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: [Kimberly Nesci, 703-

308-8059]

SUBMISSION: E-mail the completed template to: nesci.kimberly@epa.gov

TEMPLATE:

[bio-ferm GmbH]

[ 4F8342]

EPA has received a pesticide petition (4F8342) from [bio-ferm GmbH], 

[Technologiezentrum Tulln, Technopark 1, 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)

to establish an amendment/expansion of an existing tolerance exemption for the

(Options (pick one)

1. microbial pesticides [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

[4F8342]
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A. Product Name and Proposed Use Practices

[Two end-use products containing equal parts of Aureobasidium pullulans

strains DSM 14940 and DSM 14941 are currently registered by EPA. Blossom 

Protect is applied to pome fruit and walnuts only during the blossoming period to 

protect plants against bacterial fire blight and walnut blight, respectively. Botector 

is currently applied pre-harvest to grapes, pome fruit, stone fruit, and strawberries. 

Bio-ferm proposes to amend the Botector label to permit post-harvest uses, 

presently only to citrus, to protect these crops against fruit rot diseases caused by 

blue and green mold caused by Penicillium spp. and sour rot caused by Geotrichum

spp. Post-harvest application rates are projected to be up to 1 pound of Botector per 

60 gallons of water (as a 0.2% spray or dip).]

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 intraspecies 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.]
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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 a post-harvest 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 to, and reviewed and accepted by EPA in support of 

registration of Aureobasidium pullulans strain DSM 14941 (EPA Reg. No. 86174-1) 

and Aureobasidium pullulans strain DSM 14940(EPA Reg. No. 86174-2).

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 4x108 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
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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% (2x1010 CFU/g) A. pullulans strain DSM 14940, 

22% (2x1010 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 2.36x1010 CFU/g of strain DSM 14940 and 

1.5x1010 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 articletreated (strain DSM 14941). Test article-treated animals received 1.1x109 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, 
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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 7x109 CFU/g) and 56% toxicologically benign inert 

ingredients. One male animal was sacrificed for humane reasons five days postdosing 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 7x109 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 4x108 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 
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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 7x109 CFU/g each of strains 

DSM 14940 and DSM 14941. The MMAD was 4.2 μm. 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 postapplication. 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 
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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 nondietary 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 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 
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demonstrated by the micronucleus study summarized above. When A. pullulans

strains DSM 14940 and DSM 14941 are used on raw agricultural commodities preand post-harvest 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 is an existing pre-harvest tolerance exemption for Aureobasidium 

pullulans strains DSM 14940 and DSM 14941 at 40 CFR 180.1312.]

I. International Tolerances

[Aureobasidium pullulans is included in Annex IV (List of substances for 

which no MRL is required) in the EU. 

In Canada, no MRL was required to be established for A. pullulans.

Both strains are approved or provisionally approved for pre- and post-bloom use 

against fire blight in pome fruit in Austria, Belgium, France, Greece, Hungary, 

Italy, Slovakia, Slovenia, Germany, Portugal, Spain, Canada, Switzerland, and 

Turkey.

Both strains are approved or provisionally approved for use against storage diseases 

in pome fruit in Austria, Belgium, Greece, Hungary, Italy, The Netherlands, Poland, 

Slovakia, Slovenia, and Germany

Both strains are approved or provisionally approved for use against gray mold 

(Botrytis) in grapes in Austria, France, Greece, Hungary, Italy, Portugal, Slovakia, 

Slovenia, Germany, Canada, and Switzerland.]