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EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION COMPANY NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL REGISTER EPA Biopesticides and Pollution Prevention Division contact: Driss Benmhend, 703 - 308 - 95 25, benmhend.driss@epa.gov.

INSTRUCTIONS: Please utilize this outline in preparing the pesticide petition. In cases where the outline element does not apply, please insert “NA- Remove” and maintain the outline. Please do not change the margins, font, or format in your pesticide petition. Simply replace the instructions that appear in green , i.e., “ [ insert company name ] ,” with the information specific to your action. SUBMISSION: E - mail the completed template to: hollis.linda@epa.gov. TEMPLATE: DSM Food Specialties B.V. Petition number to be assigned. EPA has received a pesticide petition (To be assigned) from DSM Food Specialties B.V. , Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands, proposing, pursuant to section 408(d) of the F ederal Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 346a(d), to amend 40 CFR part 180. (Options (pick one) NA

- Remove: 1. b y establishing a tolerance for residues of 2. to establish an exemption from the requirement of a tolerance for NA

- Remove: 3. to establish an amendment/expansion of an existing tolerance exemption for the (Options (pick one) NA

- Remove: 1. microbial pesticide [insert name of active ingredient] 2. biochemical pesticide (6,11,28 - Trioxatricyclo[22.3.1.05,7]oct acosa 8,14,16,18,20 - pentaene - 25 - carboxylic acid, 22 - [(3 - amino - 3,6 - dideoxy-  -D -2

mannopyranosyl)oxy] - 1,3,26 - trihydroxy- 12 - methyl - 10 - oxo - , (1R,3S,5R,7R,8E,12R,14E,16E,18E,20E,22R,24S,25R,26S) - , CAS Registry No. 7681 - 93 - 8.

NA

- Remove: 3. plant

- pesticide [i nsert name of active ingredient] in or on [insert commodity]. Pursuant to section 408(d)(2)(A)(i) of FFDCA, as amended, DSM Food Specialties has submitted the following summary of information, data, and arguments in support of their pesticide petition

. This summary was prepared b y DSM Food Specialties and EPA has not fully

evaluated the merits of the pesticide petition. The summary may have been edited b y EPA if the terminology used was unclear, the summary contained extraneous material, or the summary unintentionally made the reader conclude that the findings reflected EPA’s position and not the position of the petitioner. I. DSM Food Specialties Petition Summary Petition number to be assigned . A. Product Name and Proposed Use Practices T he Natamycin active ingredient (AI), a polyene macrolide antimycotic biochemical substance, was discovered in a soil sample obtained near Pietermaritzburg in the state of Natal in South Africa. This biochemical substance was found to have broad, potent an tifungal properties, but no antibacterial activity. The lack of antibacterial properties is a distinctive advantage for a fungicidal pesticide because then there is no basis for concern over development of resistance by human pathogenic bacteria. Duri ng mushroom production, Natamycin will be used in a broad spectrum liquid fungicidal formulation containing approximately 10% by weight of the technical grade active ingredient (TGAI) as a stable aqueous suspension of crystals. This product, Natamycin L, has anti - germinating properties and is designed for the control of moulds and yeasts in the growth media of mushrooms. Natamycin L can be applied to the growth substrate up to four times during a typical production cycle for mushrooms. Pre - harvest interv als from a few days to more than 20 days . B. Product Identity/Chemistry 1. Identity of the pesticide and corresponding residues. Natamycin. No residues other than Natamycin were found. 2. Magnitude of residues at the time of harvest and me thod used to determine the residue. The proposed use in the present application is for the control of Verticillum3

(Dry Bubble) disease and other mushroom parasites in growing mushrooms. The application produces Natamycin residue levels in mushrooms in th e 0.15 - 0.25 ppm range. After rinsing, the residues for the same mushrooms decrease to 0.022 - 0.076 ppm. No metabolites nor degradation products of Natamycin were found. LC MS/MS methods were used for the analyses. Quantification of Natamycin residue s was accomplished relative to an external calibration curve. The limit of Quantitation (LOQ) for the Natamycin residue method was 0.01 ppm in mushrooms. The limit of detection was assigned as the lowest concentration within the calibration curve or 0.25ng/ml. The analytical method to detect and measure natamycin residues and residues of metabolites, if they were present, was fully validated. 3. A statement of why an analytical method of detecting and measuring the levels of the pesticide residue are no t needed. A worst- case estimate of the Natamycin exposure in the diet were calculated using the following: 1) Projected (and increased) mushroom intake several years into the future; 2) Use of the mushroom consumption data of the most highly exposed popul ation group; and 3) Use of the maximum residue found in the crop field trials in any treated mushroom, rather than the most probable residue. On this basis, calculations for exposure to Natamycin through consumption of treated mushrooms yielded a Margin o f Exposure (MOE) of approximately one million. This very large worst- case MOE clearly establishes the safety of Natamycin for the proposed use on mushrooms. Additionally, under normal conditions mushrooms are rinsed prior to consuming and the actual MOE will be substantially greater than a million. Because the MOE is so large even when worst- case assumptions of exposure are made, an exemption from tolerance rather than a numeric tolerance is appropriate, and an analytical method for detecting and measu ring pesticide residues in mushrooms is not needed. C. Mammalian Toxicological Profile Acute Toxicity Profile ACUTE ORAL TOXICITY

– Rat Toxicity Category III The test substance, Natamycin, was evaluated for its acute oral toxicity potential in f emale albino rats when administered as a gavage dose at a level of 2000 mg/kg. The study was terminated following the stopping rules of this procedure. One of five animals died during the study. There were no clinical signs of toxicity in survivors during the study; polyuria was observed in the animal that died on test. There was no effect on body weight gain in animals surviving to termination. Abnormal necropsy findings occurred only in the animal that died on test, and pertained to anogenital area, lungs and contents of the intestines. The acute oral LD 50 was estimated to be greater than 2000 mg/kg.4

ACUTE DERMAL TOXICITY

– Rat Toxicity Category IV The test substance, Natamycin, was evaluated for its dermal toxicity potential and relative skin irritancy when a single dose moistened with 1.0 mL of deionized water/g test substance, at a level of 5050 mg/kg, was applied to the intact skin of albino rats. No mortality occurred during the study. There were no clinical signs of toxicity or signs of dermal irritation at any time throughout the study. There was no effect on body weight gain, with the exception of one animal that failed to gain weight during the first week. The gross necropsy conducted at termination of the study revealed no observable abnormalities. The estimated LD50, as indicated by the data, was determined to be greater than 5050 mg/kg. ACUTE INHALATION TOXICITY

– Rat Toxicity Category IV The test substance, Natamycin, was evaluated for its acute inhalation toxicity potential in albino rat s. Five males and five females were exposed for four hours to an aerosol generated from the undiluted test substance (fine powder) at a level of 2.39 mg/L. There was no mortality during the study. Clinical signs included activity decrease and piloerection, which were no longer evident by Day 3. Body weights were unaffected by exposure. The gross necropsy revealed no observable abnormalities. As indicated by the data, the acute inhalation LC50 is greater than 2.39 mg/L. EYE IRRITATION

– Rabbit Toxicity Cat egory IV An acute eye irritation study was conducted on three albino rabbits using test substance Natamycin. The test substance, 0.1 mL by volume (43.7 mg), was placed into the conjunctival sac of the right eye of each animal selected for testing. All tr eated eyes were washed with room temperature deionized water for one minute immediately after recording the 24 - hour observation. A maximum average irritation score of 62.0, severely irritating, was obtained 1 hour after treatment, but there were no positive effects exhibited in any eyes at 24 hours after treatment. Hence, the initial irritation score could not be maintained and Natamycin TGAI was assigned to Toxicity Category IV. SKIN IRRTIATION

– Rabbit Toxicity Category IV A primary dermal irritati on study was conducted on three albino rabbits using test substance Natamycin. There was one intact test site per animal. Each test site was treated with 500 mg of test substance moistened with 0.5 mL of deionized water and covered with a semi - permeable dr essing. The test substance was maintained in contact with the skin for 4 hours. Observations for dermal irritation and defects were made at 1, 24, 48 and 72 hours after removal of the dressings.5

Based on the PII of 0.1, the test substance is rated slightly irritating. Based on the scores at the 72 - hour observation only, the test substance is assigned to Toxicity Category IV. SKIN SENSITIZATION

– Mice Not a Sensitizer A skin sensitization study was conducted on 3 groups of 5 female mice to determine if test substance Natamycin possesses a significant potential to cause skin sensitization. Five females were assigned to each of three groups, designated Groups I - III. The test groups were treated with an appropriate dilution (25% or 50%) in propylene glyco l vehicle, or undiluted test substance. Each animal received 25 μL to the dorsum of each ear. The animals were treated once daily for three days. After a two - day rest period, all animals were injected with tritiated methyl - thymidine in the tail vein. Five hours later, the animals were sacrificed, and the draining auricular lymph nodes removed and prepared for cell suspension and scintillation counting. A vehicle control group of five females was run concurrently, treated in the same manner with vehicle only instead of test substance or dilution. A positive control group of five females was also run concurrently, treated with 90% alpha hexylcinnamaldehyde in acetone:olive oil. The test substance produced a stimulation index of < 3 in all groups of test animals, and is not therefore considered a sensitizer (defined as producing a positive response). Genotoxicity. NATAMYCIN was tested in the Salmonella typhimurium reverse mutation assay with four histidine - requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan - requiring strain of Escherichia coli WP2uvrA . The test was performed in two independent experiments in the presence and absence of S9 - mix (Aroclor 1254 induced ra t liver S9 - mix). Based on the results of this study it is concluded that NATAMYCIN is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coil reverse mutation assay. NATAMYCIN (88.9% purity) was tested to determine t he number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor- 1254 induced rat liver S9 - mi x) . NATAMYCIN did not induce a statistically significant or biologically relevan t increase in the number of cells with chromosome aberrations in the absence and in the presence of S9 - mix, in two independently repeated experiments. NATAMYCIN is not clastogenic in human lymphocytes under the experimental conditions described in this rep ort. Reproductive and Developmental Toxicity6

Natamycin (as Delvocid 5%) was administered daily during days 6 - 18 of gestation by oral intubation to groups of 15 to 19 female Dutch Belted rabbits at concentrations of 0, 5, 15, or 50 mg/kg - day . The admini stration of up to 50 mg/kg (body weight) - day of test material for 13 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham - treated controls. From the results presented in this report the No Observed Adverse Effect Level (NOAEL) for the teratology part of the study was established to be 50 mg/kg/day. Subchronic T oxicity In a 90 day study, Natamycin was administered daily via the diet to groups of 20 Wistar rats (10 males and 10 females) at concentrations of 0, 125, 500 and 2000 ppm. Dietary exposure to Natamycin resulted in no treatment related findings in the 1 25 mg/kg/day and 500 mg/kg/day dose groups. The 2000 mg/kg/day dose group had reduced body weights (males and females), increased alanine aminotransferase activity in individual males, increased urea and inorganic phosphate values (males and females) and p otassium values (males), and reduced cholesterol and total protein values (females). From the results presented in this report the No Observed Adverse Effect Level (NOAEL) was established to be 500 ppm Natamycin, equivalent to approximately 38 and 43 mg/kg/day (corrected for food scatter) for males and females respectively. Toxicology Profile -- Existing U.S. Government findings of safety Natamycin (pimaricin) has been approved by FDA in 21 CFR Part 172, Sec. 172.155 for use as a direct food additive f or use on cheese. “The additive may be applied on cheese, as an antimycotic, in amounts not to exceed 20 milligrams per kilogram (20 parts per million) in the finished product . . .”. Natamycin as a 5% ophthalmic suspension is approved as an antifungal d rug for topical ophthalmic administration . In 21 CFR Part 573, Sec. 573.685 FDA has approved Natamycin for use in broiler chicken feeds at up to 10 grams per pound of food . Toxicology Profile

– International Findings of Safety The Food and Agricultur al Organization of the United Nations (FAO) and the World Health Organization (WHO) through the Joint FAO/WHO Expert Committee on Food Additives (JECFA), evaluated Natamycin, most recently in 2002, for safety as a direct food additive . The Committee concl uded that “Although use of Natamycin as an antifungal agent in food may result in exposure of the endogenous flora to trace quantities of antimicrobial residues, bacteria in the human gastrointestinal tract are not affected by polyenes, and the Committee c oncluded that disruption of the colonization barrier is not a concern. Fungi are found in much smaller amounts than bacteria in the human gastrointestinal tract, and the negative results in studies of acquired resistance indicate that selection of7

Natamyci n - resistant fungi is not an issue. The Committee confirmed the previously established ADI of 0

–0.3 mg/kg bw for Natamycin, which was based on observations of gastrointestinal effects in humans.” The Animal Metabolism of Natamycin As described in the WHO evaluation , studies have been done to determine the fate of Natamycin in rats and dogs. In rats, a series of studies showed that the material was not absorbed and was eliminated in the feces and urine. The results of the autoradiographic study indicate th at Natamycin is minimally absorbed into the bloodstream and excreted almost entirely in the feces. Other experiments gave similar results. In the stomach and small intestine, Natamycin was mostly untransformed, as indicated by thin - layer chromatography. Mo st degradation took place in the large intestine. The degradation products were more hydrophobic than Natamycin and were found from about 4 h after treatment. Most of the dose of 10 mg/kg was degraded about 8 h after treatment, suggesting that elimination is relatively rapid. Biotransformation was attributed to the bacterial flora in the caecum and small intestine. Overall, no more than 5

–7% of the total radioactive dose was absorbed after oral administration of [14C]Natamycin, and approximately 90% of the administered compound passed through the gastrointestinal tract without resorption and was eliminated in the faeces . In dogs, after oral administration, most of the radiolabel was eliminated in the faeces within 24 h, with less than 4% of the total dose i n urine. The WHO assessment concluded that there was very little data on humans, but one study had shown that “No Natamycin (< 1 μg/ml) could be detected in the blood after ingestion of 500 mg by human subjects”, indicating that it was unlikely to be absor bed by humans. D. Aggregate Exposure 1. Dietary exposure. Natamycin is currently permitted as an FDA food additive, and is approved as an antimycotic for use in the finished cheese at a level of 20 ppm. (See 21 CFR § 172.155). The lowest NOEL for Nat amycin is 25 mg/kg bw/day or approximately 1.75 g/person per day. The proposed use in the present application is for the control of Verticillum disease and other mushroom fungal parasites in growing mushrooms. The application produces Natamycin residue levels in mushrooms in the 0.15 - 0.25 ppm range. After rinsing, the residues for the same mushrooms decrease to 0.022 - 0.076 - ppm . i. Food. The current U.S. annual per capita mushroom consumption is 4.00 pounds. The projected maximum, mushroom consu mption for the current decade to the year 2012 is 4.86 pounds. The maximum daily intake of Natamycin residues for the proposed use of Natamycin is conservatively estimated to be 32 ng/kg.bw/day resulting in a Margin of Exposure of approximately one millio n.8 ii. Drinking water. All uses of Natamycin L will be indoor, and natamycin has only very limited (~30 - 40 ppm at neutral pH) solubility in water. Hence, exposure through drinking water is not expected. 2. Non

- dietary exposure

. The only non - die tary exposures from pesticidal uses of the Natamycin will be occupational, i.e ., commercial applicator/mixer loader exposures. Occupational exposures are not included under the Federal Food, Drug and Cosmetic Act (FFDCA) in the assessment of aggregate exp osures for the purpose of establishing tolerances and exemptions from tolerance. E. Cumulative Effects In assessing the cumulative effects of Natamycin it should be recognized that it is one of a class of polyene antimycotics naturally present in the e nvironment. Others of the class are also typically obtained from some species of Streptomyces bacteria. The amount of material likely to be present as residues of Natamycin on mushrooms results in so small an incremental exposure as to be negligible. F . Safety Determination 1. U.S. population. As discussed above, when expressed in pounds per day, per capita mushroom use is highest among men and women aged 20 - 39, and lowest for children under the age of 12. When expressed on a kilogram body weight b asis, the intakes for all individuals are all within a factor of 2, with the 29 - 39 age group still being slightly higher consumers, particularly in females. 2. Infants and children

. On a unit bodyweight basis, there is no significant difference in children’s consumption from that of the average adult. G. Effects on the Immune and Endocrine Systems Because Natamycin is minimally absorbed into the bloodstream and rapidly excreted almost entirely in the feces with minimal degradation, effects on th e immune and endocrine systems are not expected. The limited biotransformation found was attributed to the bacterial flora in the caecum and small intestine, and the degradation products were more hydrophobic than Natamycin. H. Existing Tolerances There are no existing U.S. tolerances for Natamycin as a pesticide. I. International Tolerances No international tolerances exist for use of Natamycin as a pesticide.9