Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations for Beauveria bassiana strain ANT-03

                    Docket ID Number: EPA-HQ-OPP-2013-0717
                            Date: October 23, 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 December 30, 2013 (78 FR 79359), 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 3F8176) by Technology Sciences Group, Inc., on behalf of Anatis Bioprotection Inc., 278, rang Saint-Andre', St.-Jacques-le-Mineur, Quebec JOJ 1ZO, Canada. The petition requested that 40 CFR part 180 be amended by establishing an exemption from the requirement of a tolerance for residues of Beauveria bassiana strain ANT-03. The notice referenced a summary of the petition prepared by Technology Sciences, Inc. for the petitioner, Anatis Bioprotection Inc., which is available in Docket ID Number EPA-HQ-OPP-2013-0717 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 Beauveria bassiana strain ANT-03, 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 Beauveria bassiana strain ANT-03
	
Beauveria bassiana strain ANT-03 is a naturally occurring bacterium and was originally isolated in 2000 from Lygus lineolaris found dead in a field in Sherrington, Quebec, Canada. The natural occurrence of Beauveria bassiana strains located in soils and insects is well documented in the US and worldwide. Anatis Bioprotection Inc. proposed to register a technical and an end-use product that contain Beauveria bassiana strain ANT-03 as an active ingredient that would be applied as a microbial insecticide in agricultural, horticultural and residential settings.
The end-use product containing Beauveria bassiana strain ANT-03 is proposed for use on growing crops as a contact mycoinsecticide to control or suppress foliar-feeding pests and certain grubs. 
B.  Microbial Pesticide Toxicology Data Requirements

All applicable mammalian toxicology data requirements supporting the request for an exemption from the requirement of a tolerance for residues of Beauveria bassiana strain ANT-03 in or on all food commodities have been fulfilled with data submitted by the petitioner or have been waived by the EPA. 

The toxicity and pathogenicity tests and irritation tests (acute eye and primary dermal irritation) that address potential routes of exposure to the active ingredient are all classified in Toxicity Category III or IV and revealed little to no toxicity attributed to Beauveria bassiana strain ANT-03.  Hypersensitivity incidents are to be reported, should they occur during research, development, and testing; no hypersensitivity reports were submitted. 

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 draft Biopesticides Registration Action Document provided as a reference in Section VII of this document (Ref. 3).

Acute Oral Toxicity and Pathogenicity  -  Rat (Harmonized Guideline 885.3050; MRID No. 48974203): In an acute oral toxicity and pathogenicity study, groups of ~8-week-old Sprague-Dawley rats (12/sex) were given a single oral dose of Beauveria bassiana strain ANT-03 (1.2 x 10[11] conidia/g) in sterile phosphate buffer saline (PBS) at a dose of 1.3 x 10[9] CFU/animal. The animals were observed for up to 21 days with interim scheduled sacrifices on Days 3, 7, and 14. Five males and five females were treated with inactivated test substance as controls; two males and two females were untreated "shelf controls"; and five males and five females were untreated controls. There were no treatment-related deaths, clinical signs, necropsy findings, or changes in body weight or body weight gain. The test organism was variously recovered from the blood, brain, spleen, lungs, liver, kidneys, or cecum contents through day 7, and cleared by Day 14. The test organism cleared from the mesenteric lymph nodes by day 21. Based on dissemination from an oral dose to dispersed internal regions of test animals, this organism may be infectious by oral exposure, though not pathogenic or toxic and is unlikely to grow at body temperatures. 

EPA determined this study to be acceptable and concluded that Beauveria bassiana strain ANT-03 is not acutely toxic or pathogenic orally at 1.3 x 109 CFU/animal. 

Acute Pulmonary Toxicity and Pathogenicity  -  Rat (Harmonized Guideline 885.3150; MRID No. 48974204): In an acute pulmonary toxicity and pathogenicity study, groups of ~7-8 week old Sprague-Dawley rats (15/sex) were exposed by the intratracheal route to Beauveria bassiana strain ANT- 03 (1.2 x 10[11] conidia/g) in sterile PBS at a dose of 1.1 x 10[9] CFU/animal. The animals were observed up to 21 days with interim scheduled sacrifices on Days 3, 7, and 14. Five males and five females were treated with autoclaved test substance as inactivated controls, four animals/sex were untreated "shelf controls"; and five animals/sex were untreated controls. Based on the results of this study, Beauveria bassiana strain ANT-03 was not toxic, infective, and/or pathogenic to rats when dosed at 1.1 x 10[9] CFU/animal. There were no test substance related deaths, clinical signs, or necropsy findings. No test organisms were recovered from the blood, liver, kidneys, mesenteric lymph nodes, brain, and spleen or cecum contents at any time. Except for the Day 0 sacrifice, the test substance was not seen in the lungs at any time. 

EPA determined this study to be acceptable and concluded that Beauveria bassiana strain ANT-03 is not acutely toxic, infective, or pathogenic when inhaled at 1.4 x 109 CFU/animal. 

Acute Intraperitoneal Injection Toxicity and Pathogenicity  -  Rat (Harmonized Guideline 885.3200; MRID No. 48974205):  In an acute intraperitoneal injection toxicity and pathogenicity study, fifteen male and fifteen female albino Sprague-Dawley rats were injected with Beauveria bassiana strain ANT-03 (1.2 x 10[11] conidia/g) in sterile PBS at a dose of 1.4 x 10[9] CFU/animal. The animals were treated on day 0 and observed for up to 21 days. Three rats/sex treated with the test material were sacrificed one hour after dosing and the peritoneal cavity washed with PBS for dose verification and enumeration. The remaining animals were sacrificed and necropsied on day 21. Two additional groups of five rats/sex served as untreated and inactivated MPCA controls and were also sacrificed on day 21. Based on the results of this study, Beauveria bassiana strain ANT-03 does not appear to be toxic when administered in a single dose to rats by IP injection at 1.4 x 10[9] CFU/animal. There were no treatment-related deaths, clinical signs, gross necropsy findings, or changes in body weight or body weight gain. Mesenteric lymph node weights in treated females were significantly lighter than untreated controls.

EPA determined this study to be acceptable and concluded that Beauveria bassiana strain ANT-03 is not acutely toxic, infective, or pathogenic intravenously at 1.1 x 109 CFU/animal. 

Acute Dermal Toxicity  -  Rat (OCSPP Guideline 870.1200; MRID No. 48974206): In an acute dermal toxicity study, groups of five male and five female young adult Sprague-Dawley rats were dermally exposed to Beauveria bassiana strain ANT-03 (1.2 x 10[11] conidia/g) moistened with deionized water (1.0 mL/g), applied to clipped application sites of approximately 10% of the body surface area for 24 hours at a dose level of 5050 mg/kg body weight. The animals were treated on day 0 and observed for a period of 14 days. There were no deaths or abnormal gross necropsy findings. All of the animals gained weight during both weeks of the study. There were no abnormal systemic clinical signs, and signs of dermal irritation were not detected at any time during the study. 

EPA determined this study to be acceptable and concluded that Beauveria bassiana strain ANT-03 has an acute dermal NOAEL combined of > 5050 mg/Kg bw (EPA Toxicity Category IV).  

      Acute Oral Toxicity  -  Rat (Harmonized Guideline 870.1100)  
      Acute Dermal Toxicity  -  Rat (Harmonized Guideline 870.1200)   
      Acute Inhalation Toxicity  -  Rat (Harmonized Guideline 870.1300)   
      Primary Eye Irritation  -  Rabbit (Harmonized Guideline 870.2400)    
      Primary Dermal Irritation  -  Rabbit (Harmonized Guideline 870.2500)

In lieu of toxicology testing of Beauveria bassiana strain ANT-03, the applicant provided acceptable scientific justifications/rationales (MRID Nos. 48974213 [Technical Grade Active Ingredient] and 48974302 [Bioceres End-use Product]) to address the above listed data requirements, summarized below. EPA generally agrees with the rationales provided and EPA determined the rationales to be acceptable to address the data requirements. (U.S. EPA 2014a Section VII, Ref. 1).

Oral gavage administration of the test material did not result in mortality, abnormal clinical signs, or gross pathology in rats at a single dose of 1.3 x 109 CFU/animal in an acute oral toxicity/pathogenicity study. As calculated by the reviewer, the administered dose of 1.3 x 109 CFU/rat equated to dose levels of approximately 36.7 to 65.7 mg/kg bw. In an acute pulmonary infectivity and toxicity study intratracheal administration of the test material (in phosphate buffered saline and Tween 20) at a single dose of 1.1 x 109 CFU/animal did not result in mortality, abnormal clinical signs, or gross pathology in rats. As calculated by the reviewer, the administered dose levels were approximately 31.4 to 56.2 mg/kg bw. TGAI's with Beauveria bassiana strains 447, GHA, and HF23 have been tested and classified in US EPA Toxicity Category III for primary eye irritation, and an EP formulated with Beauveria bassiana strain ATCC 74040 has also been classified in US EPA Toxicity Category III for primary eye irritation. In an acute dermal toxicity study, no skin irritation was seen when rats were dermally exposed to the test material moistened with deionized water (1.0 mL/g), applied to clipped application sites of approximately 10% of the body surface area for 24 hours at a dose level of 5050 mg/kg bw.  Because there were no observed adverse effects, the assigned US EPA Toxicity Category IV is assigned for dermal irritation. There are no inert ingredients added to the TGAI.  The inerts added to the EP are present in food, recognized as safe, have tolerance exemptions, and are not expected to produce any toxic effects.  

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).

Food Exposure and Risk Characterization: Beauveria bassiana strains are widely found in soils and insects worldwide. The EPA found that increased dietary exposure to Beauveria bassiana strain ANT-03, a naturally occurring bacterium pathogenic to insects and commonly found in agricultural settings and on fresh produce, is anticipated to be negligible because additional applications tend not to significantly increase exposure. Further, Beauveria bassiana is not known to produce any mammalian toxins, and no foodborne disease outbreaks associated with Beauveria bassiana or toxin production from Beauveria bassiana have been reported. The EPA concluded that the risk posed to adults, infants, and children is likely to be minimal because of the low acute oral toxicity/pathogenicity potential of Beauveria bassiana strain ANT-03.
 
Drinking Water Exposure and Risk Characterization: Since Beauveria bassiana is naturally present in soils and insects, exposure to Beauveria bassiana from surface water and possibly groundwater can be expected.  Exposure to residues of Beauveria bassiana strain ANT-03 in consumed drinking water may occur as a result of spray drift from aerial and ground applications or runoff of prepared fields and rangelands into surface waters. However, if Beauveria bassiana strain ANT-03 were to be transferred to surface water or groundwater intended for eventual human consumption (e.g., through spray drift or runoff) and directed to wastewater treatment systems or drinking water facilities, water treatment processes would remove any Beauveria bassiana present.  It likely would not survive the conditions water is subjected to in such systems or facilities, including chlorination, pH adjustments, filtration, and/or occasionally high temperatures (Centers for Disease Control and Prevention 2009; U.S. EPA 2004, and DeFelice et.al., 1993). In the remote likelihood that Beauveria bassiana strain ANT-03 is present in drinking water (e.g., water not subject to treatment systems or facilities), its target pest specificity and available toxicity and pathogenicity data indicate no toxicity and/or pathogenicity is likely to occur with any drinking water exposure to Beauveria bassiana strain ANT-03 that results from pesticide applications made in accordance with good agricultural practices (Section VII, Ref. 2).

Non-occupational, Residential Risk Characterization: Given the home and garden proposed use pattern for Beauveria bassiana strain ANT-03, non-occupational exposure is expected.  However, repeated exposures to Beauveria bassiana strain ANT-03 from pesticidal applications will not exceed EPA's level of concern in light of the available data that demonstrate Beauveria bassiana strain ANT-03 is not toxic (acute dermal toxicity and acute pulmonary toxicity/ pathogenicity), is non-irritating (primary dermal irritation), and is not pathogenic when used as labeled in accordance with good agricultural practices (acute pulmonary toxicity/pathogenicity, and acute injection toxicity/pathogenicity) (Section VII, Ref. 1).

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 or tolerance exemption, 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."

Beauveria bassiana strain ANT-03 is not toxic via the dietary, dermal and pulmonary routes of exposure. For the purposes of the tolerance action, therefore, the EPA has concluded that Beauveria bassiana strain ANT-03 does not have a common mechanism of toxicity with other substances. Thus, section 408(b)(2)(D)(v) of the FFDCA does not apply. 

V.  Determination of Safety for the United States Population, Infants and Children
	
In considering the establishment of a tolerance or tolerance exemption for a pesticide chemical residue, FFDCA section 408 (b)(2)(C) provides that 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 exposure (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 exposure (safety) will be safe for infants and children. This additional margin of exposure (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 safety factor when reliable data available to the EPA support the choice of a different factor.

Based on the acute toxicity and pathogenicity 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 Beauveria bassiana strain ANT-03 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 to protect infants and children and that not adding any additional margin of exposure (safety) will be safe for infants and children.

Moreover, based on the same data/information and EPA analysis as presented directly above, the Agency is able to conclude that there is a reasonable certainty that no harm will result to the United States population, including infants and children, from aggregate exposure to the residues of Beauveria bassiana strain ANT-03 when it is used -- as labeled and in accordance with good agricultural practices -- as an insecticide. 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 Beauveria bassiana strain ANT-03 demonstrate that the substance is not toxic or pathogenic potential to mammals, including infants and children. No toxicological, irritation or pathogenic endpoints were noted in mammalian testing of Beauveria bassiana strain ANT-03 and differential effects to infants and children are not expected. 

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 Beauveria bassiana strain ANT-03. Therefore, EPA is establishing an exemption from the requirement of a tolerance for residues of Beauveria bassiana strain ANT-03 in or on all food commodities when applied as a microbial insecticide and used in accordance with label directions and good agricultural practices.


VII.  References
	
 1. U.S. EPA. 2014a. Beauveria bassiana strain ANT-03 (review submitted studies for FIFRA     Section 3 registration of an MUP (TGAI) and EP with a food tolerance exemption petition. Memorandum from J. Gagliardi to D. Greenway dated September 17, 2014.
 
 2. U.S. EPA. 2014b. Environmental risk assessment for the FIFRA Section 3 registration of the TGAI, Beauveria bassiana strain ANT-03 (EPA Reg. No. 89600-R) and the EP, Bioceres (EPA Reg. No. 89600-E), containing the active ingredient Beauveria bassiana strain ANT-03. Memorandum from I. You to D. Greenway dated September 19, 2014
 
 3. U.S. EPA. 2013. Biopesticides Registration Action Document Beauveria bassiana strain ANT-03 dated October 8, 2014 (available as a "Supporting Document" within docket ID number EPA-HQ-OPP-2013-0718 at http://www.regulations.gov). 






