Document ID: EPA-HQ-OPP-2010-0099-0009
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2010-03-10T05:00Z

<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: [Sheryl Reilly, 703-308-8269]>

<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:>

<[bio-ferm GmbH]>

<[Insert petition number]>

<	EPA has received a pesticide petition ([insert petition number]) from [bio-ferm GmbH], [Konrad Lorenz Str. 20, Tulln, 3430, Austria] proposing, pursuant to section 408(d) of the Federal Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 346a(d), to amend 40 CFR part 180.>

<(Options (pick one)>
   
<	2. to establish an exemption from the requirement of a tolerance for>
	

<(Options (pick one)>
   
   	1. microbial pesticide  [Aureobasidium pullulans strains DSM 14940 and DSM 14941]

<	Pursuant to section 408(d)(2)(A)(i) of  FFDCA, as amended, [bio-ferm GmbH] has submitted the following summary of information, data, and arguments in support of their pesticide petition. This summary was prepared by [SciReg, Inc.] and EPA has not fully evaluated the merits of the pesticide petition. The summary may have been edited by 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. [bio-ferm GmbH]  Petition Summary>
   
   	[9F7623]

<A. Product Name and Proposed Use Practices>

<	[Two end-use materials containing equal parts of Aureobasidium pullulans strains DSM 14940 and DSM 14941 are being submitted to EPA for registration. Blossom Protect will be applied to pome fruit only during the blossoming period to protect plants against bacterial fire blight. Botector will be applied pre-harvest to grapes, pome fruit, stone fruit, and strawberries, and post-harvest to citrus to protect these crops against fruit rot diseases caused by Botrytis sp., Monilia sp., Penicillium sp., Nectria sp., and Pezicula sp. Application rates are projected to be up to 1.4 lbs/acre pre-bloom, as a 1% spray solution post-bloom, or as a 0.03% treatment post-harvest.]>

<B. Product Identity/Chemistry>

<	1. Identity of the pesticide and corresponding residues. [Both strains, DSM 14940 and DSM 14941, were isolated from apple leaves and classified as Aureobasidium pullulans by the German Strain Collection for Microorganisms (DSMZ). For this organism, no sexual reproduction is known. Aureobasidium is classified into the phylum Ascomycota. Based on molecular data, a close relationship to species of the order Dothideales of the class Euascomycetes was proven.

Strain DSM 14940 is a very closely related to strain DSM 14941. The two strains show similar colony characteristics on agar plates; they are identical on their 18SrRNA gene region; and they are also identical on their Internal Transcribed Spacer regions ITS1 and ITS4, which are commonly used for inter- and intra-species differentiation. Definitive differentiation from each other can only be achieved by molecular biological methods focusing on the whole genome, by conducting such analyses as RAPD PCR (Random Amplification of Polymorphic DNA Polymerase Chain Reaction) or by using recently developed strain-specific primer characteristics.

Aureobasidium pullulans strains DSM 14940 and DSM 14941 were identified at DSMZ by means of morphological characterization of the colonies on yeast medium (YM) agar plates. Further confirmation of these strains can be achieved via PCR. PCR reveals a specific band that distinguishes A. pullulans strains DSM 14940 and DSM 14941 from other isolates of A. pullulans and from other yeast-like fungi. The subject strains are neither mutant strains nor genetically modified strains.

Aureobasidium pullulans is a ubiquitous and widespread oligotrophe that can be found in environments with fluctuating water activities, such as the phyllosphere, bathrooms, food, and feeds. It can also be found in highly osmotically stressed environments, such as hypersaline waters in salterns, and rocks and monuments. Due to the production of large quantities of yeast-like propagules, this fungus disperses globally.]>

<	2. Magnitude of residues at the time of harvest and method used to determine the residue. [No magnitude of residue (MOR) studies have been conducted with the strains of Aureobasidium pullulans based on the low toxicity of the active ingredient strains (see Toxicology Profile, pages 5-10). OPPTS Guideline 885.2000, section f, Background for Residue Analysis of Microbial Pest Control Agents (MCPA), states: 

"The Agency evaluates residue data for MCPAs used on food, feed, or raw agricultural commodities only if toxic or other harmful properties were observed in the maximum hazard toxicology tests (Tier I) prescribed in toxicology test guidelines OPPTS 885.3050 through 885.3650 of this series. If Tier I toxicology tests indicate no toxic or other harmful properties, then no residue data (with the general exception of a monitoring method) would be indicated and thus a recommendation for an exemption from the requirements of a tolerance can be made."]>

<	3. A statement of why an analytical method of detecting and measuring the levels of the pesticide residue are not needed. [SciReg, Inc. is submitting a petition to establish an exemption from the requirement of a tolerance and, therefore, an analytical method is not required.]>

<C. Mammalian Toxicological Profile>

<	[The full reports that support this petition and which are summarized in this section were submitted with the active ingredient registration application for Aureobasidium pullulans strain DSM 14941.

A subcutaneous toxicity/pathogenicity study was conducted that evaluated both strains separately. No toxicity or pathogenicity was observed. Neither Aureobasidium pullulans strain DSM 14940 nor strain DSM 14941 show any acute toxicity or pathogenicity by any of the required routes of exposure. 

Acute Oral Toxicity/Pathogenicity: Twenty-four animals were dosed at 4x10[8] CFU/g of A. pullulans strain DSM 14941. Interim sacrifices were made on three animals per sex on Days 3, 7, and 14; the remaining animals were sacrificed on Day 21, the termination of the study. There were no deaths and all animals appeared normal throughout the entire study. Feces, blood, brain, lung, liver, kidney, spleen, lymph nodes, stomach, intestine, and target organs (none) were evaluated on Day 0 (feces only) and Day 3, 7, 14, and 21 (all other tissues) for test article-treated animals, untreated control animals, and inactivated test article-treated animals). A mycological summary of these evaluations follows.

Feces: All samples negative for A. pullulans
Blood: All samples negative for A. pullulans
Brain: All samples negative for A. pullulans
Lung: All samples negative for A. pullulans
Liver: All samples negative for A. pullulans
Kidney: All samples negative for A. pullulans
Spleen: All samples negative for A. pullulans
Lymph nodes: All samples negative for A. pullulans, except for one animal sacrificed on Day 7
Stomach: All samples negative for A. pullulans
Intestine: All samples negative for A. pullulans

Acute Oral Toxicity: Six animals were dosed at 2,000 mg/kg of the end-use product, Blossom Protect, comprised of 22% (2x10[10] CFU/g) A. pullulans strain DSM 14940, 22% (2x10[10] CFU/g) A. pullulans strain DSM 14941, and 56% toxicologically benign inert ingredients. There were no deaths and all animals appeared normal throughout the entire study. The LD50 was determined to be greater than 2,000 mg/kg.

Acute Subcutaneous Toxicity/Pathogenicity: 
Study 1: Sixty animals were allocated to groups of three animals per sex as follows: Untreated control, inactivated control (strains DSM 14940 and DSM 14941), and test article-treated (strains DSM 14940 and DSM 14941, three groups per strain). Test article-treated animals received 2.36x10[10] CFU/g of strain DSM 14940 and 1.5x10[10] CFU/g of strain DSM 14941. Interim sacrifices were made on three animals per sex on Days 3, 7, and 14; the remaining animals were sacrificed on Day 21, the termination of the study. There were no deaths and all animals appeared normal throughout the entire study, except for edema or slight erythema at the injection site. Feces, blood, brain, lung, liver, kidney, spleen, lymph nodes, stomach, intestine, and target organs (none) were evaluated on Day 0 (feces only) and Day 3, 7, 14, and 21 (all other tissues) for test article-treated animals, untreated control animals, and inactivated test article-treated animals). A mycological summary of these evaluations follows.

Feces: All samples negative for A. pullulans
Blood: All samples negative for A. pullulans
Brain: All samples negative for A. pullulans
Lung: All samples negative for A. pullulans
Liver: All samples negative for A. pullulans
Kidney: All samples negative for A. pullulans
Spleen: All samples negative for A. pullulans
Lymph nodes: All samples negative for A. pullulans, except for one animal sacrificed on Day 7
Stomach: All samples negative for A. pullulans
Intestine: All samples negative for A. pullulans

Study 2: Thirty-six animals were allocated to groups of three animals per sex as follows: Untreated control, inactivated control (strain DSM 14941), and test article-treated (strain DSM 14941). Test article-treated animals received 1.1x10[9] CFU/g. Interim sacrifices were made on three animals per sex on Days 1 (control and test article-treated animals) and 7 (test article-treated animals); the remaining control, inactivated control, and test article-treated animals were sacrificed on Day 28, the termination of the study. There were no deaths and all animals appeared normal throughout the entire study, except for slight to severe edema and slight erythema at the injection site. Skin, brain, liver, lung, spleen, kidney, lymph nodes, blood, cecum contents, and urine were evaluated on their respective sacrifice days. A mycological summary of these evaluations follows.

Skin: Positive for A. pullulans (three males, three females from test article-treated group sacrificed on Day 1; two females from test article-treated group sacrificed on Day 7)
Brain: All samples negative for A. pullulans
Liver: Positive for A. pullulans (one male from test article-treated group sacrificed on Day 1)
Lung: All samples negative for A. pullulans
Spleen: All samples negative for A. pullulans
Kidney: All samples negative for A. pullulans
Lymph nodes: All samples negative for A. pullulans
Blood: All samples negative for A. pullulans
Cecum contents: Positive for A. pullulans (one male and one female from test article-treated group sacrificed on Day 1; two females from test article-treated group sacrificed on Day 7)
Urine: All samples negative for A. pullulans

Acute Subcutaneous Toxicity: Ten animals were dosed at 2,000 mg/kg of the end-use product, Blossom Protect, containing 22% each of strains DSM 14940 and DSM 14941 (each strain contained 7x10[9] CFU/g) and 56% toxicologically benign inert ingredients. One male animal was sacrificed for humane reasons five days post-dosing due to abscess rupture at the injection site. All other animals survived for the duration of the 14-day study. In-life observations included a bladder formed by the injection of the test material at the injection site, dermal swelling at the injection site, and ruptured abscess at the injection site. Necropsy observations included abscesses at all injection sites. The LD50 was determined to be greater than 2,000 mg/kg.

Acute Dermal Toxicity: Ten animals were dosed at 2,000 mg/kg of Blossom Protect containing 7x10[9] CFU/g each of strains DSM 14940 and DSM 14941. One male and one female animal exhibited chromodacryorrhea, but this was not considered to be toxicologically significant. All animals appeared healthy at necropsy. The LD50 was determined to be greater than 2,000 mg/kg.

Acute Pulmonary Toxicity/Pathogenicity: Forty-two animals were assigned to control (2 groups, each containing 2 animals per sex) and treated groups (main group, 5 animals per sex; interim sacrifice groups, 3 animals per sex). Treated animals were dosed at 4x10[8] CFU/g of strain DSM 14941. Interim sacrifices were made on three animals per sex on Days 0 (3 hours post-application), 3, 7, and 14; the remaining animals were sacrificed on Day 21, the termination of the study. There were no deaths and, except on the day of dosing when all treated animals showed reduced motor activity and dyspnea, all animals appeared normal throughout the entire study. Feces, blood, brain, lung, liver, kidney, spleen, stomach, small and large intestine, and lymph nodes were evaluated on Day 0 (feces only) and Day 3, 7, 14, and 21 (all other tissues) for untreated control and test article-treated animals. A mycological summary of these evaluations follows.

Feces: All samples negative for A. pullulans
Blood: All samples negative for A. pullulans, except for one male on Day 0
Brain: All samples negative for A. pullulans
Lung: All samples negative for A. pullulans, except for two males and two females on Day 0
Liver: All samples negative for A. pullulans, except for three males on Day 0
Kidney: All samples negative for A. pullulans
Spleen: All samples negative for A. pullulans, except for two males on Day 0
Lymph nodes: All samples negative for A. pullulans, except for one animal sacrificed on Day 7
Stomach: All samples negative for A. pullulans, except for two males on Day 0
Intestine: All samples negative for A. pullulans

Acute Inhalation Toxicity: Ten animals were dosed (nose only) with a 10% suspension of Blossom Protect at 5.17 mg/L containing 7x10[9] CFU/g each of strains DSM 14940 and DSM 14941. The MMAD was 4.2 um. There were no deaths and all animals appeared healthy throughout the study. There were no necropsy findings. The LC50 was determined to be greater than 5.17 mg/L.

Eye Irritation: Three rabbits were dosed with 0.1 mL (60-62 mg) of Blossom Protect. One animal exhibited a score of 1 for conjunctival redness one hour post-application. The corneas and irises were unaffected. There were no other clinical signs. All animals appeared normal 24 hours post-application. Blossom Protect is considered to be virtually non-irritating.

Dermal Irritation: Three rabbits were dosed with 0.5 g of Blossom Protect moistened with 1.0 mL deionized water. All animals appeared normal throughout the study. Blossom Protect is considered to be virtually non-irritating.

Hypersensitivity Incidents: No reports of hypersensitivity have been associated with A. pullulans strains DSM 14940 or DSM 14941.

In vivo Micronucleus: A. pullulans strain DSM 14941 was dissolved in water and administered by oral gavage one time to groups of mice at 2,000 mg/kg. Untreated, negative (water), and positive control (cyclophosphamide, IP injection, 10 mg/kg) were also evaluated. There were no in-life clinical signs. Animals were sacrificed 24 or 48 hours post-application and the femoral bone marrow was immediately harvested. The number of polychromatic erythrocytes to total erythrocytes (mature and immature) was determined. Two thousand immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. A. pullulans strain DSM 14941 did not induce a statistically significant increase in micronucleated immature erythrocytes compared to negative and untreated control animals. All positive control groups showed a significant increase in the number of micronucleated immature erythrocytes.]>

<D. Aggregate Exposure>

<	1. Dietary exposure. [The potential for non-occupational, dietary, and non-dietary exposure to Aureobasidium pullulans by the general population, including infants and children, is likely through ingestion of treated fruit.]>

<	i. Food. [Dietary exposure to A. pullulans should not be of concern due to the low toxicity shown across the entire toxicological profile (see above). If these strains of A. pullulans are ingested, none of the studies have indicated any pathogenicity of the organism in mammals. In addition, the subject strains grow optimally at 27°C and 29°C and no growth has been noted at the normal human body temperature of 37°C.]>

<	ii. Drinking water. [Aureobasidium pullulans does not pose a risk to drinking water. Anticipated agricultural practice prohibits direct application of the end-use product directly to water. Aureobasidium pullulans is not likely to be present in aquatic environments since it is not known to inhabit aquatic environments.]>

<	2. Non-dietary exposure. [Non-dietary exposure to Aureobasidium pullulans will be much more likely through handling by formulators and applicators, but in light of the subject strains' toxicological profile, should be of little concern. Although the organism is ubiquitous, levels of exposure in broad segments of the world's population have not resulted in any significant reported adverse effects.]>

<E. Cumulative Effects>

<	[Section 408(b)(2) of the FFDCA requires that, when considering whether to establish an exemption from a tolerance, the Agency consider available information concerning the cumulative effects of the particular ingredient's residues and other substances that have a common mechanism of toxicity. These considerations include the possible cumulative effects of such residues on infants and children. Organisms that control fruit rots on various species occupy pathogen infection sites; thus, this passive mode of action does not have a common chemical-based mechanism of toxicity as can sometimes be found with typical organic chemicals. While numerous open literature studies suggest some strains of A. pullulans have deleterious effects on humans, none of these strains are the ones to be registered by bio-ferm.]>

<F. Safety Determination>

<	1. U.S. population. [Aureobasidium pullulans exhibits low mammalian toxicity as demonstrated by the toxicological profile and did not induce genetic toxicity as demonstrated by the micronucleus study summarized above. When A. pullulans strains DSM 14940 and DSM 14941 are used on raw agricultural commodities in accordance with good agricultural practice, it meets the reasonable certainty of no harm requirement.]>

<	2. Infants and children. [It is believed that the potential exposure of infants and children to residues of Aureobasidium pullulans strains DSM 14940 and DSM 14941 through their use to protect plants and fruit against diseases is no greater than that of the general population. Available information does not suggest that infants and children are more susceptible to the effects of A. pullulans than are adults.]>

<G. Effects on the Immune and Endocrine Systems>

<	[Extensive literature searches and review of Aureobasidium pullulans strains DSM 14940 and DSM 14941 toxicity data indicate there is no evidence that these strains are endocrine disruptors or that they have any known effect on immune systems.]>

<H. Existing Tolerances>

<	[There are no known agricultural tolerances or tolerance exemptions for Aureobasidium pullulans strains DSM 14940 and DSM 14941.]>

<I. International Tolerances>

<	[There are no known international tolerances for Aureobasidium pullulans strains DSM 14940 and DSM 14941, though both strains are approved for pre- and post-bloom use against fire blight in pome fruit in Austria (2004), Hungary (2007), Slovakia (2007), and Slovenia (2008).]>