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

        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

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.

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
[o]C			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[-9] gram)
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

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)

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.

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).
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).

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 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.

DAS-81419-2 Soybean co-expresses Cry1Ac and Cry1F proteins to provide protection against feeding damage by lepidopteran insect larvae.

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 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-of-evidence 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.

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-of-evidence 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

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

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

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 self-pollinating 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

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 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.

   A.  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 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 DAS-81419-2 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 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/cm[2]/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 DAS-81419-2 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 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.

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.
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 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 cotton-growing 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).

   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 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.

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 DAS-81419-2 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 fluorescens-produced 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 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
Summary of Cry1Ac Protein Expression

                                     Test

                        Cry1Ac ng/mg Tissue Dry Weight
                                    Matrix
                                     Entry
                                  Description
                                   Overall
                                     Mean
                               Std. Dev. (n=10)
                                 Min/Max Range
                                    STMR[a]
                                    HAFT[b]

                                    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
                                    STMR[a]
                                    HAFT[b]

                                    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
                                    STMR[a]
                                    HAFT[b]

                                    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

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., Spec[r], 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 DAS-81419-2

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 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

48828404

Method Validation for the Determination of Cry1F Protein in Soybean Tissues Using an Enzyme-Linked 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 DAS-81419-2 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, 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
48828417
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.

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 plant-incorporated 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, 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

48828406

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 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-cry1ac-protien.pdf
Classification: ACCEPTABLE

48828408
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
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-of-evidence 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 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-of-evidence 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).

   a. 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 δ-endotoxins Cry1Ac and Cry1F as an insect management mechanism is safe as proposed for use in soybean intended for cultivation, and human and animal consumption.

 APPENDIX B: Ecological and environmental effects  -  Bacillus thuringiensis Cry1Ac and Cry1F proteins expressed in Event DAS-81419-2 Soybean plant-incorporated 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(R) 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(R) 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 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(R) cotton (EPA Reg. No. 68467-3). Dow has requested to bridge several environmental fate and effects studies previously reviewed for the registration of WideStrike(R) 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(R) 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 cost.

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.  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 Concentration (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. 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.

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 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  (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 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-analysis of 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).

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 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.