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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-OPP2013-0704 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.
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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 DAS81419-2 soybean also expressesthe 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.
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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 plantincorporated 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-ofevidence approach where the following factors are considered: source of the trait; amino acid 

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

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

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

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

from Recombinant-DNA Plants.” The allergenicity assessment for Cry1F follows:
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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 δendotoxins Cry1Ac and Cry1F as an insect management mechanism is safe as proposed for use 

in soybean intended for cultivation, and human and animal consumption. 

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 nonoccupational exposures, including drinking water from ground water or surface water and 

exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor 

uses).

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 
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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 insectpollinated. 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. 
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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.
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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 PVGMIR9) Necessary for Its Production in MON 87701 (OECD Unique Identifier: MON 877Ø1-2) 

Soybean [PC Code 006532] http://www.epa.gov/pesticides/biopesticides/pips/bt-cry1acprotien.pdf

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.