011 at two field sites in the U.S. located in 

Richland, Iowa and York, Nebraska. Each trial site included the six soybean lines arranged in a 

randomized complete block design with four replicate blocks. 

Arthropod abundance was sampled during the vegetative and reproductive soybean stages and 

monitored using three sampling methods: pitfall trapping, sticky card trapping, and vertical beat 

sheet sampling. Arthropods collected during the study included representatives from multiple 

orders and diverse ecological roles, including phytophagous, predatory, parasitic, and 

saprophagous modes of feeding. The analyses encompassed several taxonomic groups, 

including: Oribatida (soil mites), Araneae (foraging and web spiders), Collembola (springtails), 

Hemiptera (e.g. Orius spp.), Coleoptera (e.g. ground beetles, lady beetles), Neuroptera (e.g. 

Chrysopa spp.), Lepidoptera, Diptera, and Hymenoptera (e.g. parasitoid wasps). Consistent 

across locations and sampling methods, results showed arthropod populations associated with 

DAS-81419-2 soybean were similar to those of non-transgenic soybean. Therefore, the lack of 

differences in between populations of nontarget arthropod taxa and their abundance from the 

field monitoring suggests no adverse effects on nontarget arthropod communities from 

deployment of event DAS-81419-2 soybean cultivation. 

F. Soil Fate

Since proteins are not known to persist in the environment due to the ubiquitous nature of 

proteases in microbes, the registrant submitted a rationale for demonstrating rapid degradation of 

Cry1F and Cry1Ac proteins in various crop soils. Data from a previously submitted laboratory

study showed that Cry1F and Cry1Ac expressed in cotton plants degrades quickly and does not 

accumulate in soil for WideStrike® PIP cotton (MRID No. 45556801). The soil half-life of the 

plant expressing Cry1F and Cry1Ac was estimated as 1.3 days and the Cry proteins were not 

detectable after 14 days. These results verify that the Cry1F and Cry1Ac proteins degrade rapidly 

in soils sampled from cotton growing regions.

The registrant also provided a data waiver rationale to support rapid degradation of Bt Cry1F and 

Cry1Ac proteins in soil based on the history of safe use in sprayable Bt formulations. Cry1Ac is 

present in both WideStrike® cotton event DAS-21023-5 (also described as 3006-210-23) and 

Bollgard® cotton (event MON 531, as well as a discontinued corn event: DBT418Cry1Ac corn) 

(Mendelsohn et al. 2003; Sanahuja et al. 2011). Cry1F is also present in WideStrike® cotton 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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event DAS-24236-5 (also described as 281-24-236), as well as, in Herculex® and SmartStax® 

corn (event TC1507) (Mendelsohn et al. 2003; Sanahuja et al. 2011). Other supporting 

information included previously conducted field studies showing no accumulation (i.e. 

persistence) of the proteins as a result of continuous planting of other Bt crops containing these 

proteins (Head et al. 2002; Herman et al. 2002; Shan et al. 2008). As a result, the Agency has 

determined that no additional long-term field studies are required for PIP event DAS-81419-2 

soybeans because currently registered PIPs have not been shown to persist in soil.

Although the registrant submitted a sufficient rationale of rapid degradation of Cry1F and 

Cry1Ac proteins in other crop soil systems, a soil degradation study has not been evaluated in 

soybean for this specific PIP event. A previous EPA FIFRA SAP noted that in soil degradation 

and field persistence studies, toxin degradation rates may vary with the crop, the toxin produced 

by the transformation event, the microbial community in the recipient soil microcosm, the 

statistical characterization of toxin persistence, and/or the initial dose of toxin in the experiment 

(US EPA 2001a). These differences in Cry toxin concentration may be due to the fact that 

different genetic constructs were used and/or inserted at different locations in the plant genome 

(Yu et al. 2012). Plant genotype and parental backgrounds have also been recognized as 

important factors influencing protein expression (Kranthi et al. 2005; Adamczyk and Sumerford 

2001; and Torres et al. 2006). Other important factors that affect degradation of Cry toxins 

include environmental conditions such as temperature, light, drought, and soil properties (e.g. 

pH, texture, clay content) (Adamczyk et al. 2004; Chen et al. 2005; Jiang et al. 2006; Rochester 

2006; Dong et al. 2008; Hallikeri et al. 2009; Addison and Rogers 2010; Chen et al. 2011; 

Ranjithkumar et al. 2011). To reduce this uncertainty, a soil fate study to determine the 

degradation rate (DT50) of Cry1F and Cry1Ac proteins sampled from representative soils of 

soybean growing regions is to be conducted as confirmatory data.

G. Effects on Soil Microorganisms 

Numerous published studies indicate that exposure to Cry protein produced in Bt PIP crop plants 

does not adversely affect soil microorganisms (Sanvido et al. 2007). Although a minimal 

transient increase and shift in microbial populations may result from the presence of transgenic 

plant tissue in soil, no adverse effects have been attributed to the Cry protein. In addition, the soil 

degradation studies discussed in the WideStrike cotton BRAD show Cry1F and Cry1Ac 

proteins degrade quickly in soil (US EPA 2005). 

With regard to the impact of genetically engineered crops on soil, EPA has previously noted (US 

EPA 2010a) that agricultural practices themselves cause large changes in soil and soil microbial 

composition. Furthermore, factors such as variations in seasons and weather, plant growth stage, 

and plant varieties, independent of being genetically engineered, are also responsible for 

significant shifts in soil microbial communities. Most studies with genetically engineered crops 

to date have shown minor or no effects on soil microbes beyond the variation caused by the 

factors listed above. If reports of adverse effects became available, the Agency will take 

appropriate action to mitigate potential risks.

H. Horizontal Transfer of Transgenes from Bt Crops to Soil Organisms 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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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 other microorganisms or plants has not been detected. There is no known 

mechanism for, or definitive demonstration of, DNA transfer from plants to microbes (Conner et 

al. 2003). Furthermore, several published experiments, that were conducted to assess the 

likelihood of HGT, have been unable to detect gene transfer under typical environmental 

conditions. Horizontal gene transfer to soil organisms has only been detected with very

promiscuous microbes under laboratory conditions designed to favor transfer. As a result of these 

findings and the fact that the Bt toxins engineered into event DAS-81419-2 are derived from soilinhabiting bacteria, the EPA has concluded that the risk of HGT of transgenes found in event 

DAS-81419-2 soybean expressing Cry1F and Cry1Ac proteins are low.

I. 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 Bt plant-incorporated protectants currently registered, and was 

extensively reexamined in 2010 (see US EPA 2010a), and EPA believes that these concerns have 

been satisfactorily addressed. 

The FIFRA EPA Scientific Advisory Panel meeting held on October 18-20, 2000 discussed the 

matter of gene flow and offered some issues for consideration in this matter. The panel agreed 

that the potential for gene transfer between Gossypium hirsutum (cotton) and any receptive 

plants within the U.S., its possessions and territories, was of limited probability and nearly risks 

free (US EPA 2000). Since soybean is also predominantly a self-pollinating species, the dispersal 

of pollen is limited because the anthers mature in the bud and directly pollinate the stigma of the 

same flower (OECD 2000). Thus, the risk of potential gene transfer between soybean and any

receptive plants is further negligible. Lastly, there are no known wild or weedy relatives exist in 

the U.S. with which Glycine max (L.) Merr. can form viable hybrids in nature, and soybean is not 

weedy in character or invasive (US EPA 2007). Therefore, BPPD determined that there is no 

significant risk of gene capture and expression of any Bt endotoxin by wild or weedy relatives of 

soybean in the U.S., its possessions or territories. 

J. Endangered Species Considerations

Because of the selectivity of Cry1F and Cry1Ac insecticidal crystal proteins for lepidopteran 

species and lack of evidence of effects on other nontarget species, the Agency has investigated 

concerns for Federally listed threatened and endangered insect species in the order Lepidoptera. 

Because soybean pollen is not expected to move beyond the planted soybean field and its 

immediate margins, as discussed above, any exposure to lepidopterans would be expected to 

occur within those areas. Exposure could occur via direct consumption of PIP event DAS-81419-

2 soybean plants or consumption of DAS-81419-2 pollen that falls on non-soybean plants within 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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the soybean field and its immediate margins. However, oral exposure to significant amounts of 

Cry1F and Cry1Ac via pollen consumption is not likely. Little pollen is expected to be released 

from soybean flowers, since soybean plants are predominantly self-pollinated and anthers usually

dehisce and release pollen before flowers open. Airborne pollen concentrations have been 

measured at very low levels within soybean fields (mean of 0.18 grains/cm2/day) (Yoshimura et 

al. 2006). Based on this analysis, BPPD concludes that exposure resulting from pollen falling on 

potential non-soybean food plants in the field and immediate margins is not sufficient to cause 

effects in listed lepidopterans. Therefore, any significant exposure would have to occur through 

consumption of the DAS-81419-2 soybean plants on the field or via tritrophic effects, in which 

predatory beneficial arthropod species consume soybean pest species feeding on PIP event DAS81419-2 soybean plant tissues. 

A search of EPA’s LOCATES database indicates that three species of listed lepidopterans are 

present in U.S. counties in which soybeans are grown. These are the Karner blue butterfly 

(Lycaeides melissa samuelis), St. Francis’s Satyr Butterfly (Neonympha mitchellii fransisci), and 

Mitchell’s Satyr Butterfly (Neonympha mitchellii mitchellii). The potential effects of Bt PIPs in 

corn on the Karner blue butterfly was extensively analyzed in BPPD’s Bt Crops Reassessment 

(USEPA 2001b, 2004b and 2010a and b) and in the endangered species assessment section in the 

BRAD for event MON87701 soybean expressing Cry1Ac protein (see US EPA 2010c). As 

previously discussed, soybean pollen is not expected to be deposited on plants in or around 

soybean fields in amounts sufficient to cause effects in sensitive lepidopterans. Ecology and life 

history information demonstrated that habitat requirements for larvae and adults of two of the 

identified species (Mitchell's Satyr butterfly and Saint Francis' Satyr butterfly) do not overlap 

with commercial soybean acreage, and are therefore not expected to be impacted by PIP event 

DAS-81419-2 cultivation (Bartel and Sexton 2009; Barton and Bach 2005; US FWS 1997). 

There is a possible overlap between the geographic range for Karner blue butterfly and soybean 

use sites. EPA has previously reviewed information on the proximity of Karner blue habitats, and 

none are known to exist immediately adjacent to agricultural fields (“adjacent to” was defined as 

0-3 m for corn fields, which is reasonably applied to soybean fields) (US EPA 2001b). Karner 

blue larvae feed only on lupines (Lupinus spp.) and soybean is neither identified as a larval food 

source, nor is soybean open-pollinated. Thus, pollen from event DAS-81419-2 soybean is highly

unlikely to be deposited on lupine leaves (NatureServe 2013). Furthermore, soybean is not 

identified as a nectaring source for Karner blue adults. Therefore, the risk posed to the Karner 

blue butterfly was considered to be negligible. 

Based on the above analysis, the EPA determines that there will be no direct effect to listed 

lepidopteran species as a result of the cultivation of DAS-81419-2 soybeans as proposed. 

Obligate relationships between insectivorous listed species with lepidopterans that are expected 

to be found in soybean fields, especially pest species that feed on DAS-81419-2 plants, are not 

currently known. Since the Cry1F and Cry1Ac in DAS-81419-2 soybeans targets only 

lepidopteran insects, loss of the pest insects as a result of DAS-81419-2 are expected to be offset 

by the presence of other insects that could act as food sources for listed species, including 

beneficial insects that are known not to be affected by Cry1F and Cry1Ac. Effects on species 

other than insects have also been determined to be very unlikely because of the specificity of 

Cry1F and Cry1Ac.
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

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Lastly, since the Agency previously determined Cry1F and Cry1Ac proteins are not expected to 

have adverse effects on mammals, birds, plants, freshwater and estuarine/marine fish and 

invertebrates, nontarget insects and other invertebrate species at the EEC, a “No Effect” 

determination is made for direct and indirect effects to Federally listed threatened and 

endangered species and their designated Critical Habitats. In addition, EPA does not expect that 

any threatened or endangered plant species will be affected by outcrossing to wild relatives or by

competition with such entities. Hybrid soybean does not exist in the wild in the United States, 

nor do any wild plants that can interbreed with soybean in the United States.

K. Data Needed to Confirm DAS-81419-2 Soybean Environmental Fate Assessment

Although the registrant submitted a sufficient rationale of rapid degradation of Cry1F and 

Cry1Ac proteins in other crop soil systems, the degradation rate (DT50) of Cry1F and Cry1Ac 

proteins sampled from representative soils of soybean growing regions has not been evaluated

and must be conducted as confirmatory data. The soil degradation study is to be submitted to the 

EPA within 18 months of the issuance date of the DAS-81419-2 Soybean Notice of Pesticide 

Registration. 

II. Conclusion

The biological equivalency of Cry1F and Cry1Ac proteins expressed in event DAS-81419-2 

soybean plant was confirmed with the previously characterized microbial-derived Cry1F and 

Cry1Ac protein test substances. No significant increase in exposure to NTOs was demonstrated 

based on calculated margins of exposure for protein concentrations in event DAS-81419-2 plant 

tissue matrices relative to the dose concentrations used in the NTO laboratory toxicity tests 

conducted on Cry1Ac and Cry1F. Lastly, a field monitoring study showing no differences on

nontarget arthropods on a population and community level and other supporting supplemental 

information (e.g. meta-analysis, target feeding studies, and biological specificity to the target 

pest) provided additional certainty to support the “weight-of-evidence” that there is negligible 

risk to NTOs representing diverse taxonomic groups and exposure scenarios from the cultivation of 

DAS-81419-2 soybean.

Therefore, the submitted data supports the applicant’s request and the findings from the 

environmental risk assessment conducted for WideStrike® cotton can be bridged to support a 

breeding/seed increase registration for PIP event DAS-81419-2 (US EPA 2005). Based on 

extrapolating from previous studies on Bt proteins, which tend to be very species specific in their 

activity, the Agency concludes that the cultivation of event DAS-81419-2 soybean will not result 

in adverse effects to nontarget organisms, are not likely to persist in the soil, and pose no risk of 

gene flow and development of weediness in wild relatives, and additionally will have no effect, 

direct or indirect, on Federally listed threatened and endangered species and their designated 

Critical Habitats.
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APPENDIX C: Insect Resistance Management

Cry1Ac and Cry1F proteins as DAS-81419-2 Soybean

Discussions of EPA’s risk assessments for Insect Resistance Management (IRM) are presented in 

this Appendix. Immediately below is the risk assessment completed by EPA’s A. Reynolds on 

November 7, 2013.

I. Insect Resistance Management Review 

Below is the BPPD review of Dow’s Insect Resistance Management (IRM) plan and supporting 

data for the application to register DAS-81419-2 Soybean PIP for seed production and research 

purposes, as well as supporting documentation concerning natural refuge for Bt cotton (U.S. EPA

memorandum 11/7/13; EPA File Symbol 68467-EN, MRID 49024110 replaced MRID

48828415).

CONCLUSION AND REQUIREMENTS

1) Because DAS-81419-2 soybean is intended for breeding/seed increase, Dow did not propose a 

species-specific IRM plan for the product. Rather, the company proposed to mitigate resistance 

risk by acreage limitations on a county (1,000, 10,000, or 25,000 acres) and national (250,000 

acres) scale. BPPD agrees that given the low acreage projected by Dow, it is unlikely that there 

will be a significant risk of resistance to the main soybean lepidopteran pests in the United 

States. These insects include soybean looper, velvetbean caterpillar, beet armyworm, cotton 

bollworm, green cloverworm, tobacco budworm, and cutworms (Agrotis spp.). This conclusion 

is further supported by the biology of the target insects, which are highly polyphagous (feeding 

on a number of wild hosts and cultivated crops) and (for some species) have limited 

overwintering in non-tropical areas.

2) Should a full commercial registration or expanded acreage be sought in the future, a complete, 

species-specific IRM assessment for DAS-81419-2 will be required. Data needed to support such 

a review should include additional information on pest biology, dose, simulation modeling, cross 

resistance, resistance monitoring, and potential impacts on the natural refuge strategy for Bt

cotton.

3) The use of DAS-81419-2 on a limited basis for seed increase purposes should not significantly 

impact the natural refuge strategy in place for Bt cotton PIPs. Soybean is one of the non-cotton 

crops that have been considered as part of natural refuge for tobacco budworm and cotton 

bollworm. While it can be expected that plantings of DAS-81419-2 soybean will remove some of 

the currently-available natural refuge for Bt cotton, this potential reduction will likely be small 

relative to the total amount of natural refuge present in southern cotton regions. Dow has 

proposed to further mitigate the impact on natural refuge by limiting DAS-81419-2 to 10,000 

acres in counties with > 25,000 total acres of soybean and 1,000 acres in counties with < 25,000 

total acres of soybean. 
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4) Dow must institute a stewardship program for DAS-81419-2 including resistance monitoring 

and remedial action (in the event of resistance). A resistance monitoring plan limited to 

investigations of unexpected pest damage is appropriate for a capped acreage registration. BPPD 

notes that monitoring of two potential target insects, tobacco budworm and cotton bollworm, is 

already being conducted as part of the WideStrike Bt cotton monitoring program. Dow must also

compile annual sales and acreage data for each state and provide a report to EPA if requested. 

Since DAS-81419-2 will be deployed without a structured refuge requirement, a compliance 

assurance plan and grower education program should not be necessary.

IRM Rationale and Proposed Plan for DAS-81419-2 Bt Soybean (MRID No. 49024110)

Dow’s proposed DAS-81419-2 soybean is intended for breeding, seed production, and research 

in the United States. Therefore, acreage of the product grown in the United States will be limited 

to a total of 250,000 per year. Individual counties would also be annually limited to either 20,000 

(in non-cotton growing regions), 10,000 (in cotton growing counties with at least 25,000 acres of 

soybean), or 1,000 acres (in cotton-growing counties with < 25,000 soybean acres).

Dow’s IRM rationale for DAS-81419-2 is based on the limited commercialization of the product 

in the United States. The company reasons that selection pressure should be limited by the 

acreage caps and notes that EPA has not mandated refuges for similarly-capped Bt corn breeding 

registrations. Further, the product provides two modes of action (Cry1F and Cry1Ac) for control 

of lepidopteran pests.

Soybean is one of the lepidopteran crop hosts that have been considered as natural refuge for Bt

cotton. Dow’s acreage proposal for DAS-81419-2 could therefore reduce the amount of soybean 

available for cotton refuge in areas where both crops are planted. To estimate the potential 

effects of DAS-81419-2 on natural refuge, Dow calculated the refuge contribution of soybean 

relative to the amount of cotton as such: [(acres of soybean – proposed DAS-81419-2 

acres)/(acres of soybean + cotton) * 100]. DAS acreage was set at either 10,000 or 1,000 per 

county as described above. Acreage data for county cotton and soybean were obtained from 

USDA-NASS (http://quickstats.nass.usda.gov/) for the 2010 season. Calculations were 

conducted for all counties with plantings of both soybean and cotton in the states of Alabama, 

Arkansas, Georgia, Kansas, Louisiana, Mississippi, Missouri, North Carolina, Oklahoma, South 

Carolina, Tennessee, and Georgia.

A total of 183 counties were surveyed by Dow (data contained in Tables 1-7 in MRID No.

49024110). Of these, 84 had at least 25,000 acres of soybean planted during the 2010 season. In 

these counties up to 10,000 acres of DAS-81419-2 has been proposed. Even with this potential 

reduction in refuge, the amount of available soybean refuge averaged 62% among the 84 

counties. Dow noted that this level of refuge is well above the 20% structured refuge that was 

required for Bt cotton prior to approval of natural refuge. In addition, commercial soybean fields 

are rarely treated for cotton bollworm which should further enhance production of susceptible 

insects.
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The remaining 99 counties analyzed by Dow, with less than 25,000 total soybean acres, would 

have a limit of 1,000 DAS-81419-2 acres. Some of these counties grow little soybean (< 2000 

acres) such that the relative contribution to natural refuge from the crop is likely low, regardless 

of the adoption of DAS-81419-2. Other counties grow larger amounts of soybean; however, in 

these counties full adoption of DAS-81419-2 (1,000 acres) would still have a small impact on 

natural refuge.

Dow indicated a number of other factors add to the conservatism of the IRM rationale:

 Seed production for soybean (the primary intended purpose for DAS-81419-2 in the U.S.) 

occurs in areas where the crop is favored – i.e., in counties with large amounts of soybean 

(>25,000 acres).

 Counties with not as much soybean (<25,000 acres) are less likely to be used for seed 

production. Nonetheless, a “regional” natural refuge may provide susceptible insects 

from other counties since H. zea is known to be highly mobile (Jackson et al. 2008).

 Soybean for seed production (non-commercial production) is frequently treated with 

insecticides for pod-feeding insects. Therefore, seed production soybean is likely to be 

limited in value as natural refuge regardless of the adoption of DAS-81419-2.

 In addition to soybean, H. zea is known to utilize numerous other crop and plant hosts 

including corn, peanuts, sorghum, and weed species. Accordingly, natural refuge for the 

insect is not limited solely to soybean acres and the county refuge estimates provided by 

Dow will be underestimates of the actual amounts.

 

BPPD Review

Dow’s proposal to limit the overall acreage and distribution of DAS-81419-2 in the U.S. should 

be sufficient to mitigate potential resistance development for a seed increase registration. It 

should be noted, however, that BPPD does not have sufficient information in the submitted 

materials to completely assess the risk of resistance for a full commercial registration of DAS81419-2. 

Target Pests

Dow’s submission did not indicate the primary insect pests targeted by DAS-81419-2 soybean. 

BPPD presumes this is because the product is intended for commercialization in South America; 

U.S. plantings will be solely for seed production. Nonetheless, DAS-81419-2 can be expected to 

confer protection against susceptible soybean-feeding lepidoptera while grown for seed 

production in the U.S.

Soybeans can be affected by a number of lepidopteran insects, including (among others) soybean 

looper (Pseudoplusia includens), velvetbean caterpillar (Anticarsia gemmatalis), beet armyworm 

(Spodoptera exigua), cotton bollworm/corn earworm (Helicoverpa zea), green cloverworm 

(Plathypena scabra), and cutworms (Agrotis spp.). Though not common on soybean, tobacco 

budworm (Heliothis virescens) has also been known to infest the crop (Sheck and Gould 1993). 

Other occasional lepidopteran pests include fall armyworm (Spodoptera frugiperda) and 

saltmarsh caterpillar (Estigmene acrea).
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Soybean looper occurs throughout the U.S. where soybeans are grown, but overwinters only in 

tropical regions migrating annually from Mexico, Central America, and the Caribbean (Pedigo 

1999). The insect is multivoltine with two (northern regions) to seven (southern states) 

generations per season. Soybean looper is known to feed on a wide range of vegetables, field 

crops (including corn and cotton), and weeds in addition to soybean (Capinera 2005).

Velvetbean caterpillar, similar to soybean looper, is a tropical species that migrates annually 

northward into the U.S. It can range as far north as New England, but overwinters only in the 

southern end of the Florida peninsula (Pedigo 1999; Barbara 2008). Velvetbean caterpillar is 

polyphagous and feeds on a number of field crops (soybean, alfalfa, cotton, peanut, other 

legumes) and weeds (Barbara 2008).

Green cloverworm also follows a southern overwintering/migration pattern from Gulf Coast 

areas. The insect feeds on a variety of legumes, including soybean, and has up to four 

generations per season (http://ipm.ncsu.edu/AG271/soybeans/green_cloverworm.html).

Cotton bollworm and tobacco budworm are polyphagous Noctuid species that are primary target 

pests of Bt cotton registrations (see descriptions in BPPD 2001 and 2006). Fall armyworm and 

cutworms are secondary pests targeted by some Bt corn registrations (see BPPD 2010).

IRM Risk Considerations

No data were provided in the submission to evaluate the efficacy of DAS-81419-2 against any of 

the potential U.S. target pests. Published data for another Bt soybean cultivar (TIC107 with a 

cry1A gene) showed high activity against velvetbean caterpillar and soybean looper (McRae et 

al., 2005). Cry1Ac and Cry1F are also known to have toxicity to cotton bollworm and tobacco 

budworm (BPPD 2005), while fall armyworm is affected by Cry1F (BPPD 2010). However, 

without specific data for DAS-81419-2, it is not possible to evaluate dose considerations for the 

product or determine the resistance risk for targeted insect pests.

Rather than formulate an insect-specific IRM approach for DAS-81419-2, Dow has proposed 

acreage limitations to lower selection pressure for resistance development. A number of other 

factors should further reduce the risk of resistance such that a formalized refuge strategy is 

unnecessary. These considerations include biological aspects of soybean lepidopteran pests, the 

abundance of alternative hosts (natural refuge) for these insects, the pyramiding of two toxins in 

DAS-81419-2, and anticipated limited plantings of the product for seed increase purposes.

Most of the lepidopteran insects that infest soybean are polyphagous and feed on numerous other 

wild and cultivated hosts which should provide a source of natural refuge to reduce selection 

pressure for Cry1Ac resistance. Further, soybean looper, velvetbean caterpillar, and fall 

armyworm overwinter only in tropical areas (e.g., Florida in the U. S.); population migrate 

annually from the Caribbean, Mexico and Central America northward through the U.S. Other 

key pests, cotton bollworm and green cloverworm, overwinter in southern Gulf Coast states. 

Because of this, it is less likely that any resistant individuals that evolve during the growing 

season would be able to successfully overwinter and propagate the resistant trait to future 

generations. 
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As a pyramid, DAS-81419-2 expresses two toxins (Cry1Ac and Cry1F) for lepidopteran control. 

Pyramided PIPs are thought to increase the respective durability of each toxin relative to mosaics 

of single toxin PIPs (Roush 1998, Caprio 1998, Zhao et al. 2003, others). BPPD notes that the 

potential for some cross resistance exists between Cry1Ac and Cry1F in cotton bollworm and 

tobacco budworm (Gould et al. 1995; BPPD 2005, 2010), which could limit the effectiveness of 

the pyramid for those insects. Nonetheless, pyramiding two toxins in DAS-81419-2 should 

further decrease the likelihood of resistance development.

Acreage limitations have been employed for seed/breeding registrations issued for Bt corn PIPs 

as a means to lower overall selection pressure for resistance. These restrictions have been 

implemented on county (up to 20,000 acres) and national level (250,000 acres total). Dow’s 

proposed acreage for DAS-81419-2 generally follows this standard, with up to 25,000 acres per 

county (250,000 nationally) except in counties where soybean is currently planted (see 

discussion below).

Based on the proposed acreage-limited (breeding) registration and in consideration of the 

biological aspects of the soybean pests discussed above, BPPD concludes the risk of resistance to 

Cry1Ac and Cry1F in DAS-81419-2 should be low. Therefore, a refuge strategy is not 

recommended at this time for DAS-81419-2. However, should any request be made to expand 

the scope of DAS-81419-2 to a commercial registration (with large or unlimited acreage), BPPD 

recommends a formal IRM assessment to better evaluate the resistance risk and develop 

appropriate mitigation measures. Such a review should include appropriate data for dose 

expression, simulation modeling, and cross resistance.

Considerations for Bt Cotton Natural Refuge

In addition to the primary target pests of soybean, BPPD is also concerned about other insect 

pests that are targeted by Bt cotton but may also be exposed to Bt soybean. These insects, 

tobacco budworm (TBW, Heliothis virescens) and cotton bollworm (CBW, Helicoverpa zea), are 

highly polyphagous and are known to exploit numerous crop and wild plant hosts. Cry1Ac and 

Cry1F are expressed in both DAS-81419-2 and WideStrike Bt cotton (Bollgard II also expresses 

Cry1Ac).

The established resistance management plan for Bt cotton involves the use of “natural refuge” in 

which non-PIP hosts (such as weeds and other non-cotton cultivated crops) provide sources of 

susceptible insects to dilute any potential resistance genes arising from transgenic cotton. One of 

the non-cotton crops included in calculations of natural refuge is soybean. Should a significant 

portion of the soybean crop be devoted to a Bt variety, the potential amount of natural refuge 

available to TBW and CBW could be reduced.

To evaluate potential impacts to cotton natural refuge, Dow conducted an analysis of counties in 

southern states growing soybean. The company tabulated soybean acreage in 2010 (USDA 

NASS data) and projected the impact DAS-81419-2 would have on the natural refuge

contribution of soybean. In the surveyed counties with large soybean acreage (>25,000), full 

adoption of DAS-81419-2 would still leave a sizable (average 62%) contribution to natural 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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refuge from non-Bt soybean. As could be expected, the potential reduction in natural refuge was 

greater in counties with less than 25,000, particularly those with less than 5,000 acres. Soybean 

contribution in 19 counties (of 99 total) would be reduced to less than 10% with the full addition 

of DAS-81419-2. In counties with less than 1,000 soybean acres (3 total), refuge contribution 

from soybean could potentially be eliminated. Generally, soybean refuge was lower in Georgia 

and Texas than the other surveyed states. This was largely because these states each have a 

number of counties with low overall soybean acreage but relatively high (> 20,000) cotton 

planting.

Dow’s analysis was limited by considering only soybean as a contributor to cotton natural 

refuge. BPPD agrees with the company that numerous other crops (e.g., tobacco, peanut, corn, 

sorghum, alfalfa) and wild or weedy hosts that support TBW and CBW are present in cottongrowing regions. As such, Dow’s assessment of the impact of DAS-81419-2 on natural refuge 

was likely highly conservative and underestimated the amount of refuge relative to cotton.

Nonetheless, it can be expected that plantings of DAS-81419-2 soybean will remove some of the 

currently-available natural refuge for Bt cotton. But this potential reduction will likely be small 

relative to the total amount of natural refuge available in southern cotton regions. As part of the 

natural refuge assessment in 2006-2007, BPPD reviewed extensive host utilization data 

developed by Monsanto (see BPPD 2006, 2007; Jackson et al. 2008). TBW and CBW were 

sampled for host preference by testing insect bodies for the presence of bound gossypol (TBW) 

or insect wings for the ratio of C3 to C4 plant isotopes (CBW). These data showed that TBW and 

CBW in cotton-growing areas fed much more frequently on non-cotton hosts than on cotton. In 

many of the sampled states, 50% or more of sampled TBW developed on crops other than cotton. 

While soybean is a component of these non-cotton hosts, it is unlikely that it is the sole (or even 

a majority) source of natural refuge, given the relatively low county acreages (< 25,000 soybean 

acres in most counties). This is the case in Georgia, where low soybean acreage (relative to 

cotton) in Dow’s analysis appeared to indicate a small natural refuge contribution from soybean 

(< 10% in many counties). However, Monsanto’s host utilization data clearly showed a high 

proportion of natural refuge in the state, as very few sampled TBW (0 to 10%) developed on 

cotton (MRID No. 46717201, reviewed in BPPD 2006, 2007).

BPPD’s assessment of the impact of DAS-81419-2 on Bt cotton natural refuge is contingent 

upon the acreage projections provided by Dow (as discussed in the previous section). A full 

commercial registration of DAS-81419-2 without acreage limitations would require a more 

detailed assessment potentially including revised natural refuge calculations and simulation 

modeling.

Stewardship Activities

BPPD’s review indicated that other aspects of IRM including resistance monitoring and remedial 

action plan are needed for DAS-81419-2. A full resistance monitoring plan with pest sampling 

and detection bioassays should not be warranted for a seed increase registration; rather an 

approach based on investigations of unexpected pest damage reports should be appropriate. In 

addition, both TBW and CBW are monitored for Cry1Ac and Cry1F susceptibility as part of the 

WideStrike cotton monitoring program (see BPPD 2005). A remedial action plan, in the event of 
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

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documented resistance, could mimic those in place for Bt corn or cotton or could rely on a “stop 

sale” approach if resistance develops. It was also noted that Dow will need to compile DAS81419-2–specific annual sales and total acreage data for each state and provide a report to EPA if 

requested. Since DAS-81419-2 will be deployed without a structured refuge requirement, a 

compliance assurance plan and grower education program should not be necessary.
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APPENDIX D: References

Product Characterization and Human Health

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Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 76 of 78

Appendix E. Bibliography of data submitted for registration of Bacillus 

thuringiensis Cry1Ac and Cry1F Proteins expressed in Event DAS-81419-2 

Soybean.

Table E. 1. Data Submitted for Cry 1Ac and Cry1F proteins expressed in DAS-81419-2 

Soybean 

MRID

No. Citation Receipt Date

49024100

Dow AgroSciences LLC (2012) Submission of Pesticide Use, Product Chemistry and 

Toxicity Data in Support of the Application for Registration of DAS-81419-2 Soybean. 

Transmittal of 10 Studies.

18-Dec2012

49024101 Unknown (2012) Supplement to MRID 488284-01: Molecular Characterization of 

DAS/81419-2 Soybean. Unpublished study prepared by Dow AgroSciences. 5p.

18-Dec2012

49024102

Embrey, S. (2012) Characterization for Soy Leaf Tissue Samples (TSN303074, 

TSN303075). Project Number: 120416. Unpublished study prepared by Dow AgroSciences 

LLC. 2p.

18-Dec2012

49024103

Unknown (2012) Supplement to MRID 48828409: Similarity Assessment of Cry1Ac Protein 

to Known Toxins by Bioinformatics Analysis: (DAS-81419-2 Soybean). Project Number: 

120416. Unpublished study prepared by Dow AgroSciences LLC. 1p.

18-Dec2012

49024104

Unknown (2012) Supplement to MRID 48848216: Characterization of the Cry1Ac Protein 

Derived from Transgenic Soybean Event DAS-81419-2. Project Number: 110840. 

Unpublished study prepared by Dow AgroSciences LLC. 3p.

18-Dec2012

49024105

Unknown (2012) Supplement to MRID 48828417: Characterization of the Cry1F Protein 

Derived from Transgenic Soybean Event DAS-81419-2. Unpublished study prepared by 

Dow AgroSciences LLC. 3p.

18-Dec2012

49024106

Unknown (2002) Certificate of Analysis for Test/Reference/Control Substances 

(TSN103748): (DAS-81419-2 Soybean). Project Number: BIOT023159. Unpublished study 

prepared by Dow AgroSciences LLC. 7p.

18-Dec2012

49024107

Unknown (2012) Supplement to MRID 488284-10: An Acute Oral Toxicity Study with the 

Northern Bobwhite: (DAS-81419-2 Soybean). Unpublished study prepared by Dow 

AgroSciences LLC. 1p.

18-Dec2012

49024108

Unknown (2002) Certificate of Analysis for Test/Reference/Control Substances 

(TSN103748): (DAS-81419-2 Soybean). Project Number: BIOT023159. Unpublished study 

prepared by Dow AgroSciences LLC. 7p.

18-Dec2012

49024109

Unknown (2012) Supplement to MRID 48828413: Potential Impact of Cry1F and Cry1Ac in 

DAS-81419-2 Soybean on Non-Target Organisms and Threatened and Endangered Species. 

Unpublished study prepared by Dow AgroSciences LLC. 2p.

18-Dec2012

49024110

Storer, N. (2012) Insect Resistance Management Implications of Seed-Production and 

Research Registration for DAS-81419-2 Soybean - Revised. Project Number: IRM/2012/08. 

Unpublished study prepared by Dow AgroSciences LLC. 19p.

18-Dec2012

49044800

Dow AgroSciences, LLC (2013) Submission of Product Chemistry, Efficacy, Toxicity and 

Exposure/Risk Data in Support of the Application for Registration of DAS-81419-2 

Soybean. Transmittal of 9 Studies.

25-Jan2013
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 77 of 78

49044801

Storer, N. (2012) Insect Resistance Management Implications of Seed-Production and 

Research Registration for DAS-81419-2 Soybean - Revised. Project Number: IRM/2012/08. 

Unpublished study prepared by Dow AgroSciences, LLC. 19p.

25-Jan2013

49044802

Guttikonda, S. (2013) Response to EPA Comment on Molecular Characterization of DAS81419-2 Soybean Study - Provide Information Concerning the Source of the Promoters and 

Terminators; Provide the Actual construction of the Cry Proteins; Related to MRID 488284-

01. Project Number: IRSOY/2013/01. Unpublished study prepared by Dow AgroSciences, 

LLC. 8p.

25-Jan2013

49044803

Han, L. (2013) Response to EPA Comment on Biological Equivalency of (DAS-81419-2) 

and Microbe-Produced Cry1Ac and Cry1F Proteins Study - Provide Explanation to Establish 

the Equivalence of Dow''s Cry1Ac and Cry1F Proteins to those Expressed in the Soybean; 

Identify the Leaf Powder Diluent Used in the Insect Bioassays: Related to MRID 488284-03. 

Project Number: IRSOY/2013/02. Unpublished study prepared by Dow AgroSciences, LLC. 

8p.

25-Jan2013

49044804

Han, L. (2013) Response to EPA Comment on Similarity Assessment of Cry1Ac Protein to 

Known Toxins Bioinformatics Analysis Study - Identify the Protein Toxin Source; Explain 

the Error on Page 11 of 16: Related to MRID 488284-09. Project Number: IRSOY/2013/03. 

Unpublished study prepared by Dow AgroSciences, LLC. 5p.

25-Jan2013

49044805

Han, L. (2013) Response to EPA Comment on Characterization of the Cry1Ac Protein 

Derived from Transgenic Soybean Event DAS-81419-2 Study - Address Amino Acid 

Sequence Similarity between Dow''s Cry1Ac Microbial Protein and Soybean-Produced 

Cry1Ac; Provide a Replacement for the Illegible Figure on Page 55: Related to MRID 

488284-16. Project Number: IRSOY/2013/04. Unpublished study prepared by Dow 

AgroSciences, LLC. 8p.

25-Jan2013

49044806

Han, L. (2013) Response to EPA Comment on Characterization of the Cry1F Protein 

Derived from Transgenic Soybean Event DAS-81419-2 Study - Address Amino Acid 

Sequence Similarity between Dow''s Cry1F Microbial Protein and Soybean-Produced Cry1F: 

Related to MRID 488284-14. Project Number: IRSOY/2013/05. Unpublished study prepared 

by Dow AgroSciences, LLC. 7p.

25-Jan2013

49044807

Herman, R. (2013) Response to EPA Comment on Cry1F(Synpro) ICP and Cry1Ac(Synpro) 

ICP: An Acute Oral Toxicity Study with the Northern Bobwhite - Provide Data 

Demonstrating the Equivalence of Dow''''s Test Material #101811 to Plant-produced Protein; 

Provide a Justification of the Test Dosage: Related to MRID 488284-10. Project Number: 

IRSOY/2013/06. Unpublished study prepared by Dow AgroSciences, LLC. 14p.

25-Jan2013

49044808

Han, L. (2013) Response to EPA Comment on Cry1F(Synpro) and Cry1Ac(Synpro) 

Insecticidal Crystal Proteins: An 8-Day Dietary Study with the Rainbow Trout 

Oncorhynchus mykiss, WALBAUM - Provide Data Demonstrating the Equivalence of 

Dow''s Test Material #101811 to Plant-produced Protein: Related to MRID 488284-11. 

Project Number: IRSOY/2013/07. Unpublished study prepared by Dow AgroSciences, LLC. 

13p.

25-Jan2013

49044809

Han, L. (2013) Response to EPA Comment on Potential Impact of Cry1F and Cry1Ac in 

DAS-81419-2 Soybean on Non-target Organisms and Threatened and Endangered Species 

Study - Provide Gene Flow and Weediness Information to Address Environmental Fate 

Concerns; Address Soil Degradation: Related to MRID 488284-13. Project Number: 

IRSOY/2013/08. Unpublished study prepared by Dow AgroSciences, LLC. 6p.

25-Jan2013

49081500 Dow AgroSciences, LLC (2013) Submission of Pesticide Use Data in Support of the 

Application for Registration of DAS-81419-2 Soybean. Transmittal of 1 Study.

14-Mar2013

49081501 Han, L. (2013) Amended Response to the EPA Comment on Characterization of the Cry1F 14-Mar-
Bt Cry1Ac and Cry1F proteins expressed in DAS-81419-2 Soybean 

Biopesticides Registration Action Document January 2014

Page 78 of 78

Protein Derived from Transgenic Soybean Event DAS-81419-2 Study - Address Amino Acid 

Sequence Similarity Between Dow's Cry1F Microbial Protein and Soybean-Produced Cry1F. 

Project Number: IRSOY/2013/05. Unpublished study prepared by Dow AgroSciences, LLC. 

7p.

2013