   Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations for Bacillus 
                          thuringiensis Cry1F Protein

                    Docket ID Number: EPA-HQ-OPP-2013-0704
                            Date: January 13, 2014
                                       
Section 408(c)(2)(A)(i) of FFDCA allows the 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 the EPA determines that the exemption is "safe." Section 408(c)(2)(A)(ii) of 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 FFDCA section 408(c)(2)(B), in establishing or maintaining in effect an exemption from the requirement of a tolerance, the EPA must take into account the factors set forth in FFDCA section 408(b)(2)(C), which require the EPA to give special consideration to exposure of infants and children to the pesticide chemical residue in establishing a tolerance exemption, 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, FFDCA section 408(b)(2)(D) requires that the EPA consider "available information concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity."
The EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide residues. First, the EPA determines the toxicity of pesticides. Second, the 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.
I.  Summary of Petitioned-for Tolerance Exemption
In the Federal Register of November 22, 2013 (78 FR 70007), the EPA issued a notice pursuant to FFDCA section 408(d)(3), 21 U.S.C. 346a(d)(3), announcing the filing of a pesticide tolerance petition (PP 2F8066) by Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN, 46268. The petition requested that 40 CFR part 174 be amended by establishing an exemption from the requirement of a tolerance for residues of Bacillus thuringiensis Cry1F protein in the food commodity soybean. The notice referenced a summary of the petition prepared by the petitioner, Dow AgroSciences LLC, which is available in Docket ID Number EPA-HQ-OPP-2013-0704 via http://www.regulations.gov.
II. Toxicological Profile
Consistent with section 408(b)(2)(D) of FFDCA, the EPA reviewed the available scientific data and other relevant information on Bacillus thuringiensis Cry1F protein, and considered its validity, completeness, and reliability, as well as the relationship of this information to human risk. The EPA also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children.
A.  Overview of Bacillus thuringiensis Cry1F protein
	
Since 2001, Bacillus thuringiensis Cry1F protein has been used in registered pesticide products to provide protection from feeding damage caused by a number of lepidopteran pests. In conjunction with some of these registered pesticide products, the EPA established the following exemptions from the requirement of a tolerance:

   (1) Bacillus thuringiensis Cry1F protein in cotton (40 C.F.R. § 174.504)  -  see the Federal Register of April 25, 2007 (72 FR 20434).

   (2) Bacillus thuringiensis Cry1F protein in corn (40 C.F.R. § 174.520)  -  see the Federal Register of April 25, 2007 (72 FR 20435).
         
Dow AgroSciences LLC developed DAS-81419-2 soybean (Glycine max) to express Bacillus thuringiensis Cry1F protein for use as a plant-incorporated protectant (PIP). Event DAS-81419-2 soybean was created by Agrobacterium - mediated transformation using the 2T-DNA plasmid vector pDAB9582, and produces both Bacillus thuringiensis protein δ-endotoxins Cry1Ac and Cry1F. An exemption from the requirement of a tolerance for residues of Bacillus thuringiensis Cry1Ac protein in all food commodities, when used as a PIP, was established at 40 CFR 174.510 (72 FR 20435; April 25, 2007). 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. The proteins are intended to provide protection from feeding damage caused by the larvae of a number of lepidopteran pests. Dow AgroSciences LLC's DAS-81419-2 soybean also expresses the Phosphinothricin Acetyltransferase (PAT) enzyme, which is exempt from the requirement of a tolerance when used as a PIP inert ingredient in all food commodities (40 CFR 174.522; April 25, 2007; 72 FR 20435). This enzyme confers tolerance of the soybean plants to the herbicide, glufosinate. In its July 24, 2012 submission to the EPA, Dow AgroSciences LLC, proposed to register an end-use pesticide product (EPA File Symbol 68467-EN) that contains Bacillus thuringiensis Cry1F and Cry1Ac proteins as the active ingredients for commercial use as a PIP.  Accompanying the pesticide registration application was Pesticide Petition 2F8066 requesting that an exemption from the requirement of a tolerance be established for residues of Bacillus thuringiensis Cry1F protein in soybean when used as a PIP.

In support of its tolerance exemption request, an analytical method for enforcement purposes was submitted by Dow AgroSciences LLC.  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 dr weight (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.



B.  Toxicology Data Requirements

All applicable mammalian toxicology data requirements supporting the request for an exemption from the requirement of a tolerance for residues of Bacillus thuringiensis Cry1F protein in or on the food and feed commodities of soybean have been fulfilled with data submitted by the petitioner or by data and information available to the Agency resulting from its previous reviews. 

The overall conclusions from all toxicological information submitted by the petitioner are briefly described below, and more in-depth synopses of study results can be found in the associated Biopesticides Registration Action Document and its supporting materials provided as a reference in Section VII of this document. 

No indication of toxicity toward mammals was noted in the review of the two Cry proteins, Cry1Ac and Cry1F, expressed in soybean. 

It should be noted that the allergenicity of Cry1Ac protein was evaluated previously as evidenced in MRID No. 478417-09 (US EPA, 2010a). Based upon sequence comparisons to known allergens and digestibility assays (MRID No. 478417-08), 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. 

Mammalian Toxicity and Allergenicity Assessment, Bacillus thuringiensis Cry1F Protein 

An acute oral toxicity study in mice (MRID No. 455423-14) indicated that Cry1F is non-toxic to humans and other mammals. Cry1F protein is a δ-endotoxin from Bacillus thuringiensis that has been used extensively in plant-incorporated protectants as a means of insect pest management. 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 (PP 2F8066) is included with the submission from Dow AgroSciences 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. 455423-18 ) The Cry1F protein was digested rapidly (< 5 min) in SGF 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). 

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. 

III.  Aggregate Exposure  

In examining aggregate exposure, FFDCA section 408 directs the 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).

The Agency 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 nonoccupational
sources.

As previously discussed, data indicated that Cry1F protein is non-toxic to humans and other mammals, andCry1F 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 Agency 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, corn, and cotton, 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, corn, and cotton and 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 even 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, corn, and cotton 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. Cry1F protein expression is low in the pollen of soybean, cotton and corn.  Additionally, soybean flowers in a cleistogamous fashion wherein the anthers generally release pollen within a closed or covered floral tube. There is no additional inhalation exposure from the presence of Cry1F in soybean due to the closed and self-pollinating nature of the soybean flower.  Pollen movement from soybean is minimal and highly localized when it happens (i.e., the pollen does not move readily on wind currents). This is the case also for the heavy and sticky pollen of cotton, which is insect-pollinated.  While pollen exposure is potentially high for wind-pollinated corn, it is not significant because of low Cry1F protein expression in corn pollen and lack of any human toxicity or allergenicity associated with the Cry1F protein.  

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.

IV.  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 EPA consider "available information concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity."

Because the Bacillus thuringiensis Cry1F protein does not have a toxic effect, this statutory provision to consider cumulative effects of substances with common mechanisms of toxicity does not apply.  


V.  Determination of Safety for the United States Population, Infants and Children
	
FFDCA section 408(b)(2)(C) provides that, in considering the establishment of a tolerance or tolerance exemption for a pesticide chemical residue, the 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 the EPA shall apply an additional tenfold (10X) 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 database on toxicity and exposure, unless the EPA determines that a different margin of safety will be safe for infants and children. This additional margin of safety is commonly referred to as the Food Quality Protection Act Safety Factor. In applying this provision, the EPA either retains the default value of 10X, or uses a different additional or no safety factor when reliable data are available to support a different additional or no safety factor. 

Based on the toxicity and allergenicity data/information discussed in Section II of this document, the EPA concludes that there are no threshold effects of concern to infants, children, or adults when Bacillus thuringiensis Cry1F protein is used as labeled in accordance with good agricultural practices. As a result, the EPA concludes that no additional margin of exposure (safety) is necessary.

Moreover, based on the same data and the EPA analysis as presented directly above, the Agency concludes that there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to the residues of Bacillus thuringiensis Cry1F protein when it is used as labeled and in accordance with good agricultural practices as a plant-incorporated protectant in soybean. Such exposure includes all anticipated dietary exposures and all other exposures for which there is reliable information. The EPA has arrived at this conclusion because, considered collectively, the data and information available on Bacillus thuringiensis Cry1F protein do not demonstrate toxic, pathogenic, and/or infective potential to mammals, including infants and children.

VI.  Conclusions
The EPA concludes that there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to residues of Bacillus thuringiensis Cry1F protein. Therefore, an exemption from the requirement of a tolerance is established for residues of Bacillus thuringiensis Cry1F protein in the food and feed commodities of soybean when used as plant-incorporated protectants, and in accordance with label directions and good agricultural practices.

VII.  References
	
Lerouge, P. Cabanes-Macheteau, M., Rayon, C., Fichette-Lainé, A-C., Gomord, V., and Faye, L., (1998) N-Glycoprotein biosynthesis in plants: recent developments and future trends. Plant Molecular Biology 38: 31-48.

Sjoblad, Roy D., et al., (1992) Toxicological Considerations for Protein Components of Biological Pesticide Products. Regulatory Toxicology and Pharmacology 15:3-9.

U.S. Environmental Protection Agency (2005) BIOPESTICIDES REGISTRATION ACTION DOCUMENT (Bacillus thuringiensis Cry1F (synpro and Cry1Ac (synpro) Construct 281/3006 Insecticidal Crystal Proteins as expressed in cotton) (Chemical PC Codes 006512 and 006513, respectively) May 2005 Office of Pesticide Programs 
http://www.epa.gov/oppbppd1/biopesticides/ingredients_keep/tech_docs/brad_006512.pdf  

US Environmental Protection Agency (2010a) 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 

US Environmental Protection Agency (2010b) BIOPESTICIDES REGISTRATION ACTION DOCUMENT Cry1Ab and Cry1F Bacillus thuringiensis (Bt) Corn Plant-Incorporated Protectants  http://www.epa.gov/pesticides/biopesticides/pips/cry1f-cry1ab-brad.pdf 

US Environmental Protection Agency (2014) 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]

Wozniak, C. A. (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.



