US Environmental Protection Agency Office of Pesticide Programs BIOPESTICIDE REGISTRATION ACTION DOCUMENT Aspergillus flavus (NRRL 21882) (PC Code 006500) March 24, 2004 Page 1 of 57 BIOPESTICIDES REGISTRATION ACTION DOCUMENT Aspergillus flavus (NRRL 21882) (PC Code 006500) U.S. Environmental Protection Agency Office of Pesticide Programs Biopesticides and Pollution Prevention Division Aspergillus flavus (NRRL 21882) (PC Code 006500) Aspergillus flavus NRRL 21882 May 24, 2004 Page ii of 57 Biopesticides Registration Action Document FINAL DRAFT TABLE OF CONTENTS Aspergillus flavus NRRL 21882 (PC Code 006500) I. EXECUTIVE SUMMARY/FACT SHEET ........................... Page 6 of 57 II. OVERVIEW .................................................. Page 10 of 57 A. Product Overview ........................................ Page 10 of 57 B. Use Profile .............................................. Page 10 of 57 C. Estimated Usage ......................................... Page 11 of 57 D. Data Requirements ....................................... Page 11 of 57 E. Regulatory History ........................................ Page 12 of 57 III. SCIENCE ASSESSMENT ....................................... Page 15 of 57 A. Physical and Chemical Properties Assessment ................ Page 15 of 57 1. Product Identity and Mode of Action ................... Page 15 of 57 2. Physical And Chemical Properties Assessment .......... Page 19 of 57 3. Analytical Methods for Peanuts and Pesticide ............ Page 19 of 57 B. Human Health Assessment ................................. Page 21 of 57 1. Food Clearances/Tolerances .......................... Page 21 of 57 2. Toxicology Assessment ............................... Page 21 of 57 3. Dietary Exposure and Risk Characterization ............ Page 31 of 57 4. Occupational and Residential Exposure and Risk Characterization ................................ Page 32 of 57 5. Drinking Water Exposure and Risk Characterization ..... Page 33 of 57 6. Acute and Chronic Dietary Risks for Sensitive Subpopulations, Particularly Infants and Children .... Page 33 of 57 7. Aggregate Exposure from Multiple Routes Including Dermal, Oral, and Inhalation ...................... Page 34 of 57 8. Cumulative Effects .................................. Page 34 of 57 9. Determination of Safety for the U.S. Population, including Infants and Children .................... Page 35 of 57 C. Environmental Assessment ................................. Page 35 of 57 1. Ecological Effects Hazard and Risk Assessment .......... Page 35 of 57 2. Environmental Assessment and Risk ................... Page 43 of 57 D. Efficacy Data - Product Performance ........................ Page 43 of 57 IV. PUBLIC INTEREST FINDING .................................. Page 45 of 57 Aspergillus flavus NRRL 21882 May 24, 2004 Page iii of 57 Biopesticides Registration Action Document FINAL DRAFT V. RISK MANAGEMENT AND REGISTRATION DECISION .......... Page 46 of 57 A. Determination of Eligibility ................................ Page 46 of 57 B. Regulatory Position ....................................... Page 46 of 57 1. Conditional/Unconditional Registration Eligible use ...... Page 46 of 57 2. Tolerance Reassessment .............................. Page 46 of 57 3. Ineligible Uses ...................................... Page 46 of 57 4. CODEX Harmonization .............................. Page 46 of 57 5. Non-food Re/Registrations ............................ Page 46 of 57 6. Risk Mitigation ..................................... Page 47 of 57 7. Endangered Species Statement ........................ Page 47 of 57 C. Labeling Rationale ........................................ Page 47 of 57 1. TGAI /MP Product Labeling .......................... Page 47 of 57 2. End-use Product Labeling ............................ Page 48 of 57 3. Application Rate .................................... Page 49 of 57 D. Labeling ................................................. Page 49 of 57 1. TGAI or Manufacturing Use Product .................. Page 49 of 57 2. End-Use Product Name: .............................. Page 50 of 57 VI. ACTIONS REQUIRED BY REGISTRANTS ...................... Page 51 of 57 VII. APPENDICES ............................................... Page 53 of 57 APPENDIX A - USE SITES ................................... Page 53 of 57 APPENDIX B - BIBLIOGRAPHY ............................. Page 54 of 57 Aspergillus flavus NRRL 21882 May 24, 2004 Page iv of 57 Biopesticides Registration Action Document FINAL DRAFT List of Tables Tables are numbered according to the Section of the BRAD in which they appear. Table III.A.1.a: Product Characterization - A. flavus NRRL 21882 (TGAI) and afla- guard™ (EP). Table III.A.1.b: Physical & Chemical Properties - A. flavus NRRL 21882 (TGAI) and afla-guard™ (EP). Table III.B.2.a: Recovery of Aspergillus flavus NRRL 21882 in samples from male rats treated IP. Table III.B.2.b: Recovery of Aspergillus flavus NRRL 21882 in samples from female rats treated IP. Table III.B.2.c: Summary Tier I Acute Mammalian Toxicity - Aspergillus flavus NRRL 21882 and afla-guard™. Table III.B.2.d: Tier I - Data Waivers: Acute Mammalian Toxicity of Aspergillus flavus NRRL 21882 (TGAI) and afla-guard™ (EP). Table III.C.1.a: Eco-Toxicology Summary/Studies Evaluated. Table III.C.1.b: Eco-Toxicology Summary: Data Waivers. Table VI.a: Data required for Aspergillus flavus NRRL 21882 and aflaguard™. Table VII.a: Use Site Conditional registration - Aspergillus flavus NRRL 21882 and afla-guard™. Aspergillus flavus NRRL 21882 May 24, 2004 Page v of 57 Biopesticides Registration Action Document FINAL DRAFT BIOPESTICIDE REGISTRATION ACTION DOCUMENT TEAM Office of Pesticide Programs: Biopesticides and Pollution Prevention Division Dennis Szuhay Chief, Microbial Pesticides Branch John Kough Microbiologist, Senior Scientist Zigfridas Vaituzis Microbiologist, Senior Scientist Joel Gagliardi Microbial Ecologist Hilary Hill Entomologist Shanaz Bacchus Chemist, Regulatory Action Leader Chris Jay Pfeifer Environmental Protection Specialist, Regulatory Action Leader Aspergillus flavus NRRL 21882 May 24, 2004 Page 6 of 57 Biopesticides Registration Action Document FINAL DRAFT I. EXECUTIVE SUMMARY/FACT SHEET Active Ingredient and Proposed Use The active ingredient Aspergillus flavus NRRL 21882, also known as A. flavus NRRL 21882 or NPRL 45(PC Code 006500), belongs to the naturally occurring genus of fungi, Aspergillus, which is ubiquitous in the environment. This specific strain, Aspergillus flavus NRRL 21882 or NPRL 45, was isolated from a peanut seed in Georgia, United States (US) in 1991. It does not produce aflatoxins, cyclopiazonic acid or known intermediates in the aflatoxin biosynthetic pathway. When the pesticide is applied once per season to the soil at the pre- pegging phase of peanut plant growth, it is expected to displace aflatoxin-producing A. flavus strains naturally found on peanuts. Some other Aspergillus strains have been domesticated to provide human consumable products. For example, Aspergillus oryzae is used in the fermentation of soy sauce and miso, and the digestive enzyme alpha galactosidase found in Beano is produced from Aspergillus niger. NPRL 45 is not likely to exchange genetic material with other known aflatoxin producing strains of naturally occurring A. flavus, based on its characteristic lack of vegetative compatibility with the latter. The Technical Grade Active Ingredient (TGAI), Aspergillus flavus NRRL 21882, EPA Reg. No. 75624-R, is manufactured in Japan (EPA Establishment # 75792-JPN-001). It is formulated in the US into the End-use Product (EP) afla-guard™ (EPA Reg. No. 75624-E), which contains 0.01% of the active ingredient, Aspergillus flavus NRRL 21882. The proposed application rate is 20 pounds EP (approximately 0.002 pound or less than 1 gram active ingredient) per acre. All manufacturing regulations must be met to assure the quality and integrity of the product. Quality control measures, discussed in Section III. A. of this BRAD, are in place to ascertain that human pathogens, potential metabolites, such as aflatoxins, and unintentional ingredients are within regulatory levels. Toxicology, Human Exposure and Risks Summaries of the toxicological effects, from reviews of submitted studies, are found in Table III.B.2.c (see Section III.B.2. of this BRAD: Toxicology - Health Effects). No toxic, infective or pathogenic effects were observed in two acute oral exposure tests in rodents. Based on submitted studies, both the TGAI and the End-use Product are considered Toxicity Category IV for acute oral effects. The results of the acute pulmonary exposure study showed no infectivity or pathogenicity, and clearance was observed from all tissues of surviving treated animals. Infective and pathogenic effects were observed in the intraperitoneal study and there was one unscheduled death, but the fungal active ingredient cleared all surviving rodent tissues by the end of the study on day 22. The implications of this study to pesticide handler exposure are summarized below under Occupational and Residential exposure and discussed in Section III.B.4. of this BRAD. No hypersensitivity incidents have been reported by workers who have conducted laboratory experiments and field trials for more than 11 years. The properties of Aspergillus species are known, and Personal Protective Equipment (PPE) are required to mitigate worker exposure. Aspergillus flavus NRRL 21882 May 24, 2004 Page 7 of 57 Biopesticides Registration Action Document FINAL DRAFT Based on low toxicity potential observed in the studies, low application rates, no hypersensitivity reports and the clearance of the microbe from rodent tissues during the toxicology tests, data waiver requests for primary dermal, hypersensitivity and immune response studies were waived (see Section III.B.2. of this BRAD: Toxicology - Health Effects). The rationale for the request to waive data for the primary eye irritation study was supplemental, but upgradeable. However, the End-use Product is applied once during the season at approximately 1 gram of active ingredient per acre, and drift is expected to be minimal because of the adherence of the pesticide to the carrier. In addition, non-occupational eye exposure is not expected because of the application of the EP to commercial peanut fields and not residential areas. Provided eye protective equipment to mitigate eye exposure is on the label for the proposed use, this data waiver request is granted. Additional data or justification must be submitted to meet Agency guideline requirements, should the applicant wish to amend the registration to remove PPE for eye protection from the label. Food Tolerances This is the first proposed food/feed use of Aspergillus flavus NRRL 21882 for which an exemption from tolerance has been requested. The summaries of the reviewed studies, published literature and scientific and exposure rationales in support of this exemption from tolerance are included in this Biopesticide Registration Action Document (BRAD). A final rule establishing the exemption from tolerance for residues of Aspergillus flavus NRRL 21882 on peanut food and feed commodities will be published in the Federal Register concomitant with the issuance of the conditional registration of this pesticide. FQPA Considerations The Agency has considered Aspergillus flavus NRRL 21882 in light of the safety factors of the Food Quality Protection Act (FQPA) of 1996 and has made a determination of reasonable certainty of no harm to the U.S. population in general, and to infants and children in particular. The ubiquitous occurrence of Aspergillus flavus strains suggests that the fungus and its metabolites are normally expected to be present in/on food commodities regardless of treatment with Aspergillus flavus NRRL 21882 (Section III.B.3 of this BRAD). Nevertheless, screening of starter cultures by Vegetative Compatibility assays, plating, and growth in an enrichment culture, ascertain product identity of the pesticidal microbial active ingredient. The Agency also considered the potential for contamination by aflatoxins or unintentional ingredients associated with the pesticidal active ingredient, A. flavus NRRL 21882. Quality control and quality assurance methods are in place to ascertain that the pesticide itself is free of aflatoxins and that all batches containing aflatoxins and unintentional ingredients above regulatory levels must be incinerated or destroyed by appropriate technology. Lack of aflatoxins is determined by thin layer chromatography (TLC) or High Pressure Liquid Chromatography (HPLC). Kojic acid (KA), a metabolite associated with this group of fungi, is also found in other naturally occurring fungi, such as koji molds used for production of soy sauce. Levels of KA associated with this microbial active ingredient are much lower than the No Observed Adverse Aspergillus flavus NRRL 21882 May 24, 2004 Page 8 of 57 Biopesticides Registration Action Document FINAL DRAFT Effects Level (NOAEL) of 250 mg/kg. There is no US Food and Drug Administration (US FDA) action level for KA, and the acute oral toxicology studies demonstrated low toxicity potential (Toxicity Category IV). Batches of the pesticide with potential contaminants or unintentional ingredients above regulatory levels are to be destroyed. Thus, contamination of peanuts by the pesticide itself, or by its metabolites, is not likely if quality control measures assure product integrity. As discussed in detail in Section III.B.2-9 of this BRAD, no toxicity endpoints were indicated to justify setting a numerical tolerance for Aspergillus flavus NRRL 21882. Based on the Toxicity Category IV classification for acute oral toxicity, and regulatory programs already in place that address Aspergillus flavus and aflatoxin residues on peanuts, a safety factor is not required for residues of Aspergillus flavus NRRL 21882 on peanuts. In this assessment no acute, subchronic, chronic, immune, endocrine, or nondietary exposure issues have been identified which may have any incremental adverse effects on infants, children and the general U.S. population as discussed in this document. Dietary exposure via potential transfer of residues of Aspergillus flavus NRRL 21882, or its metabolites, to edible peanut food/feed commodities is not likely to pose an incremental risk above that which now exists from naturally occurring Aspergilli strains of fungi. Actually, treatment with the pesticide is likely to decrease aflatoxin levels in peanuts by 71-98% as demonstrated in efficacy trials (see Section III.D. of this BRAD). Residues of Aspergillus flavus NRRL 21882 and its potential metabolites are not expected to survive the heating (blanching, roasting) associated with preparing edible peanuts or processing peanuts into its byproducts, peanut butter and peanut meal. The fungal active ingredient and potential metabolites are also not likely to separate into peanut oil due to the high heat and solvents used in processing. Such residues are also not expected to be different on peanut hay in treated fields than in untreated fields, because the total levels of Aspergilli in the soil do not change in the long-term following treatment with the pesticide (Section III.D of this document). Furthermore, levels of A. flavus and potential metabolites of toxicological concern, such as the potent liver carcinogens, aflatoxins, on peanuts are regulated by the US Department of Agriculture (USDA) and the US FDA during marketing of peanut food/feed commodities (Section III.B.3 of this BRAD). The pesticide is not intended for direct application to water or to crops grown in water, and runoff is expected to be minimal to non-existent based on application of the pesticide to drought-ridden areas or to peanut crops which are not generally irrigated. Aspergillus flavus NRRL 21882 is not registered on any other food/feed commodity, such that cumulative exposure is not expected . Thus, dietary exposure (including drinking water), cumulative and aggregate exposure of Aspergillus flavus NRRL 21882 to consumers via consumption of treated peanuts, peanut butter and peanut oil, and secondary transfer to meat and milk via peanut meal or peanut hay, are not expected to be greater, but may even be less, than current existing levels. Aspergillus flavus NRRL 21882 May 24, 2004 Page 9 of 57 Biopesticides Registration Action Document FINAL DRAFT Occupational and Residential Exposure and Risk While there was some infectivity and pathogenicity potential, and 1 unscheduled death, in the acute intraperitoneal (IP) study in rodents, clearance was observed from all tissues by the end of the 22 day study. Generally, FQPA considerations only take into account non-occupational exposure, but, in this case, the Agency considered the impact of potential effects via routes of exposure similar to the IP study in relation to both occupational (worker) and non-occupational exposure. Potential exposure to workers and pesticide handlers of A. flavus NRRL 21882 is not expected to pose any incremental risk above that which currently exists. The pesticide demonstrates low toxicity, infectivity and pathogenicity potential by the acute oral or pulmonary routes. Occupational and non-occupational dermal and inhalation are expected, but mitigated as follows. Pesticide drift and non-occupational, as well as worker exposure, are minimized by (1) application as a large granular pesticide, in which the active ingredient adheres to the carrier; and (2) low application rates (less than 0.002 lb or approximately 1 gram of active ingredient per acre). Mitigation of exposure and risks to workers and pesticide handlers can be achieved by use of appropriate Personal Protective Equipment (PPE) and a Restricted-Entry Interval (REI) of 4 hours. In addition to the low exposure scenario noted above, non-occupational exposure is mitigated by application of the pesticide to commercial, agricultural sites, and not to residential areas. Thus, non-occupational or residential dermal and inhalation exposures are expected to be no greater than those expected from background A. flavus levels. (Section III.B.4 of this BRAD). Ecological and Environmental Exposure and Risks Ecological and environmental exposure and risk are summarized in Section III.C. of this BRAD. Evaluations of acceptable avian oral and inhalation infectivity/pathogenicity and honeybee exposure studies indicate low potential toxicity/pathogenicity effects of the pesticide and that the pesticide is not likely to pose any incremental adverse concerns to these non-target organisms. Data requirements were waived based on justifications that there were no recorded evidence of adverse effects to most non-target insects. In addition, total A. flavus levels do not increase following the single seasonal application of the pesticide. The low application rate of this naturally occurring soil colonizer to drought ridden peanut fields suggests minimal to no accumulation in water. Thus, the justifications to waive test data for freshwater fish, estuarine and marine vertebrates and invertebrates, and terrestrial non-target plants and endangered species are acceptable for the proposed uses. Data Gaps and Requirements/Labeling All deficiencies and labeling must meet Agency requirements (Section V.C of this document). Standard analysis of 5 production batches and efficacy data from a large scale field trial are required as conditions of registration (Section VI of this BRAD). If more extensive use patterns are sought for treatment of other non-agricultural or agricultural sites or crops, additional information and data will be required on a case-by-case basis.